Abstract The signal recognition particle (SRP) is a ribo-nucleoprotein required for targeting a subset of nascentpre-secretory proteins to the endoplasmic reticulum mem-brane. Of the six SRP polypeptides, the most highly con-served is Srp54p, a modular protein consisting of an amino-terminal (N) domain of unknown function, a centralGTPase (G) domain, and a carboxyl-terminal (M) domainimplicated in the recognition of both signal sequences andSRP RNA. To identify regions of Srp54p that interact withother SRP subunits or regulatory proteins, we carried outsystematic mutagenesis of the fission yeast homolog, prin-cipally using a “clustered charged-to-alanine” strategy. Ofthe 35 alleles examined, 13 are unable to support growth,two confer cold-sensitivity, five confer heat-sensitivity,and 15 produce no discernible phenotype. The lethal andconditional mutations map throughout the protein to sev-eral conserved regions, confirming that these motifs playcritical roles in Srp54p function. The effects of the amino-acid substitutions are analyzed with reference to the re-cently determined tertiary structures of the N/G domainand the intact protein from a thermophilic bacterium.

Key words GTPase · RNA-protein interaction · Protein targeting

Introduction

The signal recognition particle (SRP) recognizes the sig-nal sequences on a subset of nascent pre-secretory proteinsas the first step in targeting ribosomes translating thesepolypeptides to the endoplasmic reticulum (ER) membrane(reviewed in Walter and Johnson 1994). SRP is a stable ri-bonucleoprotein composed of one RNA molecule of ap-proximately 300 nucleotides (SRP RNA) and six polypep-tides organized into two heterodimeric proteins (Srp9/14pand Srp68/72p) and two monomeric proteins (Srp19p andSrp54p) (Walter and Blobel 1980, 1982; Siegel and Wal-ter 1985; Lütcke et al. 1993). Of these, Srp54p has beenascribed the greatest number of functions, including keyroles in the recognition of signal peptides and binding tothe SRP receptor in the ER membrane (Krieg et al. 1986;Kurzchalia et al. 1986; Tajima et al. 1986; Siegel and Wal-ter 1988b; Römisch et al. 1990; Zopf et al. 1990; High andDobberstein 1991). Although Srp54p is known to directlycontact SRP RNA, this interaction is facilitated by Srp19p(Siegel and Walter 1988 b, c; Hann et al. 1992; Selinger etal. 1993).

In contrast to the other SRP polypeptides, the sequenceof the Srp54 protein, a member of the GTPase superfam-ily, has been highly conserved throughout evolution. Forexample, Srp68p and Srp72p are only 18% and 23% iden-tical, respectively, between mammals and Saccharomycescerevisiae, as compared to the 55% identity between theSrp54p sequences (Brown et al. 1994). In eubacteria, thereexist homologs of SRP RNA (designated 4.5S) and theSrp54 protein, but not of the other subunits of the complex(Römisch et al. 1989; Fleischmann et al. 1995). Based oncomparative sequence analysis, Srp54p can be divided intothree distinct regions: an amino-terminal N domain con-taining a high concentration of charged amino acids, a cen-tral G domain containing the GTPase consensus motifs G-1 through G-4 as well as additional invariant residues,and a carboxy terminal M domain containing several am-phipathic α-helices rich in methionine residues (Bernsteinet al. 1989; Römisch et al. 1990; Zopf et al. 1990). These

Received: 29 June / 9 November 1998

Enrique Martínez-Force · Sujata Lakhe-ReddyJo Ann Wise

Systematic mutagenesis of the fission yeast Srp54 protein

E. Martínez-Force1 · S. Lakhe-Reddy · J. A. Wise (½)Department of Molecular Biology and Microbiology,Case Western Reserve University,School of Medicine,10900 Euclid Avenue,Cleveland, OH 44106-4960, USAe-mail: jaw17@po.cwru.eduTel.: +1-216-368 1876Fax: +1-216-368 2010

Present address:1Instituto de la Grasa, Consejo Superior de InvestigacionesCientíficas, Avenida Padre García Tejero, 4, E-41012-Sevilla, Spain

Communicated by S. W. Liebman

Curr Genet (1999) 35: 88–102 © Springer-Verlag 1999

ORIGINAL PAPER

assignments were borne out by recent X-ray crystallo-graphic data (Freymann et al. 1997; Keenan et al. 1998),which also provided dramatic insights into the function ofthe Srp54 protein (discussed below).

We previously analyzed the effects in vivo of a seriesof mutations in the GTPase signature motifs of the Schizo-saccharomyces pombe Srp54p homolog. The results ofthese studies suggested a model in which GTP hydrolysisis required for release of the particle from the ER mem-brane (Althoff et al. 1994b). More recent biochemical anal-yses were consistent with this proposal, and further sug-gested that hydrolysis of GTP by Srp54p and SRα oper-ates a “concerted switch” to ensure unidirectionality dur-ing ER targeting (reviewed in Millman and Andrews 1997).To identify additional amino acids important for the func-tion of Srp54p, we have carried out an extensive mutationalanalysis of the entire protein. Because we were particu-larly interested in obtaining conditional mutants in regionslikely to be involved in interactions with other components,clustered charged-to-alanine scanning was selected as theprincipal mutagenesis strategy. The seven conditional mu-tants identified, which map to diverse motifs throughoutthe protein, should prove useful in future genetic strategiesaimed at illuminating interactions among components ofthe SRP cycle.

Materials and methods

Materials. Restriction enzymes were purchased from either Bethes-da Research Laboratories or New England Biolabs. [α-35S]dATP forDNA sequencing, as well as the Sculptor in vitro MutagenesisSystem, was supplied by Amersham Corp. Oligonucleotides used forsequencing and mutagenesis were synthesized by either OperonTechnologies, Inc. or Oligos Etc., Inc.

Oligonucleotide-directed mutagenesis. Mutagenesis of the srp54gene was carried out with the Sculptor kit on pSP54-A, a plasmidderived from pSP54-U (Althoff et al. 1994b). Single-stranded pha-gemid pSP54-A template DNA was prepared from Escherichia colistrain TG1 using M13KO7 helper phage (Promega). The time of Ex-onuclease III digestion of the parental strand was increased to 45 mindue to the long distance between the NciI site and the mutagenesistargets. The sequences of the oligonucleotides used for clusteredcharged-to-alanine and site-specific mutagenesis are shown in Ta-ble 1. Potential mutant plasmids were isolated from E. coli andscreened initially by size (to eliminate plasmids with large deletions)and restriction mapping with DraI (to eliminate plasmids which hadrearranged with little loss or gain of size). In 17 out of 27 cases, themutagenic oligonucleotide was designed so that the mutation wouldcreate or destroy a restriction enzyme site (usually HaeIII; see Ta-ble 2). All of the mutations were confirmed by sequencing with theSequenