Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 6

Ribosome Associated Factors Recruited for Protein Export and Folding

AMANDA RAINE

ISSN 1651-6192ISBN 91-554-6182-4urn:nbn:se:uu:diva-4839

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2005

List of papers

I Raine A, Ullers R, Pavlov M, Luirink J, Wikberg JE, Ehren-berg,M. (2003). Targeting and insertion of heterologous mem-brane proteins in E. coli. Biochimie 85, 659-659

II Ullers R, Houben EN, Raine A, ten Hagen-Jongman CM, Ehren-berg M, Brunner J, Oudega B, Harms N & Luirink J. (2003). In-terplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome. J Cell Biol 161, 679-684

III Raine A, Ivanova N, Wikberg JE & Ehrenberg M. (2004) Simul-taneous binding of trigger factor and signal recognition particle to the E. coli ribosome. Biochimie 86, 495-500

IV Raine A, Wikberg JE & Ehrenberg M. Characterisation of TF binding to ribosome nascent chain complexes. Manuscript

Reprints were made with permissions from the publishers.

CONTENTS

INTRODUCTION ....................................................................................................9

THE RIBOSOME ....................................................................................................10Overview............................................................................................................10Selected structural features of the 50S subunit .............................................10Properties of the peptide exit tunnel ..............................................................11

MEMBRANE TARGETING..................................................................................12Overview............................................................................................................12Post-translational targeting .............................................................................12Co-translational targeting................................................................................14Conservation of the SRP pathway..................................................................14Structure and function of SRP and its receptor.............................................16

Signal sequence binding .............................................................................18SRP on the ribosome....................................................................................18GTP regulated targeting .............................................................................19A model describing the initial steps in SRP targeting ............................22

The Sec translocon ............................................................................................24The ribosome and the translocon...............................................................24

CHAPERONE ASSISTED PROTEIN FOLDING...............................................26The DnaKJE and GroEL/ES chaperone systems ..........................................26A network of chaperones with overlapping function..................................27Trigger Factor: a ribosome associated chaperone.........................................28

The molecular details of Trigger Factor....................................................29Trigger Factors substrate binding properties...........................................30

RESULTS AND DISCUSSION .............................................................................31SRP dependent targeting and insertion of heterologous membrane proteins (paper I) ..............................................................................................31Heterologous membrane proteins insert into a SecY/ YidC interface (paper I) ..............................................................................................................32E. coli SRP does not have elongation arrest activity .....................................34L23 on E. coli ribosomes interact with the nascent chain, SRP and TF (paper II).............................................................................................................34Quantification of SRP and TF binding to naked E. coli ribosomes (paper III)........................................................................................................................35Simultaneous binding of TF and SRP to E. coli ribosomes (paper III) .......37Characterisation of TF binding to ribosome nascent chain complexes (paper IV) ...........................................................................................................38

CONCLUSIONS.....................................................................................................40

SUMMARY IN SWEDISH ....................................................................................41

ACKNOWLEDGMENTS ......................................................................................42

REFERENCES.........................................................................................................43

Abbreviations

cryo EM Cryo electron microscopy IMP Inner membrane protein PPIase Peptidyl-prolyl cis/trans isomerase PT Peptidyl transferase RNC Ribosome nascent chain complex SA Signal anchor sequence SRP Signal recognition particle TM Transmembrane domain TF Trigger factor Bop bacteriopsin MC4 Melanocortin receptor 4 Lep Leader peptidase RpoB RNAse polymerase B subunit

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INTRODUCTION

On the ribosome the genetic code is translated into proteins. A large number of mRNAs code for proteins that are to be translocated across or inserted into the cell membrane. In Escherichia coli the transport of newly translated polypeptides to the site for translocation (the translocon) at the inner mem-brane may occur by different mechanisms. Proteins that are to be translo-cated across the membrane are principally delivered to the membrane post-translationally, and independent of the ribosome. By contrast, proteins that are to be inserted into the inner membrane are targeted to the translocon co-translationally, and the context of the ribosome is an absolute requirement Accurate folding of proteins into the functional state is crucial to all cells. This is reflected in the many chaperone systems that have evolved through-out the kingdoms to promote proper protein folding. There are folding chaperones that operate post-translationally in the cytosol but similarly to membrane targeting, the folding of the nascent polypeptide may already be assisted at the ribosome Apparently, both protein folding and membrane targeting may occur co-translationally and assisted by factors interacting with the ribosome. Interac-tions between the ribosome, the emerging polypeptide, and the factors in-volved in co-translational folding and membrane targeting, are all localised in the same vicinity on the ribosomal 50S subunit. In the past few years it has become more and more evident that the destiny of the emerging poly-peptide is affected by the events taking place at this particular area on the ribosome.

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THE RIBOSOME

OverviewOn the ribosome the translation of the genetic information in the mRNA into proteins is catalysed. These “protein factories” are huge in size and complex-ity and although they have been studied for over forty years there are still puzzling questions and mysteries as to how this amazing macromolecule and its arsenal of auxiliary factors functions. Here I will only briefly discuss some features of the ribosome that are relevant for co-translational protein folding and membrane targeting. The ribosome is a ribonucleotide particle with two separate subunits that has sedimentation coefficients of 50S and 30S in prokaryotes and is joined throughout the elongation of a protein. The small subunit binds mRNA and mediates the interaction between mRNA codons and tRNA anticodons. The large subunit catalyses the production of the peptide and contains the pepti-dyl transferase (PT) center where the peptide bonds are formed.

Selected structural features of the 50S subunit In recent years, great insights into the molecular structure and organisation of ribosomes have been obtained by x-ray crystallography and electron mi-croscopy (EM). The crystal structure of the large subunit of Haloarcula marismortui shows that a 100 Å long passage begins below the PT center and reaches the surface at the back of the subunit [1, 2]. Nascent peptides are passed down this tunnel as they are synthesized and around 35 amino acids in extended conformation will be covered by the ribosome. On the 50S sur-face six ribosomal proteins surround the exit of the peptide tunnel: L19, L22, L23, L24, L29 and L31e [1] (figure 1.). Ribosomes are attached to the mem-brane at sites named translocons when membrane proteins (and secretory proteins in eukaryotes) are being synthesized [3]. The structure of the ribo-some in complex with the translocon pore obtained by cryo EM, shows that the interface between the translocon and the ribosome is localised to the area around the peptide exit site [4]. Moreover nascent signal peptides appear to be close to L23 and L29 as judged by cross-linking experiments [5]. Hence this area appears to be optimal for interaction with the nascent chain. In-deed, the attachment site for signal recognition particle (SRP), a ribosome associated factor promoting co-translational membrane targeting and for trigger factor (TF), a ribosome associated folding chaperone has been mapped to this vicinity [5-8].

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Properties of the peptide exit tunnel The part of the peptide exit tunnel near the PT center is lined by RNA until a constriction is formed by ribosomal proteins L4 and L22 [1]. At present, the significance of this constriction is not well defined but recent data suggest it to be involved in mediating translational arrest of a set of regulatory nascent peptides [9-14]. An interesting question is how the peptide moves down the tunnel. Passive diffusion may be the answer but there have also been sug-gestions, based on cryo EM data, that the tunnel may promote peptide pas-sage in a peristaltic fashion involving L4 and L22 [15]. Furthermore, FRET measurements of nascent chains inside the ribosome in combination with cross-linking experiments indicate that the ribosome can sense the nature of a membrane protein inside the tunnel and that L22 as it reaches down to the surface of the 50S, communicates this information to the translocon [16, 17]. However, for the majority of peptides to travel down easily, the tunnel wall ought to be of a “non stick” character. Consistent with this, the tunnel wall is mostly hydrophilic and although there are hydrophobic groups along the wall, it is absent of any large hydrophobic patches that could stick to nascent peptides. Below the L4/22 constriction the dimension of the tunnel is large enough to accommodate an -helix and closer to the exit it becomes even wider [2]

Figure 1. View of the ribosomal 50S subunit facing the peptide exit. Proteins and rRNA thought to make contact with SRP and the translocon are high-lighted. Adapted from [18].

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MEMBRANE TARGETING

OverviewThe concept of signal sequences was introduced in the early 1970s by Blobel et al. They hypothesised (although they had no experimental support for it at the time) that mRNAs for secretory proteins code for an N-terminal signa-ture sequence that is recognised by a soluble factor, which targets the ribo-some-nascent-chain complex to the ER membrane. Their speculations proved to be right and the soluble component that was binding to signal sequences on the ribosome was later identified as SRP [19]. The early work on protein translocation was primarily performed in eu-karyotic systems and the mammalian SRP was found to be a ribonucleotide particle comprising six proteins on a 7S RNA [20]. Its receptor on the ER membrane consists of two subunits, a smaller one that is an integral mem-brane protein and a larger subunit that is attached to the smaller subunit on the cytoplasmic side of the ER membrane [21, 22]. SRP binds to nascent sig-nal sequences on the ribosomes and transiently arrests translation (in eu-karyotes) [23]. Then the SRP-ribosome complex binds to the SRP receptor on the ER membrane and the ribosome-nascent-chain complex is released onto the Sec-translocon, a process that is regulated by GTP hydrolysis on both SRP and its receptor [24-27]. Translation is resumed down into the translo-con pore and depending the nature of the nascent chain it is translocated through or transferred into to the lipid bilayer [3] (figure 2). The targeting sequences are N-terminal cleavable signal sequences possessing certain threshold hydrophobicity [28, 29]. In the case of many integral membrane proteins the first transmembrane domain serves as the “signal anchor se-quence”, anchor meaning that it is not translocated to the periplasmic space as a cleavable signal sequence but is retained in the membrane. The E. coliSRP was later identified by searching for sequence homologues of the mammalian SRP subunits [30-32] and today SRP homologues have been found in all sequenced genomes.

Post-translational targetingWhereas the mammalian SRP is responsible for co-translational delivery of both secretory and integral membrane proteins to the ER, several targeting routes are available in E. coli. Proteins that are translocated across the inner membrane, such as periplasmic and outer membrane proteins are mainly transported to the translocon post-translationally or late co-translationally, when a substantial amount of the peptide has been synthesized.

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Figure 2. Schematic overview of the major membrane targeting routes in E. coli. Post-translational targeting is mediated by the SecB/SecA pathway. Co-translational targeting is mediated by the SRP pathway.

These proteins are kept in a state competent for translocation by the export chaperone SecB. SecB functions as a homotetramer [33] and binds to the mature part of the pre-secretory protein [34]. The signal sequence is not directly involved in binding to SecB but may, at least in some proteins, retard folding of the ma-ture part of the protein and thereby indirectly increase the opportunity for successful SecB targeting [35]. It should be noted that there have been con-

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flicting opinions of whether SecB binds to the signal sequence or to the ma-ture domain of the protein [3, 33, 36]. At any rate, SecB keeps the presecre-tory protein in a loosely folded conformation [37] and transfers its substrate to SecA, an ATPase at the cytoplasmic side of the translocon that recognises the signal sequence. ATP binding triggers SecA insertion together with the N-terminal part of the protein into the translocon and ATP hydrolysis re-leases the secretory protein. Repeated cycles of SecA binding to parts of the protein remaining on the cytoplasmic side, ATP binding and hydrolysis is thought to plunge the secretory protein stepwise through the translocon [38] In chloroplasts, SRP targets proteins to the thylakoid membrane post-translationally [39]. Interestingly, a role for mammalian SRP was recently described in the post translational targeting of proteins having a C-terminal membrane insertion sequence that does not become available until protein synthesis is terminated [40]. Moreover, E. coli SRP has been found essential for the Sec independent membrane insertion of an IMP with its only TM at the very C-terminal end [41].

Co-translational targeting In E. coli, all components of the SRP pathway are absolutely essential for viability but curiously, it appears to play a smaller role in targeting than in e.g mammalian systems. In E. coli SRP is more specialised towards targeting of inner membrane proteins (IMP) [42] and thus perturbation of SRP does not affect protein secretion . Perhaps, in fast growing bacteria with high translation rates in which SRP do not possess elongation arrest activity it is advantageous to uncouple translocation from translation when possible. Although SRP is the default pathway for IMPs under normal conditions, some IMPs may also utilize the SecB system and other chaperones to some extent as a rescue pathway. For a subset of polytopic inner membrane pro-teins this may not be feasible due to a propensity to rapidly and severly mis-fold, and they would thus be dependent on a strictly co-translational mode of export to the membrane. This high tendency to misfold may not be en-tirely due to the presence of multiple highly hydrophobic TM domains since periplasmic parts of IMP proteins have also been shown to be determinants for strict SRP dependency [43]. The toxicity of aggregated IMPs in the cell is underscored by the fact that in strains with reduced levels of SRP the heat shock regulated proteases Lon and ClpQ become essential for viability [44].

Conservation of the SRP pathway As mentioned, SRP is a ubiquitous molecule, and although parts of the SRP system exhibit remarkably high sequence homology, large differences in the composition exist between SRPs of the three kingdoms. In eukaryotes SRP comprises six protein subunits (SRP9, 14, 19, 54, 68 and 72 denoted accord-

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ing to molecular weight) and a 7S RNA. The whole particle can be divided into two domains: the S domain that is involved in signal sequence recogni-tion and receptor interaction and the Alu domain that is implicated in SRPs elongation arrest function [30, 45, 46]. SRP of gram negative bacteria is of a less complex architecture and consists of a single protein (Ffh, the homo-logue of the signal sequence binding subunit SRP54) and a smaller 4.5 S RNA corresponding to helix 8 of 7S RNA [30, 32]. In Archaea the SRP is of intermediary complexity comprising the 7S RNA and the homologues of SRP54 and SRP19 [47]. Interestingly, some gram positive bacteria such as B. subtilis have retained the Alu domain and histone like proteins (HBsu) is replacing SRP9/14 [48]. The most conserved part of SRP is thus the SRP54 subunit and the helix 8 of the RNA (figure 3). The SRP receptor in eukaryotes is composed of two proteins: SR the pe-ripheral subunit and SR the membrane integrated subunit [21, 49, 50]. In E.coli the SRP receptor (FtsY) consists of the homologue of SR only and no membrane integrated subunit has been found [51].

.Figure 3. SRPs from the three kingdoms. The helix 8 and SRP54 are the most conserved elements of the SRP particles.

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Structure and function of SRP and its receptor Despite the large differences in complexity between the bacterial and mam-malian systems the basic features of SRP targeting is well conserved. The discussion in this section is primarily focused on the prokaryotic and the common characteristics of the SRP pathway. SRP54 or Ffh in in E. coli can be divided into three domains: the N-terminal four helix bundle (N domain) that packs tightly against a central GTPase domain (G-domain) with a classical Ras-like GTPase fold. The two of them are often referred to as the NG domain and in the G domain there are four conserved motifs involved in forming the nucleotide binding site [27, 52, 53]. In addition, the G domain contains a motif unique to the SRP GTPases called the insertion box domain (IBD). Likewise, FtsY is in part composed of a NG domain that is homologous to the NG domain of SRP54 (figure 4). The NG domains make up the inter-molecular interface in the FtsY-Ffh complex and the intra-molecular interface between the N and G domains has a hinge like function to enable relative orientations between the N and G domains [52-55]. The third domain of Ffh; the C-terminal M domain is responsible for signal sequence binding and is a helical domain with a methionine rich hydropho-bic groove covered by a flexible finger loop [56, 57]. The NG and M domains are connected by a linker that has been disordered in crystal structures of Ffh, entailing difficulties in unambiguous determination of the M domain orientation relative to the NG domain in free SRP [56]. Clearly, this points at flexibility in the orientation between the NG and M domains. Nonetheless, a more recent structure of S. solfacatarius SRP54 suggest the free Ffh (SRP54) to be” L shaped”, with the NG domain making up the long arm of the L and the M domain is the short arm [58] (figure 6). In this structure the RNA bound to the M domain runs in parallel with the NG domain and the RNA may contact the G domain. In addition, there is a small hydrophobic contact between the distal end of the N domain and the M domain close to the finger loop, suggesting that the free SRP adopts a relatively compact conformation. Interestingly, cross-linking combined with mass spectrometry imply that the M domain has a different position in the Ffh and FtsY complex and is in proximity to the FtsY N domain [59] . However, the significance of this ob-servation is difficult to evaluate in the absence of the RNC. In any case, the orientation and putative interactions between the M domain and the NG domain in Ffh are likely to change in response to the additional components involved in the targeting cycle. Moreover, structural changes are likely to occur in the NG domain upon nucleotide and receptor binding (as will be discussed in the next section) and presumably also in correspondence to signal sequence binding in the M domain. The N-terminal part of FtsY (denoted the A-domain) has a high negative net charge [60] and is involved in the attachment to the membrane [61] but the

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mechanism of association is unclear. FtsY is, in contrast to its eukaryotic counterpart, only transiently bound to the membrane and may be capable of early interaction with the SRP-RNC complex although the consequences of complex formation already in the cytosol are not known. In eukaryotes, the 7S RNA of SRP plays an obvious role as a protein scaffold and the Alu domain is binding to the elongation factor binding site, causing translation arrest. However the significance of the conserved part corre-sponding to the E. coli 4.5S RNA is less obvious. It may be involved in stabi-lizing the structure of SRP at different points in targeting and there is data suggesting that it plays a small role in signal sequence binding. Notably, the 4.5S RNA have been shown to accelerate the rates of Ffh-FtsY complex asso-ciation and dissociation ~ 200 fold without substantially altering the equilib-rium dissociation constant of the complex. As large conformational changes are predicted to accompany complex formation, a model was proposed where the RNA in a catalytic manner acts to transiently tether Ffh and FtsY thus lowering the energy barrier of the transition state(s) when the complex-promoting conformations are adopted [62].

Figure 4. Ffh and FtsY have highly conserved NG domains. The G domains have raslike GTPase folds and insertion box domains (IBD) that are unique to the SRP GTPases. SRPs M-domain is responsible for signal sequence bind-ing and contains the binding site for the 4.5S RNA. FtsY has a negatively charged A-domain involved in membrane binding.

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SIGNAL SEQUENCE BINDING

SRP binds to signal sequences as they emerge from the ribosome. The classi-cal signal peptides typically have a 9-12 residue long hydrophobic core flanked by positive charges in the N terminal and a site for signal peptidase cleavage in the C-terminal. IMPs do generally not possess a cleavable signal sequence; instead the first TM domain is used as a targeting sequence (signal anchor sequence). Despite the fact that signal peptides do not show evidence of any specific consensus sequence, SRP is able to bind them with high speci-ficity. In E. coli SRP demonstrates even higher specificity toward certain sequences as it selectively rejects binding of most pre-secretory proteins. Signal anchor (SA) sequences are generally longer and more hydrophobic than SecB dependent signal sequences. Higher threshold hydrophobicity is certainly one factor that determines SRP specificity toward IMPs in E. colibut there is also experimental data suggesting that basic residues in the N terminus promotes SRP binding to signal peptides whose hydrophobicity is just below the lower limit for interaction with SRP [63]. Structural data show that the 4.5S RNA is near the hydrophobic groove in the M domain and may to some degree participate in signal sequence binding, perhaps by electro-static interactions with basic residues flanking the hydrophobic core [64]. On the contrary, mammalian SRP interacts with the wide range of signal pep-tides. The eukaryotic M domain has an extension of ~100 residues proximal to the hydrophibic groove that is not present in the prokaryotic SRP suggest-ing that this region is of importance for the broader substrate specificity in eukaryotes. Besides hydrophobicity, secondary structure is of importance for targeting. The signal sequence adopts an -helical structure in hydrophobic surroundings and disruption of the -helical conformation prevents co-translational targeting. Moreover, when residing in the translocon and in-side the ribosomal exit channel the signal sequence is in the form of an -helix further emphasizing the importance of the secondary structure [16, 65-67]. A “methionine bristle” hypothesis has been put forward to account for the flexibility in the signal sequence binding pocket [30]. SRP54 and its homo-logues have a particularly high number of methionine residues clustered in the M domain. The side chain of methionine in the hydrophobic binding site is proposed to form flexible bristles that may contribute to the ability of binding a wide variety of signal sequences.

SRP ON THE RIBOSOME

Crosslinking experiments have mapped the binding site of SRP at the tip of the N domain, to ribosomal protein L23 close to the peptide exit site [6, 8]. Moreover, the homologue of L29 was shown to interact with the mammalian SRP54 subunit but a contact between E. coli Ffh and L29 has not yet been verified [5, 8]. Given that the nascent signal sequence is close to L23 and L29,

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SRP is optimally located on the ribosome [5, 68]. Mammalian SRP binds with subnanomolar affinities to RNCs that do not diminish as the signal sequence grows longer indicating that the signal sequence has significant affinity for L23 [69]. The 12 Å cryo EM map of mammalian SRP binding to the 80S ribo-some carrying a nascent signal sequence confirms SRPs positioning on L23 [70]. Additional SRP54 contacts were formed by the M domain contacting helix 59 and helix 24 of the 25S rRNA at the opposite side of the peptide exit which similar to L25/35 (L23p/29p) are contact points shared with the Sec-translocon. A fourth connection involves the 7S RNA and 25S RNA close to L16 (L13p), also on the opposite side of the peptide exit relative to the L25/L35 contact point. The Alu domain is close to the elongation factor binding site, consistent with its role in translation arrest. Furthermore, it was seen that the NG domain is rotated 50° and shifted away from the aligned position with SRP RNA helix 8 that was observed in the S. solfactarius struc-ture. .

GTP REGULATED TARGETING

GTPases confers regulation of cellular processes by GTP binding and hy-drolysis at distinct stages of a mechanism. However the SRP GTPases exhib-its properties that distinguish them from the classical “molecular switch” type of regulators and form a distinct subfamily in the GTPase subfamily The SRP GTPases have low affinity for both GTP and GDP (~1 µM) [71] and appear to be stable in the nucleotide free state [72] although the physiologi-cal relevance of the apo form is unknown considering that the cellular con-centration of GTP is such that both SRP and its receptor should generally be GTP bound. Even so, the RNC complex stimulates GTP binding in mammal-ian SRP as judged by cross-linking [73], and phospholipids have been sug-gested to stimulate GTP hydrolysis in FtsY [74]. GTP binding is a prerequi-site for interaction between SRP and FtsY [75], and subsequent hydrolysis is accompanied by their dissociation [76] (figure 8). Given that the nucleotide free modes are stable and dissociation of GDP (and GTP) is fast there is no requirement for a guanine exchange factor (GEF). A peculiar and unique character of the SRP GTPases is that they mutually act as GTPase activating proteins (GAPs) for each other [77]. The crystal structure of the coupled NG domains of T. aquaticus Ffh and FtsY show that they form a pseudo symmet-rical heterodimer with an extensive surface of interaction [78, 79]. Major conformational changes relative to their respective free NG domains were observed involving conserved motifs in the active sites and of the N domain position relative to the G domain. Most strikingly, the two non-hydrolysable GTP analogues face each other and align such that their respective -phosphates hydrogen bonds to the ribose ring of the facing nucleotide (fig-ure 5). Thus catalytic groups are brought into the active site in cis and the substrates interact in trans. The structure offers hints but the questions linger as to how reciprocal activation occurs and how idling is prevented. It has

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been hypothesized that signal sequence binding to the M-domain may inflict structural changes communicated to the NG domain to prevent GTP hy-drolysis. As the crystal structure of the SRP and FtsY complex did not in-clude the M-domain this idea awaits experimental evidence. Extensive mu-tational analysis indicates that the Ffh-FtsY interaction is dynamic with sev-eral conformational changes taking place before activation of GTP hydroly-sis [80, 81]. These may serve as potential regulation points in the targeting reaction and analogous conformations inflicted by various point mutations may in the wildtype be stabilised by the interactions with the RNC and the translocon ensuring precise timing of signal sequence binding, release and prevention of futile cycles of GTP hydrolysis.

Figure 5. The NG domains of FtsY and Ffh (T. aquaticus) form a pseudo het-erodimer with an extensive surface of interaction. The bound nucleotides are aligned head to tail and are hydrogen bonded to each other.

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Figure 6. The recently solved crystal structures of E. coli TF, resembling a “crouching dragon” and SRP from S. solfactarius.

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A MODEL DESCRIBING THE INITIAL STEPS IN SRP TARGETING

Based on the available structures and biochemical data, Wild et al compiled the following model for the initial steps in the targeting cycle [82]. The scheme is divided into four steps ending with SRP docking to its receptor.

i) The free SRP adopts a compact conformation similar to what was observed in the S. solfactarious crystal structure and has low affinity for nucleotides. The molecule is L shaped and the RNA is running in parallel with the NG domain. The finger loop of the M domain is closing the hydrophobic groove preventing signal sequence binding. There is a direct contact between the N domain and the M domain and in addition the RNA may contact the G do-main. ii) In the sampling mode, SRP binds to the ribosome and searches for a sig-nal sequence. The affinity for the ribosome is moderate and the on and off rates are fast. The contact involves at least rpL23 and rpL29 and the distal loops of the SRP N domain. The contact between the N domain and M do-main would be lost due to ribosome binding and a rotation of the NG do-main would perhaps be induced already at this stage, leading to a structure more amenable for signal sequence binding. Although this is a reasonable model, it is only speculative as there are no structural data available of SRP in the sampling mode. iii) In the targeting mode, the signal sequence binding into the hydrophobic groove of the M domain is transmitted to the NG domain and the SRP changes to the fully open conformation observed in the cryo EM structure with the NG domain shifted away from the parallel alignment with the RNA. The N and M domains are located at opposite sides of the peptide exit by means of the N domain binding to L23/L29 and the M domain contacting H24 in rRNA. This causes the signal sequence bound hydrophobic groove of the M domain to be positioned straight over the peptide exit. The affinity for the ribosome is now very high and GTP affinity is perhaps increased to se-cure interaction with the receptor (figure 7) iv) The docking mode involves the twin like formation of the NG domains of SRP and its receptor. A repositioning of SRP on the ribosome has been ob-served in response to SR binding as judged by cross-linking data. It is tempt-ing to speculate that this would facilitate the next step in the targeting cycle namely the docking onto the translocon. At present, little is known about this docking mode or the subsequent transport and release of the signal se-quence to the translocon (figure 8).

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Figure 7. Cartoon of SRP in a ”targeting mode”, binding to the ribosome and the nascent signal sequence at the peptide exit. Adapted from [83].

Figure 8. GTP controlled targeting of the SRP-RNC complex in E. coli. FtsYexists in the cytosol as well as bound to the inner membrane and can thus bind the SRP-RNC prior to membrane binding. Then how are futile cycles of GTP hydrolysis prevented. When is hydrolysis triggered? How does the docking event take place? Are SRP and FtsY physically displaced at the translocon? When does SRP release the signal sequence, before or after GTP hydrolysis? Many questions remain to be answered.

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The Sec translocon Protein secretion and membrane integration is mediated by a conserved protein conducting channel; the Sec- translocon which is built by hetero-trimers of the integral membrane proteins SecY, SecE and SecG [84]. Assem-bly of a number of heterotrimers is believed to form the translocon pore but the exact compositon is still a matter of debate. Association with the ribo-some or SecA provides the energy for the translocation process and the pro-tonmotive force stimulates translocation. The interior environment of the translocon is aqueous implying that there exists a mechanism for preventing ion flow through the channel during translocation. Moreover a lateral gate must exist where TMs can escape out to the lipid bilayer. The recent x-ray structure of a protein conducting channel has identified structural elements that may be involved in these mechanisms [85]. The translocon pore narrows at the center creating funnel like shapes on both sides of a “plug” that may be displaced when a polypeptide enters. At the narrowest part the walls are lined by hydrophobic residues suggested to act as a gasket around the trans-locating polypeptide to maintain the membrane barrier. Although SecYEG is the functional core, several other proteins are found to be associated. Proteins SecD, SecF and YajC forms a complex that can be copurified with the translocon [86, 87]. The role of these auxiliary proteins in translocation and membrane insertion is ill defined. Another recently dis-covered protein that associates with the SecY translocon is YidC, an essential protein related to Alb3 and Oxa1; proteins involved in membrane protein biogenesis in chloroplasts and mithochondria [88]. Notably, YidC acts both in conjunction with the Sec-translocon and in Sec-independent membrane insertion of small phage coat proteins. Recent studied have indicated cyto-chrome o oxidase and the F0F1-ATPase as native substrates for YidC-dependent/Sec-independent membrane insertion [89, 90]. A wealth of cross-linking data suggest that YidC in association with SecYEG facilitates lateral transfer of TM domains into the lipid bilayer [91-95]. YidC has been pro-posed to act as an integral membrane chaperone in IMP biogenesis but the mechanism of YidC mediated membrane protein assembly remains elusive.

THE RIBOSOME AND THE TRANSLOCON

Ribosomes have a high intrinsic affinity for the Sec-translocon which is re-tained between ribosomes and translocons of different origins, emphasizing the evolutionary conservation of the binding site [96]. The cryo EM recon-struction of a translating 80S ribosome in complex with the translocon iden-tified several contact points. Intriguingly, the most extensive contacts were formed by L25 and L35 (L23/L29) and a lesser contact with RNA helix 24; both shown to be binding sites for SRP. The overlapping binding sites of SRP and the translocon clearly prove that rearrangement of SRP on the ribo-some has to take place before docking onto the ribosome. Binding of the

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ribosome to the translocon induces a 90° rotation of expansion segment 27 of 25S RNA close to the channel attachment site. This repositioning may be of significance for the evacuation of SRP concurrent with translocon docking [4].

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CHAPERONE ASSISTED PROTEIN FOLDING

Correct folding of newly synthesized proteins must take place in the ex-tremely crowded environment of the cytosol. The native conformation of a protein is the thermodynamically most stable. Shielding of hydrophobic surfaces from the aqueous environment forces the formation of folding in-termediates that may be kinetically trapped and prone to aggregation. A class of proteins named chaperones has evolved to facilitate the folding of proteins, prevent misfolding and aggregation.

The DnaKJE and GroEL/ES chaperone systems In E. coli there are two major systems involved in post-translational chaper-oning of protein folding i) the DnaKJE system and ii) the GroEL/ES system. Both systems assist the folding of newly synthesized polypeptides released from the ribosome (figure 9). Strictly speaking, the line between post and co-translational folding is not absolute since these chaperones also interact with nascent polypeptides, although they do not make any physical contact with the ribosome. The folding of proteins promoted by these systems are regu-lated by ATP hydrolysis. DnaK belongs to the well conserved Hsp70 family [97] and binds to short hydrophobic peptide stretches of polypeptides in extended conformation. In the ATP bound state, the kinetics of substrate binding is fast. The co-chaperone DnaJ which also can bind extended polypeptides and bring them to DnaK stimulates ATP hydrolysis in DnaK, inducing a conformation that firmly binds the substrate. Nucleotide exchange, promoted by another co-chaperone: GrpE, cycles DnaK back to the ATP bound state and the poly-peptide is released [98-102] . Repeated rounds of binding and rebinding can occur before a polypeptide folds into its native form and the polypeptide may also be handed over to the GroEL/ES system after having aquired some degree of structure. GroEL/ES belong to the family of ring shaped chaperonins. GroEL has a complex architecture of 14 identical subunits that forms two stacked rings of seven subunits each. Misfolded and partially folded proteins can bind into this cavity which exposes hydrophobic surfaces towards the center. The co-factor GroES then binds GroEL in the presence of 7 ATPs, inducing a con-formational change that releases the polypeptide into the cavity until ATP is hydrolysed. Then GroES leaves and GroEL returns to the conformational state exposing hydrophobic surfaces [103-106]. If the polypeptide is folded into its native conformation it will no longer rebind to GroEL. If not, several cycles of GroEL binding may be required.

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A network of chaperones with overlapping function Chaperones, whether they primarily promote protein export or folding, have in common their promiscuous binding to non native polypeptides. Even though they may preferably bind to certain ligands, they appear to have a a great deal of overlapping substrate specificity. For instance, if SecB is de-pleted, DnaK and DnaJ can substitute for SecB in protein export of several proteins. In accordance with this, SecB is proposed to function as a general folding chaperone besides its role in protein export [107, 108]. Moreover, GroEL has been shown to be involved in protein export of -lactamase [109]. There are numerous examples of overlapping chaperone binding and func-tion in the literature. An unfolded polypeptide binds to whatever chaperone is available. If the chaperone with the highest affinity for the non native pro-tein is not available, another chaperone with lower affinity that is present in sufficiently high concentration may temporarily take over its function [107] .

Figure 9. The major protein folding chaperone systems in prokaryotes; DnaKJE, GroEL/ES and trigger factor.

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Trigger Factor: a ribosome associated chaperone Chaperone assisted protein folding may be initiated co-translationally and there are chaperones whose functions as folding catalysts are dependent on the close contact with the ribosome. In prokaryotes, the trigger factor (TF) has been identified as such a chaperone. Trigger Factor was originally pro-posed a role in protein export since it was discovered in early studies to sta-bilize a translocation competent form of a secretory protein [110-116]. This hypothesis was later disregarded when it was discovered that depletion of TF did not affect protein secretion.TF is a 48 kDa protein that is abundant in the cell (2-3 times in excess over ribosomes). Interestingly, neither TF nor DnaK appear to be essential for cell viability if depleted separately. On the contrary, simultaneous deletion of the DnaK and TF genes results in synthetic lethality at temperatures above 30 C and massive aggregation of more than 340 protein species [117-119]. This was taken as evidence that TF and DnaK is functionally redundant. DnaK has indeed been shown to interact with nascent polypeptides and in the absence of TF it appears to bind to shorter nascent chain than during normal conditions [118]. In addition cells depleted of TF have increased DnaK levels [117] Folding assays with multidomain proteins such as -galactosidase and fire-fly luciferase, translated in a cell free system depleted of TF and DnaK showed that the newly synthesised proteins folds rapidly and spontaneously although with low yields of active protein. Rapid and non controlled folding apparently leading to non productive intra and intermolecular interactions is likely to be the cause. However, when TF and DnaKJE was present sepa-rately or in combination, the completion of folding was delayed but the yield of active proteins was markedly improved, implying that DnaK and TF both prevents the emerging polypeptide to fold by a rapid default pathway that leads to a high degree of misfolding [120]. Another interesting observation is that inactivation of the TF gene appears to accelerate export of outer membrane proteins such as OmpA and further-more suppress the requirement of SecB to keep these proteins in a transloca-tion competent state . Overproduction, on the other hand depresses target-ing of OmpA, suggesting that TF sequesters nascent polypeptides long enough to necessitate the aid of a targeting factor such as SecB to keep the protein translocation competent after release from TF [121]. Inactivation of other chaperone such as DnaK normally impairs protein export, further in-dicating that these chaperones promote protein folding of nascent proteins in complementary but mechanistically distinct ways.

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THE MOLECULAR DETAILS OF TRIGGER FACTOR

Limited proteolysis revealed TF as a modular protein with three distinct domains [122]. The 144 residue N-terminal domain constitutes the ribosome binding part of the molecule[123]. A conserved ribosome binding motif GFRKxGxxP [7] has been localised to the tip of the N-terminal domain which is proposed to make out the “ribosomal anchor” The central domain M-domain (residues 145-247) has cis/trans peptidyl pro-lyl isomerase (PPIase) activity and is related to the PPIase family of FK506 binding proteins [124]. TF is a peculiar chaperone considering that it has both PPIase activity and chaperone functions. For some proteins the rate limiting step in protein folding is the isomerization of peptidyl prolyl bonds, but the significance of TFs PPIase function is puzzling since TF has been demonstrated to bind peptides and polypeptide substrates independently of proline residues [125, 126]. Studies attempting to dissect the functions of the different domains of TF have revealed that the N-domain by itself has chap-erone activities which are enhanced by the C-and M-domains [127]. The M-domain however does not display any chaperone activities in the absence of the N-domain even though the PPIase activity towards short peptides is retained in the single M-domain. Furthermore, a TF single amino acid mu-tant lacking PPIase activity exhibits the same chaperone properties as wild-type TF [128]. Taken together, a substantial amount of evidence indicates that TF promotes folding independently of its PPIase activity. The C-terminal domain (residues 248-432) was for a long time a mystery and no function could be assigned to it. New insights into the molecular struc-ture of TF have shed some light on its function and will be discussed below. The crystal structure of TF was recently solved and gave new important insights as to how TF functions as a chaperone [129, 130]. The crystal struc-tures of E. coli and V. cholerae TF reveal the molecule to have an unusual elongated shape reminiscent of a “crouching dragon” (figure 6). The ribo-some binding N-domain constitutes the “tail” of the molecule and the M-domain is located at the opposite side of the molecule and makes out the “head”. The C-terminal and connecting regions form the middle part of the molecule with the “arms” and “back. A hydrophobic cavity is formed be-tween the N-terminal tail and the C-terminal arms that may be able to ac-commodate larger peptides than previously assumed. Co-crystallisation of the N-domain with the H. marismortui 50S subunit and subsequent superpo-sition of the entire chaperone suggest that TF crouches over the peptide exit providing a shielded space for the emerging polypeptide. In line with bio-chemical data showing that the PPIase activity is dispensable for TFs chap-erone function, the M-domain head is positioned outside the folding cavity implying that that the peptide must first escape the folding cave before it can be exposed to TFs PPIase activity. However, the TF structure may not be static on the ribosome and conformational changes should not yet be ruled

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out. Nevertheless, the structure gives an hint of how TF may support protein folding on a molecular level. By providing a protected space where the nas-cent peptide can interact with TFs large hydrophobic surface, peptide stretches susceptible to intramolecular interactions and misfolding may be protected until an entire folding unit is out of the ribosome. The conserved GFRKxGxxP sequence is situated in the flexible loop in the N-domain and was identified as a ribosome motif by mutational analysis. Cross-linking experiments pointed out L23 and L29 as its ribosomal binding partners [7]. L29 was however shown dispensable for TF binding leaving L23 as the major attachment point for TF on the ribosome. The crystal struc-ture of TF in complex with the 50S subunit confirms the biochemical data and shows that TF binds in the triple junction of L23, L29 and domain III of 23S rRNA. The interactions with L29 and 23S RNA are small and are not likely to contribute significantly to the binding energy. Although TF bends over the peptide exit site there are no other physical contacts between other domains than the N-domain and the ribosome. A propensity to form dimers has been observed for TF at high concentra-tions [131]. However the dimeric form of TF is most likely incompatible with ribosome binding [129, 130], raising the question to what role dimerisation might play. Experimental data indicating a role for dimeric TF in protein folding separately from the ribosome was recently presented [132]. On the other hand, the idea that dimerisation may prevent promiscuous binding to proteins in the cytosol has been brought up [130], leaving this issue still un-resolved.

TRIGGER FACTORS SUBSTRATE BINDING PROPERTIES

TF binding to short peptides have been investigated using a large number of membrane bound 13 mer peptides derived from a set of different proteins. Comparative analysis of the peptide sequences that were most efficient in TF binding proved them to be enriched in basic and aromatic residues whereas peptides with a negative netcharge were not favoured in TF binding. No specific positioning of the aromatic and basic residues was preferred and proline residues were not overrepresented in the peptides binding TF well. Inspection of the TF binding sites in the proteins from which they were de-rived showed that they occur rather frequently and most of the binding sites were buried in the hydrophobic interior of the proteins. No correlation be-tween TF binding and secondary structure in those regions in the particular proteins could be seen. Comparison with the DnaK binding motif (5 hydro-phobic residues flanked by positively charged residues) [133, 134] show that the binding specificity differs in that TF prefers aromatic residues while DnaK prefers aliphatic residues and TF does not favour any specific posi-tioning of the basic residues while DnaK prefer them flanking the hydro-phobic stretches.

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RESULTS AND DISCUSSION

SRP dependent targeting and insertion of heterologous membrane proteins (paper I) In this study we have used in vitro translation, targeting and cross-linking to investigate the molecular environment of two nascent heterologous mem-brane proteins in the cytosol, and in the membrane of E. coli. The cross-linking patterns could be compared with those obtained from native E. coliproteins such as FtsQ and leader peptidase, that have been the subjects of similar studies. Heterologous membrane protein expression in E. coli is in many cases inefficient [135, 136]. We wanted to address the possibility that the bottleneck lies at the level of membrane targeting and insertion of such proteins in E. coli.Truncated mRNAs of bacterioopsin (Bop) and melanocortin receptor 4 (MC4R) were translated in an E. coli cell lysate and subsequently treated with bifunctional cross-linkers to detect interactions with targeting factors in the cytosol. Immunoprecipitation identified SRP and TF as cross-linking partners , consistent the notion that both TF and SRP scans the exit site for nascent peptides and that SRP specifically interacts with hydrophobic se-quences present in N-terminal signal sequences or in transmembrane do-mains (TMs). To further demonstrate SRP dependent membrane targeting of a heterolo-gous membrane protein, we translated full length Bop mRNA in an in vitrotranslation system assembled from purified E. coli components and thus devoid of targeting factors. In the absence of SRP and FtsY only minute amounts of Bop was inserted into inverted membrane vesicles (IMVs). As the concentration of SRP was titrated up the insertion of Bop was amelio-rated, demonstrating dependence of targeting factors for efficient association with the membrane. That the SRP dependent membrane insertion occurs co-translationally was demonstrated by producing ribosomal complexes that expose full-length Bop peptides and present them to IMVs and targeting factors. Only residual amounts of full length Bop were inserted into the membrane in this experiment. Ribosome complexes carrying 98 Bop were on the contrary rather efficiently targeted and inserted into IMVs indicating that there is a window for successful insertion into the membrane, mediated by the SRP pathway. The propensity of the nascent chain to misfold on the ribosome may influence the window of opportunity for successful mem-brane targeting. Alternatively, it is plausible that SRP targeting can occur but it is the insertion into the the translocon that is compromised. The nascent chain of Bop, as it has multiple TM domains and short connecting regions would be expected to expose putative SRP binding sequences reasonably

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close to the ribosome at any nascent chain length. For a more rigorous as-sessment of co-translational Bop targeting it would have been useful to in-clude experiments with added SecB/SecA.

Heterologous membrane proteins insert into a SecY/ YidC interface (paper I) To inspect the molecular environment of these nascent peptides in the mem-brane we used a photo cross-linking approach where the cross-linker is site specifically incorporated into the nascent chain, a method that is widely used for studying membrane protein biogenesis. A stop codon mutation was introduced into the sequence coding for the 97 and 118 aminoacid long N-terminal fragments of MC4R and the 70 and 98 amino acid N- terminal frag-ments of Bop. The stop codon mutations were placed in the signal anchor (SA) sequences which are first TMs. Translation was then carried out in the presence of a suppressor tRNA, that had been chemically charged with a UV inducible cross-linker (Tmd-Phe), together with inverted E. coli membrane vesicles to allow for membrane targeting. In the vesicle fraction, we identi-fied SecY as the major cross-linking partner for the shorter (70 residues) nas-cent chain of Bop. The shorter nascent chain of MC4R (97 residues) however, displayed only weak cross-links to translocon components. When the longer nascent chain of Bop (98 residues) was analysed it was evident that the cross-link to SecY had diminished but a strong cross-link to to YidC ap-peared. The same strong cross-link to YidC but only a faint cross-link to SecY was observed for the longer MC4R (118 residues). These results suggest that the nascent chain of these heterologous membrane proteins initially inserts into the membrane in the vicinity of SecY, the major component of the translocon pore. As the nascent peptide grows it comes in closer contact with YidC indicating that the SA sequence may reside in the interface be-tween SecY and YidC, consistent with a role for YidC in lipid partitioning of TM domains (figure 10). The same type of cross-linking patterns were observed for nascent chains of FtsQ, a single transmembrane protein with an “N-terminal in, C-terminal out “ topology [95]. Leader peptidase, another E. coli protein that has been the subject of extensive cross-linking analysis has an “N-terminal in, C-terminal out” topology similar to the proteins used in this study. For short nascent chains of leader peptidase (50 residues the minimum requirement for stable interaction with the membrane for this particular protein) where the Tmd-Phe probe was inserted at various positions along the TM domain, it was shown that cross-links to YidC were already present to this short pep-tide in varying intensity depending on the positioning of the cross-linker probe [92]. SecY cross-links were also prominent to this peptide. The contact with YidC was persistent during chain elongation but the SecY contact di-minished. Based on those observations it was suggested that YidC partici-

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pates in both the reception and partitioning of some proteins [92]. Thus it is possible that YidC contacts Bop and MC4R earlier but not detected here dependent on the position of the cross-linker probe. Taken together, the data obtained here indicate that at least the initial steps of heterologous membrane protein targeting and insertion proceeds nor-mally. Of course it can not be excluded that later steps/other steps in the membrane protein biogenesis is limiting. Notably, in several cases have functional expression of heterologous membrane proteins in E. coli been improved by adding native fusion tags such as MBP [137, 138]. MBP is a protein that is normally SecB/SecA targeted to the membrane. Precisely how MBP increases functional expression of these proteins and how they are targeted to the membrane is not known but it may be speculated that it is the targeting to the membrane that is improved by utilizing SecB targeting. Per-haps the capacity of the SRP system is to easily titrated out to allow for even low over-expression level of membrane proteins. Apparently, inner mem-brane proteins are recognised by the translocon and processed accordingly even though a large periplasmic protein is fused to it.

Figure 10. A cartoon which illustrates the initial steps of Bop insertion into E.coli membranes. The 70 residue long nascent chain of Bop inserts into the membrane close to SecY. As the peptide becomes longer a contact with YidC is established while SecY cross-links diminish, in line with a role for YidC in lateral transfer of TM domains. The TM domains are coloured in dark grey and the approximate position of the cross-linker probe is marked by as aster-isk.

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E. coli SRP does not have elongation arrest activity SRP from eukaryotes carry the Alu domain that has been proposed to be responsible for the transient arrest of elongation that occurs when SRP binds a nascent signal peptide. The cryo EM structure of mammalian SRP in com-plex with the ribosome confirms that the Alu binding domain is involved in binding to the elongation factor binding site on the 80S ribosome. Given that E. coli SRP lacks the Alu domain it has been assumed not to possess elonga-tion arrest ability. In one study however, indirect evidence was presented and interpreted as the existence of SRP mediated elongation arrest in E. coli[139]. We measured the elongation rates of Bop in the absence and presence of E. coli SRP and found no evidence of such translation arrest. The signifi-cance of SRP mediated elongation arrest is unclear and appears not to be essential for proper targeting in vitro, and defective translation arrest only slightly affect translocation in vivo in yeast [140]. The most obvious reason for a translation arrest activity would be to increase the window of opportu-nity for successful targeting to the membrane in a large eukaryotic cell. It has been proposed that E. coli does not require translation arrest activity due to its smaller cell size. However, the fact translation rates are much faster in E. coli than in eukaryotes is not in favour of that line of reasoning.

L23 on E. coli ribosomes interact with the nascent chain, SRP and TF (paper II). Photo cross-linking scanning was used to map the interactions of the nascent membrane protein FtsQ with TF and SRP. A Tmd-Phe probe was inserted along the SA sequence of a 77 residue long nascent chain of FtsQ. Ffh cross-links appeared in more or less all positions in the nascent chain but TF cross-links appeared more shifted to the C-terminal part of the nascent sequence. Although it is difficult to draw any useful conclusions from these scans re-garding TF and SRP binding to the ribosome, another interesting finding became evident. The nascent chain was found extensively cross-linked to 8-12 kD proteins that could be immunoprecipitated with antisera against ribo-somal proteins L23 and L29. Moreover, when the FtsQ77 with a Tmd-Phe probe in the SA sequence were produced in a reconstituted translation sys-tem, addition of SRP strikingly eliminated the cross-link between L23 and FtsQ77. Hence, L23 and SRP appear to compete for the nascent signal se-quence. This finding illustrates that L23 is a strategic position for factors interacting with nascent chains. Purified non-translating ribosomes mixed with SRP were then incubated with a bifunctional cross-linker to identify cross-links between Ffh and ribosomal proteins. Subsequent immunoblot-ting with antibodies towards L23 indeed revealed a contact between Ffh and L23. Consistent with this finding, it had previously been shown that mam-malian SRP binds to the eukaryotic counterparts of L23 and L29 (L25 and

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L35) on the 80S ribosome. On eukaryotic ribosome-nascent-chain complexes cross-links to SRP were shifted from L25 to L35 after complex formation between SRP and SR, strongly indicative of repositioning of SRP on the ribo-some at this stage in targeting [6].

In our study no cross-links between Ffh and L29 could be detected. This does not exclude such a contact as it may be due to unfavourable positioning of cross-linking residues when using bifunctional cross-linkers. However, whereas another study using a site specific UV induced cross-linker engi-neered into surface positions of Ffh to search for interactions with ribosomal partners on the E. coli ribosome corroborates that Ffh binds to L23, no cross-links between Ffh and L29 were detected either to naked ribosomes nor ribo-some nascent chain complexes [8]. Moreover, FtsY did not affect the cross-link between L23 and Ffh [8]. Interestingly this, indicates that the position-ing of SRP on the ribosomes may differ slightly between species. Although the fact that Ffh and FtsY can functionally replace SRP54 and SR in vitro sug-gests that the contact mode with the ribosome is highly conserved, some differences in the interactions of mammalian and E. coli SRP with the ribo-some may exist, especially when considering the structural restraints that the much larger mammalian SRP must impose compared to a smaller E. coliSRP. At present it is not known if signal sequences attach to L23 with higher affin-ity than nascent sequences in general. The fact that mammalian SRPs affinity for the RNC does not diminish as the nascent chain grows longer is in agreement with signal sequence binding to L23. A speculative thought is that signal sequence binding to L23, analogous to the translation arrest activ-ity, keeps the signal sequence close to the ribosome for a prolonged period of time conferring an increased window of opportunity for SRP binding. Signal sequence sequestering by L23 could furthermore explain the observed SRP dependent targeting of proteins with targeting sequences at the very C-terminal end that does not become exposed until after termination of transla-tion.

Quantification of SRP and TF binding to naked E. coli ribosomes (paper III) In this study we measured the affinities for SRP and TF to non translating ribosomes using a spin down assay and radioactively labelled factors. By rapidly pelleting ribosomes in a micro ultracentrifuge, ribosome bound fac-tors were separated from unbound factors. The equilibrium binding con-stants may be determined by this method provided that ribosomes are spun down much faster than the factors and that pellet incorporation into the pellet is irreversible. The KD values were determined at two different tem-peratures, enabling determination of the corresponding enthalpies of bind-ing. SRP was found to bind ribosomes with a KD value of 90 nM (at 4°C)

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irrespective of the presence of nucleotides (table 1). The KD value obtained here is close to the value obtained for mammalian SRP binding to non trans-lating wheat germ ribosomes (70 nM) measured by fluorescence spectros-copy [69]. The KD value for TF binding at the same temperature was deter-mined to 1.1 µM. This value corresponds to the KD value obtained in another study (1 uM at 20°C) by competing wild type TF with fluorescence labelled mutants of TF [141]. The method used here does not account for the kinetics of TF and SRP bind-ing to the ribosome. The kinetics of TF binding to non translating ribosome have been investigated by others using fluorescence labelled TF and was found to be slow [141]. Thus the TF-ribosome complex is long lived, perhaps to enable TF to stay bound to the ribosome throughout the synthesis of a protein. Unfolded protein substrates on the other hand have been found to bind TF with fast kinetics (in the absence of ribosomes) . Since TF is an abun-dant protein which is present well in excess over ribosomes, and that it pre-sumably should stay bound to the ribosome throughout the synthesis of at least a single folding domain, fast kinetics of ribosome binding is probably not optimal. The kinetics of SRP binding to the naked ribosome has not been determined but ought to be fast considering the sub stoichiometric amounts of SRP in the cell. Hence, although TF and SRP both bind to ribosomes, their binding modes appear to be very different. TF binds to naked ribosomes with moderate affinity and slow kinetics and presumably interact rather promiscuously with the nascent chain. On the contrary, SRP most likely binds to the ribosome rapidly, with higher affinity and is more specific in its interaction with the polypeptide substrate.

Table 1. KD values for SRP and TF binding to ribosomes.

Complex

KD (µM)

4°C 20°C

H (kJ/mole)

SRP:70S 0.09 ± 0.02 0.17 ± 0.02 26.8

SRP:GTP:70S 0.11 ± 0.02

SRP:GDP:70S 0.09 ± 0.02

SRP:MFTI:RNC 0.18 ± 0.03

TF:70S 1.1 ± 0.3 2.0 ± 0.03 25.2

.

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Simultaneous binding of TF and SRP to E. coli ribosomes (paper III)Since TF and L23 both have been proved to bind to ribosomal protein L23 and considering that L23 is a rather small protein it was more or less as-sumed that their respective binding sites are overlapping. Cross-linking analysis in paper II indicated that this was the case. We initially set out to corroborate this conclusion by using a more quantitative approach with radio-labelled TF and SRP as described in the previous section. To our sur-prise, we found that TF does not displace SRP from naked ribosomes. Nei-ther did SRP displace TF in the reverse experiments. Hence it appears that TF and SRP simultaneously binds to E. coli ribosomes and thus have sepa-rate binding sites on L23. An independent study using several other techni-cal approaches corroborates this conclusion [142] and it was moreover sug-gested that the loss of cross-links observed in the combined presence of TF and SRP (observed in paper II) may be due to structural changes induced by the simultaneous binding. In line with this, cross-linking between SRP and TF on the ribosome illustrated that they are within 10Å from each other on the ribosome [142]. Furthermore, Buskiewicz et al showed that FtsY in the presence of a non hydrolysable GTP analogue, binds to SRP on the ribosome and displaces TF. In contrast we failed to detect any such displacement of TF by FtsY in our experiments. However, as we used GTP, our experiments should be repeated in the presence of a non hydrolysable GTP analogue before any final conclusion can be drawn. A displacement of TF induced by the SRP-FtsY complex is an attractive model for evacuating the ribosome surface from TF before targeting to the translocon. However, the presence of a nascent chain may complicate the picture and clearly more data needs to be collected before settling this discussion.

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Characterisation of TF binding to ribosome nascent chain complexes (paper IV) TF binding to non translating ribosomes has been quantified by us and oth-ers as described above. The affinity of TF for the ribosome is expected to increase in the presence of a nascent chain but quantitative information re-garding TF-RNC binding has been scarce. One limiting factor is the technical difficulties with obtaining TF free RNCs of high quality from crude cell-free translation systems. In this study we have used a defined in vitro translation system assembled from purified E. coli factors to produce ribosomes stalled on truncated mRNAs coding for different nascent peptides. The fractions of ribosomes displaying a nascent chain were typically around 50-65 %, esti-mated by incorporation of a radioactive amino acid. These were used to-gether with radioactively labelled TF in equilibrium binding studies as de-scribed in the previous section. Since TF also binds to non translating ri-bosomes present in the reaction, albeit with known affinity, a two site bind-ing model was used. As substrates in the TF binding experiments we used nascent chains of RpoB, a protein that has been shown to be aggregation prone in TF and DnaK depleted cells. We found the affinity for TF to increase with nascent chain length and a 133 residue long nascent chain of RpoB was found to bind TF with 30-fold higher affinity than non translating ribosomes. In com-parison, the shortest nascent chain used (60 residues) only had 2-fold higher affinity compared to naked ribosomes (table 2). This indicates that when an increasing number of binding motifs in the nascent chain is displayed out-side the ribosome, multiple contacts with TFs extensive folding cave is estab-lished, in accordance with the recently solved crystal structure [130]. Other studies have shown that TF can be cross-linked to a nascent chain as short as 57 residues [28] and in the scanning of a nascent SA sequence de-scribed in paper II, TF cross-links was found predominantly in the region of the nascent chain close to the ribosome. This is not consistent with our find-ing that a 60 residue nascent chain of RpoB was too short to bind well to TF. Another cross-linking study using nascent chains of the secretory protein OmpA is more in line with our results [68]. OmpA nascent chains were found cross-linked to L23 until a length of 89 residues at which TF cross-links appeared. These became more pronounced when the nascent chain length was increased further. Interestingly, we found that a 100 residue long nascent chain of the mem-brane protein leader peptidase binds to TF with more than 5-fold lower af-finity than the nascent RpoB of similar length. Inspection of the amino acid sequence does not give any obvious explanation to this difference in affinity, although it can not be excluded that Lep100 has fewer binding motifs for TF than RpoB100. Another possibility is that L23 has higher affinity for Lep100 than for RpoB100, thus competing more efficiently with TF for binding to

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Lep100. A third option is that an extended structure of the nascent chain is important for TF binding. SRP is thought to bind nascent signal sequences in

-helical conformation which is induced already inside the ribosome. Per-haps the hydrophobic environment in TFs peptide binding cave also pro-motes the -helical conformation of the signal sequence, with reduced affin-ity as a consequence. Clearly, a comprehensive analysis of TF binding to different ribosome nascent chain complexes has to be performed to give a more complete picture.

Table 2. KD values for TF binding to RNCs

Complex % nascent chain KD

RpoB60 60- 65 500 ± 50

RpoB80 60-65 108 ± 10

RpoB103 60-65 47 ± 11

RpoB133 45-55 32 ± 5

Lep100 60-65 300 ± 50

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CONCLUSIONS

In vitro translation, membrane targeting and cross-linking experiments showed that heterologous nascent membrane proteins interact with E. coliSRP and translocon components. The cross-linking patterns were compara-ble to those previously observed for native E. coli membrane proteins. Thus the initial steps in targeting and insertion of heterologous membrane pro-teins may proceed similar to native membrane proteins.

Measurements of the in vitro elongation rates of a membrane protein showed that E. coli SRP does not have elongation arrest activity.

In cross-linking experiments, E. coli SRP was found to bind ribosomal pro-tein L23 at the peptide exit site. Moreover, a nascent signal sequence was found to cross-link to L23. This interaction was competed out by SRP show-ing that L23 is an optimal position for interaction with emerging polypep-tides.

The KD values for SRP and TF binding to naked E. coli ribosomes were de-termined to 90 nM and 1 µM respectively. Competition binding experiments reveal that TF and SRP bind simultaneously to naked ribosomes and thus have separate binding sites on L23.

A quantitative analysis of TF binding to ribosome nascent chains complexes shows that the affinity increases as the nascent chain becomes longer. We conclude that when an increasing number of binding motifs in the nascent chain is displayed outside the ribosome, multiple contacts with TFs exten-sive folding cave is established.

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SUMMARY IN SWEDISH

Korrekt proteinveckning och transport av proteiner till cellmembranet är av största vikt för cellens överlevnad. Båda dessa processer kan starta redan på ribosomen med hjälp av vissa ribosomassocierade faktorer; SRP och TF. SRP läser av platsen där en ny peptidkedja kommer fram på ribosomens yta och känner igen vissa sekvenser som är specifika för proteiner som ska transpor-teras till cellmembranet. TF är ett protein som assisterar proteiner när de ska veckas till sin slutgiltiga form. Likt SRP läser TF av ribosomens yta efter nya proteiner att hjälpa och förhindra att felaktig veckning uppstår. I denna avhandling har vi dels undersökt hur två stycken främmande mem-branproteiner levereras till och inserteras i E. coli cell membran. Vi har funnit att de processas på samma sätt som E. colis egna membranproteiner. Detta är av värde att veta för rekombinant produktion av främmande membran-proteiner i E. coli. Vi har även karaktäriserat SRP och TFs bindning till ribosomer med hjälp av krosslänkande tekniker samt kvantitativa bindingstudier. Både SRP och TF binder till det ribosomala proteinet L23 som finns nära peptid utgången. Där är de strategiskt placerade för att kunna interagera med den framträdande polypeptiden. Kvantitativa studier av TFs affinitet för framträdande poly-peptider undersöktes. Dessa resultat ger information om mekanismen för TF assisterad proteinveckning.

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ACKNOWLEDGMENTS

My sincere gratitude to:

Måns, thank you for being such a good friend and supervisor.

Jarl, thank you for all support and encouragement over the years.

All my friends in the Ehrenberg laboratory and at ICM: Especially thanks to: Ayman, for regularly bringing the cakes from Egypt- they are great together with strong coffee! Elli, for being such a sweet person, Emmelie, for all the dog discussions, Johan, for making sure there is always fresh coffee, Martin- for being such a nice room mate and always helping out with computers and other problems, Ray, for proofreading the thesis in the last minute, Natalia, for being a nice and fun room mate- I´ve actually almost started to enjoy the salsa music, Misha, for everything you have taught me, Lamine, for being so kind and for your dirty sense of humour. Eva, I enjoyed making the fMet prep with you! Suparna, for always being kind and helpful. Vasili, for fun talks-I like the story about your cat!. Anyway, I owe you all a lot and thank you for sharing components and for your efforts with all the prep work. Now that I´m finished with this thesis I promise I’ll turn into a nice person again!

Ronald Ullers and Joen Luirink for collaborations.

All my old and new friends at the Pharmaceutical Pharmacology depart-ment, Erika R, Tina, Anna K, Karolina, Jonas, Helgi, Johan, Mattias, Lisa, Ilona, Ramona, Ruta, Maija, Felikss, Ilse, Peteris, Svetlana. Thanks to Erika J and Agneta H for helping out with the administrative matters.

All friends outside BMC, nobody mentioned but nobody forgotten!

My family, you are the best!!!!!!!!!

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REFERENCES

1. Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. (2000) The structural basis of ribosome activity in peptide bond synthesis, Science. 289,920-30. 2. Moore, P. B. & Steitz, T. A. (2003) The structural basis of large ribosomal subunit function, Annu Rev Biochem. 72, 813-50. 3. Johnson, A. E. & van Waes, M. A. (1999) The translocon: a dynamic gate-way at the ER membrane, Annu Rev Cell Dev Biol. 15, 799-842. 4. Beckmann, R., Spahn, C. M., Eswar, N., Helmers, J., Penczek, P. A., Sali, A., Frank, J. & Blobel, G. (2001) Architecture of the protein-conducting chan-nel associated with the translating 80S ribosome, Cell. 107, 361-72. 5. Ullers, R. S., Houben, E. N., Raine, A., ten Hagen-Jongman, C. M., Ehren-berg, M., Brunner, J., Oudega, B., Harms, N. & Luirink, J. (2003) Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome, J Cell Biol. 161, 679-84. 6. Pool, M. R., Stumm, J., Fulga, T. A., Sinning, I. & Dobberstein, B. (2002) Distinct modes of signal recognition particle interaction with the ribosome, Science. 297, 1345-8. 7. Kramer, G., Rauch, T., Rist, W., Vorderwulbecke, S., Patzelt, H., Schulze-Specking, A., Ban, N., Deuerling, E. & Bukau, B. (2002) L23 protein functions as a chaperone docking site on the ribosome, Nature. 419, 171-4. 8. Gu, S. Q., Peske, F., Wieden, H. J., Rodnina, M. V. & Wintermeyer, W. (2003) The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome, Rna. 9, 566-73. 9. Nakatogawa, H., Murakami, A., Mori, H. & Ito, K. (2005) SecM facilitates translocase function of SecA by localizing its biosynthesis, Genes Dev. 19,436-44. 10. Murakami, A., Nakatogawa, H. & Ito, K. (2004) Translation arrest of SecM is essential for the basal and regulated expression of SecA, Proc Natl Acad Sci U S A. 101, 12330-5. 11. Nakatogawa, H., Murakami, A. & Ito, K. (2004) Control of SecA and SecM translation by protein secretion, Curr Opin Microbiol. 7, 145-50. 12. Nakatogawa, H. & Ito, K. (2004) Intraribosomal regulation of expression and fate of proteins, Chembiochem. 5, 48-51. 13. Nakatogawa, H. & Ito, K. (2002) The ribosomal exit tunnel functions as a discriminating gate, Cell. 108, 629-36. 14. Nakatogawa, H. & Ito, K. (2001) Secretion monitor, SecM, undergoes self-translation arrest in the cytosol, Mol Cell. 7, 185-92. 15. Gabashvili, I. S., Gregory, S. T., Valle, M., Grassucci, R., Worbs, M., Wahl, M. C., Dahlberg, A. E. & Frank, J. (2001) The polypeptide tunnel sys-tem in the ribosome and its gating in erythromycin resistance mutants of L4 and L22, Mol Cell. 8, 181-8.

44

16. Woolhead, C. A., McCormick, P. J. & Johnson, A. E. (2004) Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins, Cell. 116, 725-36. 17. Liao, S., Lin, J., Do, H. & Johnson, A. E. (1997) Both lumenal and cytoso-lic gating of the aqueous ER translocon pore are regulated from inside the ribosome during membrane protein integration, Cell. 90, 31-41. 18. Pool, M. R. (2003) Getting to the membrane: how is co-translational pro-tein targeting to the endoplasmic reticulum regulated?, Biochem Soc Trans. 31, 1232-7. 19. Walter, P., Ibrahimi, I. & Blobel, G. (1981) Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein, J Cell Biol. 91, 545-50. 20. Walter, P. & Blobel, G. (1982) Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum, Nature. 299, 691-8. 21. Gilmore, R., Walter, P. & Blobel, G. (1982) Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor, J Cell Biol. 95, 470-7. 22. Gilmore, R., Blobel, G. & Walter, P. (1982) Protein translocation across the endoplasmic reticulum. I. Detection in the microsomal membrane of a receptor for the signal recognition particle, J Cell Biol. 95, 463-9. 23. Mason, N., Ciufo, L. F. & Brown, J. D. (2000) Elongation arrest is a physiologically important function of signal recognition particle, Embo J. 19,4164-74. 24. Miller, J. D., Wilhelm, H., Gierasch, L., Gilmore, R. & Walter, P. (1993) GTP binding and hydrolysis by the signal recognition particle during initia-tion of protein translocation, Nature. 366, 351-4. 25. Connolly, T. & Gilmore, R. (1989) The signal recognition particle recep-tor mediates the GTP-dependent displacement of SRP from the signal se-quence of the nascent polypeptide, Cell. 57, 599-610. 26. Gilmore, R. & Blobel, G. (1983) Transient involvement of signal recogni-tion particle and its receptor in the microsomal membrane prior to protein translocation, Cell. 35, 677-85. 27. Keenan, R. J., Freymann, D. M., Stroud, R. M. & Walter, P. (2001) The signal recognition particle, Annu Rev Biochem. 70, 755-75. 28. Valent, Q. A., Kendall, D. A., High, S., Kusters, R., Oudega, B. & Luirink, J. (1995) Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides, Embo J. 14, 5494-505. 29. von Heijne, G. (1998) Life and death of a signal peptide, Nature. 396, 111, 113. 30. Bernstein, H. D., Poritz, M. A., Strub, K., Hoben, P. J., Brenner, S. & Wal-ter, P. (1989) Model for signal sequence recognition from amino-acid se-quence of 54K subunit of signal recognition particle, Nature. 340, 482-6. 31. Poritz, M. A., Strub, K. & Walter, P. (1988) Human SRP RNA and E. coli 4.5S RNA contain a highly homologous structural domain, Cell. 55, 4-6.

45

32. Romisch, K., Webb, J., Herz, J., Prehn, S., Frank, R., Vingron, M. & Dob-berstein, B. (1989) Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding do-mains, Nature. 340, 478-82. 33. Watanabe, M. & Blobel, G. (1989) Cytosolic factor purified from Es-cherichia coli is necessary and sufficient for the export of a preprotein and is a homotetramer of SecB, Proc Natl Acad Sci U S A. 86, 2728-32. 34. MacIntyre, S. & Henning, U. (1990) The role of the mature part of secre-tory proteins in translocation across the plasma membrane and in regulation of their synthesis in Escherichia coli, Biochimie. 72, 157-67. 35. Liu, G., Topping, T. B. & Randall, L. L. (1989) Physiological role during export for the retardation of folding by the leader peptide of maltose-binding protein, Proc Natl Acad Sci U S A. 86, 9213-7. 36. Watanabe, M. & Blobel, G. (1995) High-affinity binding of Escherichia coli SecB to the signal sequence region of a presecretory protein, Proc Natl Acad Sci U S A. 92, 10133-6. 37. Weiss, J. B., Ray, P. H. & Bassford, P. J., Jr. (1988) Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro, Proc Natl Acad Sci U S A. 85, 8978-82. 38. Fekkes, P. & Driessen, A. J. (1999) Protein targeting to the bacterial cyto-plasmic membrane, Microbiol Mol Biol Rev. 63, 161-73. 39. Schunemann, D. (2004) Structure and function of the chloroplast signal recognition particle, Curr Genet. 44, 295-304. 40. Abell, B. M., Pool, M. R., Schlenker, O., Sinning, I. & High, S. (2004) Sig-nal recognition particle mediates post-translational targeting in eukaryotes, Embo J. 23, 2755-64. 41. Froderberg, L., Houben, E., Samuelson, J. C., Chen, M., Park, S. K., Phil-lips, G. J., Dalbey, R., Luirink, J. & De Gier, J. W. (2003) Versatility of inner membrane protein biogenesis in Escherichia coli, Mol Microbiol. 47, 1015-27. 42. Ulbrandt, N. D., Newitt, J. A. & Bernstein, H. D. (1997) The E. coli signal recognition particle is required for the insertion of a subset of inner mem-brane proteins, Cell. 88, 187-96. 43. Newitt, J. A., Ulbrandt, N. D. & Bernstein, H. D. (1999) The structure of multiple polypeptide domains determines the signal recognition particle targeting requirement of Escherichia coli inner membrane proteins, J Bacte-riol. 181, 4561-7. 44. Bernstein, H. D. & Hyndman, J. B. (2001) Physiological basis for conser-vation of the signal recognition particle targeting pathway in Escherichia coli, J Bacteriol. 183, 2187-97. 45. Siegel, V. & Walter, P. (1986) Removal of the Alu structural domain from signal recognition particle leaves its protein translocation activity intact, Nature. 320, 81-4. 46. Thomas, Y., Bui, N. & Strub, K. (1997) A truncation in the 14 kDa protein of the signal recognition particle leads to tertiary structure changes in the RNA and abolishes the elongation arrest activity of the particle, Nucleic Acids Res. 25, 1920-9.

46

47. Eichler, J. & Moll, R. (2001) The signal recognition particle of Archaea, Trends Microbiol. 9, 130-6. 48. Nakamura, K., Yahagi, S., Yamazaki, T. & Yamane, K. (1999) Bacillus subtilis histone-like protein, HBsu, is an integral component of a SRP-like particle that can bind the Alu domain of small cytoplasmic RNA, J Biol Chem. 274, 13569-76. 49. Meyer, D. I. & Dobberstein, B. (1980) A membrane component essential for vectorial translocation of nascent proteins across the endoplasmic reticu-lum: requirements for its extraction and reassociation with the membrane, JCell Biol. 87, 498-502. 50. Tajima, S., Lauffer, L., Rath, V. L. & Walter, P. (1986) The signal recogni-tion particle receptor is a complex that contains two distinct polypeptide chains, J Cell Biol. 103, 1167-78. 51. Luirink, J., ten Hagen-Jongman, C. M., van der Weijden, C. C., Oudega, B., High, S., Dobberstein, B. & Kusters, R. (1994) An alternative protein tar-geting pathway in Escherichia coli: studies on the role of FtsY, Embo J. 13,2289-96. 52. Freymann, D. M., Keenan, R. J., Stroud, R. M. & Walter, P. (1997) Struc-ture of the conserved GTPase domain of the signal recognition particle, Na-ture. 385, 361-4. 53. Montoya, G., te Kaat, K., Moll, R., Schafer, G. & Sinning, I. (1999) Crys-tallization and preliminary x-ray diffraction studies on the conserved GTPase domain of the signal recognition particle from Acidianus ambiva-lens, Acta Crystallogr D Biol Crystallogr. 55, 1949-51. 54. Montoya, G., Svensson, C., Luirink, J. & Sinning, I. (1997) Crystal struc-ture of the NG domain from the signal-recognition particle receptor FtsY, Nature. 385, 365-8. 55. Freymann, D. M., Keenan, R. J., Stroud, R. M. & Walter, P. (1999) Func-tional changes in the structure of the SRP GTPase on binding GDP and Mg2+GDP, Nat Struct Biol. 6, 793-801. 56. Keenan, R. J., Freymann, D. M., Walter, P. & Stroud, R. M. (1998) Crystal structure of the signal sequence binding subunit of the signal recognition particle, Cell. 94, 181-91. 57. Clemons, W. M., Jr., Gowda, K., Black, S. D., Zwieb, C. & Ramakrishnan, V. (1999) Crystal structure of the conserved subdomain of human protein SRP54M at 2.1 A resolution: evidence for the mechanism of signal peptide binding, J Mol Biol. 292, 697-705. 58. Rosendal, K. R., Wild, K., Montoya, G. & Sinning, I. (2003) Crystal struc-ture of the complete core of archaeal signal recognition particle and implica-tions for interdomain communication, Proc Natl Acad Sci U S A. 100, 14701-6. 59. Chu, F., Shan, S. O., Moustakas, D. T., Alber, F., Egea, P. F., Stroud, R. M., Walter, P. & Burlingame, A. L. (2004) Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry, Proc Natl Acad Sci U S A. 101, 16454-9. 60. de Leeuw, E., Poland, D., Mol, O., Sinning, I., ten Hagen-Jongman, C. M., Oudega, B. & Luirink, J. (1997) Membrane association of FtsY, the E. coli SRP receptor, FEBS Lett. 416, 225-9.

47

61. Millman, J. S., Qi, H. Y., Vulcu, F., Bernstein, H. D. & Andrews, D. W. (2001) FtsY binds to the Escherichia coli inner membrane via interactions with phosphatidylethanolamine and membrane proteins, J Biol Chem. 276,25982-9. 62. Peluso, P., Shan, S. O., Nock, S., Herschlag, D. & Walter, P. (2001) Role of SRP RNA in the GTPase cycles of Ffh and FtsY, Biochemistry. 40, 15224-33. 63. Peterson, J. H., Woolhead, C. A. & Bernstein, H. D. (2003) Basic amino acids in a distinct subset of signal peptides promote interaction with the signal recognition particle, J Biol Chem. 278, 46155-62. 64. Batey, R. T., Rambo, R. P., Lucast, L., Rha, B. & Doudna, J. A. (2000) Crystal structure of the ribonucleoprotein core of the signal recognition par-ticle, Science. 287, 1232-9. 65. McKnight, C. J., Briggs, M. S. & Gierasch, L. M. (1989) Functional and nonfunctional LamB signal sequences can be distinguished by their bio-physical properties, J Biol Chem. 264, 17293-7. 66. Bruch, M. D., McKnight, C. J. & Gierasch, L. M. (1989) Helix formation and stability in a signal sequence, Biochemistry. 28, 8554-61. 67. Rothe, C. & Lehle, L. (1998) Sorting of invertase signal peptide mutants in yeast dependent and independent on the signal-recognition particle, Eur J Biochem. 252, 16-24. 68. Eisner, G., Koch, H. G., Beck, K., Brunner, J. & Muller, M. (2003) Ligand crowding at a nascent signal sequence, J Cell Biol. 163, 35-44. 69. Flanagan, J. J., Chen, J. C., Miao, Y., Shao, Y., Lin, J., Bock, P. E. & John-son, A. E. (2003) Signal recognition particle binds to ribosome-bound signal sequences with fluorescence-detected subnanomolar affinity that does not diminish as the nascent chain lengthens, J Biol Chem. 278, 18628-37. 70. Halic, M., Becker, T., Pool, M. R., Spahn, C. M., Grassucci, R. A., Frank, J. & Beckmann, R. (2004) Structure of the signal recognition particle interacting with the elongation-arrested ribosome, Nature. 427, 808-14. 71. Jagath, J. R., Rodnina, M. V., Lentzen, G. & Wintermeyer, W. (1998) In-teraction of guanine nucleotides with the signal recognition particle from Escherichia coli, Biochemistry. 37, 15408-13. 72. Rapiejko, P. J. & Gilmore, R. (1997) Empty site forms of the SRP54 and SR alpha GTPases mediate targeting of ribosome-nascent chain complexes to the endoplasmic reticulum, Cell. 89, 703-13. 73. Bacher, G., Lutcke, H., Jungnickel, B., Rapoport, T. A. & Dobberstein, B. (1996) Regulation by the ribosome of the GTPase of the signal-recognition particle during protein targeting, Nature. 381, 248-51. 74. de Leeuw, E., te Kaat, K., Moser, C., Menestrina, G., Demel, R., de Kruijff, B., Oudega, B., Luirink, J. & Sinning, I. (2000) Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase ac-tivity, Embo J. 19, 531-41. 75. Jagath, J. R., Rodnina, M. V. & Wintermeyer, W. (2000) Conformational changes in the bacterial SRP receptor FtsY upon binding of guanine nucleo-tides and SRP, J Mol Biol. 295, 745-53.

48

76. Connolly, T., Rapiejko, P. J. & Gilmore, R. (1991) Requirement of GTP hydrolysis for dissociation of the signal recognition particle from its recep-tor, Science. 252, 1171-3. 77. Powers, T. & Walter, P. (1995) Reciprocal stimulation of GTP hydrolysis by two directly interacting GTPases, Science. 269, 1422-4. 78. Egea, P. F., Shan, S. O., Napetschnig, J., Savage, D. F., Walter, P. & Stroud, R. M. (2004) Substrate twinning activates the signal recognition par-ticle and its receptor, Nature. 427, 215-21. 79. Focia, P. J., Shepotinovskaya, I. V., Seidler, J. A. & Freymann, D. M. (2004) Heterodimeric GTPase core of the SRP targeting complex, Science. 303,373-7. 80. Shan, S. O., Stroud, R. M. & Walter, P. (2004) Mechanism of association and reciprocal activation of two GTPases, PLoS Biol. 2, e320. 81. Shan, S. O. & Walter, P. (2005) Co-translational protein targeting by the signal recognition particle, FEBS Lett. 579, 921-6. 82. Wild, K., Halic, M., Sinning, I. & Beckmann, R. (2004) SRP meets the ribosome, Nat Struct Mol Biol. 11, 1049-53. 83. Halic, M. & Beckmann, R. (2005) The signal recognition particle and its interactions during protein targeting, Curr Opin Struct Biol. 15, 116-25. 84. Veenendaal, A. K., van der Does, C. & Driessen, A. J. (2004) The protein-conducting channel SecYEG, Biochim Biophys Acta. 1694, 81-95. 85. Van den Berg, B., Clemons, W. M., Jr., Collinson, I., Modis, Y., Hart-mann, E., Harrison, S. C. & Rapoport, T. A. (2004) X-ray structure of a pro-tein-conducting channel, Nature. 427, 36-44. 86. Duong, F. & Wickner, W. (1997) The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling, Embo J. 16, 4871-9. 87. Duong, F. & Wickner, W. (1997) Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme, Embo J. 16, 2756-68. 88. Luirink, J., Samuelsson, T. & de Gier, J. W. (2001) YidC/Oxa1p/Alb3: evolutionarily conserved mediators of membrane protein assembly, FEBS Lett. 501, 1-5. 89. Yi, L., Jiang, F., Chen, M., Cain, B., Bolhuis, A. & Dalbey, R. E. (2003) YidC is strictly required for membrane insertion of subunits a and c of the F(1)F(0)ATP synthase and SecE of the SecYEG translocase, Biochemistry. 42,10537-44. 90. van der Laan, M., Bechtluft, P., Kol, S., Nouwen, N. & Driessen, A. J. (2004) F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis, J Cell Biol. 165, 213-22. 91. Houben, E. N., ten Hagen-Jongman, C. M., Brunner, J., Oudega, B. & Luirink, J. (2004) The two membrane segments of leader peptidase partition one by one into the lipid bilayer via a Sec/YidC interface, EMBO Rep. 5, 970-5.92. Houben, E. N., Urbanus, M. L., Van Der Laan, M., Ten Hagen-Jongman, C. M., Driessen, A. J., Brunner, J., Oudega, B. & Luirink, J. (2002) YidC and

49

SecY mediate membrane insertion of a Type I transmembrane domain, J Biol Chem. 277, 35880-6. 93. Raine, A., Ullers, R., Pavlov, M., Luirink, J., Wikberg, J. E. & Ehrenberg, M. (2003) Targeting and insertion of heterologous membrane proteins in E. coli, Biochimie. 85, 659-68. 94. Beck, K., Eisner, G., Trescher, D., Dalbey, R. E., Brunner, J. & Muller, M. (2001) YidC, an assembly site for polytopic Escherichia coli membrane pro-teins located in immediate proximity to the SecYE translocon and lipids, EMBO Rep. 2, 709-14. 95. Urbanus, M. L., Scotti, P. A., Froderberg, L., Saaf, A., de Gier, J. W., Brunner, J., Samuelson, J. C., Dalbey, R. E., Oudega, B. & Luirink, J. (2001) Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC, EMBO Rep. 2, 524-9. 96. Prinz, A., Behrens, C., Rapoport, T. A., Hartmann, E. & Kalies, K. U. (2000) Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA, Embo J. 19, 1900-6. 97. Frydman, J. (2001) Folding of newly translated proteins in vivo: the role of molecular chaperones, Annu Rev Biochem. 70, 603-47. 98. Bukau, B. & Horwich, A. L. (1998) The Hsp70 and Hsp60 chaperone machines, Cell. 92, 351-66. 99. Mayer, M. P. & Bukau, B. (1998) Hsp70 chaperone systems: diversity of cellular functions and mechanism of action, Biol Chem. 379, 261-8. 100. McCarty, J. S., Rudiger, S., Schonfeld, H. J., Schneider-Mergener, J., Nakahigashi, K., Yura, T. & Bukau, B. (1996) Regulatory region C of the E. coli heat shock transcription factor, sigma32, constitutes a DnaK binding site and is conserved among eubacteria, J Mol Biol. 256, 829-37. 101. Gamer, J., Multhaup, G., Tomoyasu, T., McCarty, J. S., Rudiger, S., Schonfeld, H. J., Schirra, C., Bujard, H. & Bukau, B. (1996) A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32, Embo J. 15, 607-17. 102. Szabo, A., Langer, T., Schroder, H., Flanagan, J., Bukau, B. & Hartl, F. U. (1994) The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE, Proc Natl Acad Sci U S A. 91,10345-9. 103. Fenton, W. A. & Horwich, A. L. (2003) Chaperonin-mediated protein folding: fate of substrate polypeptide, Q Rev Biophys. 36, 229-56. 104. Saibil, H. (2000) Molecular chaperones: containers and surfaces for fold-ing, stabilising or unfolding proteins, Curr Opin Struct Biol. 10, 251-8. 105. Martin, J. (2004) Chaperonin function--effects of crowding and con-finement, J Mol Recognit. 17, 465-72. 106. Grallert, H. & Buchner, J. (2001) Review: a structural view of the GroE chaperone cycle, J Struct Biol. 135, 95-103. 107. Randall, L. L. & Hardy, S. J. (2002) SecB, one small chaperone in the complex milieu of the cell, Cell Mol Life Sci. 59, 1617-23. 108. Ullers, R. S., Luirink, J., Harms, N., Schwager, F., Georgopoulos, C. & Genevaux, P. (2004) SecB is a bona fide generalized chaperone in Escherichia coli, Proc Natl Acad Sci U S A. 101, 7583-8.

50

109. Bochkareva, E. S. & Girshovich, A. S. (1992) A newly synthesized pro-tein interacts with GroES on the surface of chaperonin GroEL, J Biol Chem. 267, 25672-5. 110. Guthrie, B. & Wickner, W. (1990) Trigger factor depletion or overpro-duction causes defective cell division but does not block protein export, JBacteriol. 172, 5555-62. 111. Lecker, S., Lill, R., Ziegelhoffer, T., Georgopoulos, C., Bassford, P. J., Jr., Kumamoto, C. A. & Wickner, W. (1989) Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro, Embo J. 8, 2703-9. 112. Cunningham, K., Lill, R., Crooke, E., Rice, M., Moore, K., Wickner, W. & Oliver, D. (1989) SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA, Embo J. 8, 955-9. 113. Lill, R., Crooke, E., Guthrie, B. & Wickner, W. (1988) The "trigger factor cycle" includes ribosomes, presecretory proteins, and the plasma membrane, Cell. 54, 1013-8. 114. Crooke, E., Guthrie, B., Lecker, S., Lill, R. & Wickner, W. (1988) ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle, Cell. 54, 1003-11. 115. Crooke, E., Brundage, L., Rice, M. & Wickner, W. (1988) ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes, Embo J. 7, 1831-5. 116. Crooke, E. & Wickner, W. (1987) Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form, Proc Natl Acad Sci U S A. 84, 5216-20. 117. Deuerling, E., Schulze-Specking, A., Tomoyasu, T., Mogk, A. & Bukau, B. (1999) Trigger factor and DnaK cooperate in folding of newly synthesized proteins, Nature. 400, 693-6. 118. Teter, S. A., Houry, W. A., Ang, D., Tradler, T., Rockabrand, D., Fischer, G., Blum, P., Georgopoulos, C. & Hartl, F. U. (1999) Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperon-ing nascent chains, Cell. 97, 755-65. 119. Deuerling, E., Patzelt, H., Vorderwulbecke, S., Rauch, T., Kramer, G., Schaffitzel, E., Mogk, A., Schulze-Specking, A., Langen, H. & Bukau, B. (2003) Trigger Factor and DnaK possess overlapping substrate pools and binding specificities, Mol Microbiol. 47, 1317-28. 120. Agashe, V. R., Guha, S., Chang, H. C., Genevaux, P., Hayer-Hartl, M., Stemp, M., Georgopoulos, C., Hartl, F. U. & Barral, J. M. (2004) Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed, Cell. 117, 199-209. 121. Lee, H. C. & Bernstein, H. D. (2002) Trigger factor retards protein ex-port in Escherichia coli, J Biol Chem. 277, 43527-35. 122. Zarnt, T., Tradler, T., Stoller, G., Scholz, C., Schmid, F. X. & Fischer, G. (1997) Modular structure of the trigger factor required for high activity in protein folding, J Mol Biol. 271, 827-37.

51

123. Hesterkamp, T., Deuerling, E. & Bukau, B. (1997) The amino-terminal 118 amino acids of Escherichia coli trigger factor constitute a domain that is necessary and sufficient for binding to ribosomes, J Biol Chem. 272, 21865-71. 124. Stoller, G., Rucknagel, K. P., Nierhaus, K. H., Schmid, F. X., Fischer, G. & Rahfeld, J. U. (1995) A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor, Embo J. 14, 4939-48. 125. Scholz, C., Mucke, M., Rape, M., Pecht, A., Pahl, A., Bang, H. & Schmid, F. X. (1998) Recognition of protein substrates by the prolyl isomerase trigger factor is independent of proline residues, J Mol Biol. 277, 723-32. 126. Patzelt, H., Rudiger, S., Brehmer, D., Kramer, G., Vorderwulbecke, S., Schaffitzel, E., Waitz, A., Hesterkamp, T., Dong, L., Schneider-Mergener, J., Bukau, B. & Deuerling, E. (2001) Binding specificity of Escherichia coli trig-ger factor, Proc Natl Acad Sci U S A. 98, 14244-9. 127. Kramer, G., Rutkowska, A., Wegrzyn, R. D., Patzelt, H., Kurz, T. A., Merz, F., Rauch, T., Vorderwulbecke, S., Deuerling, E. & Bukau, B. (2004) Functional dissection of Escherichia coli trigger factor: unraveling the func-tion of individual domains, J Bacteriol. 186, 3777-84. 128. Kramer, G., Patzelt, H., Rauch, T., Kurz, T. A., Vorderwulbecke, S., Bukau, B. & Deuerling, E. (2004) Trigger factor peptidyl-prolyl cis/trans isomerase activity is not essential for the folding of cytosolic proteins in Es-cherichia coli, J Biol Chem. 279, 14165-70. 129. Ludlam, A. V., Moore, B. A. & Xu, Z. (2004) The crystal structure of ribosomal chaperone trigger factor from Vibrio cholerae, Proc Natl Acad Sci U S A. 101, 13436-41. 130. Ferbitz, L., Maier, T., Patzelt, H., Bukau, B., Deuerling, E. & Ban, N. (2004) Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins, Nature. 431, 590-6. 131. Patzelt, H., Kramer, G., Rauch, T., Schonfeld, H. J., Bukau, B. & Deuer-ling, E. (2002) Three-state equilibrium of Escherichia coli trigger factor, BiolChem. 383, 1611-9. 132. Liu, C. P., Perrett, S. & Zhou, J. M. (2005) Dimeric trigger factor stably binds folding-competent intermediates and cooperates with the DnaK-DnaJ-GrpE chaperone system to allow refolding, J Biol Chem.133. Rudiger, S., Germeroth, L., Schneider-Mergener, J. & Bukau, B. (1997) Substrate specificity of the DnaK chaperone determined by screening cellu-lose-bound peptide libraries, Embo J. 16, 1501-7. 134. Rudiger, S., Buchberger, A. & Bukau, B. (1997) Interaction of Hsp70 chaperones with substrates, Nat Struct Biol. 4, 342-9. 135. Tate, C. G. & Grisshammer, R. (1996) Heterologous expression of G-protein-coupled receptors, Trends Biotechnol. 14, 426-30. 136. Grisshammer, R. & Tate, C. G. (1995) Overexpression of integral mem-brane proteins for structural studies, Q Rev Biophys. 28, 315-422. 137. Tucker, J. & Grisshammer, R. (1996) Purification of a rat neurotensin receptor expressed in Escherichia coli, Biochem J. 317 ( Pt 3), 891-9. 138. Weiss, H. M. & Grisshammer, R. (2002) Purification and characteriza-tion of the human adenosine A(2a) receptor functionally expressed in Es-cherichia coli, Eur J Biochem. 269, 82-92.

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139. Avdeeva, O. N., Myasnikov, A. G., Sergiev, P. V., Bogdanov, A. A., Brimacombe, R. & Dontsova, O. A. (2002) Construction of the 'minimal' SRP that interacts with the translating ribosome but not with specific membrane receptors in Escherichia coli, FEBS Lett. 514, 70-3. 140. Luirink, J. & Sinning, I. (2004) SRP-mediated protein targeting: struc-ture and function revisited, Biochim Biophys Acta. 1694, 17-35. 141. Maier, R., Eckert, B., Scholz, C., Lilie, H. & Schmid, F. X. (2003) Interac-tion of trigger factor with the ribosome, J Mol Biol. 326, 585-92. 142. Buskiewicz, I., Deuerling, E., Gu, S. Q., Jockel, J., Rodnina, M. V., Bu-kau, B. & Wintermeyer, W. (2004) Trigger factor binds to ribosome-signal-recognition particle (SRP) complexes and is excluded by binding of the SRP receptor, Proc Natl Acad Sci U S A. 101, 7902-6.

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 6

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A doctoral dissertation from the Faculty of Pharmacy, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy. (Prior to January, 2005, the series was published under the title "Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy".)

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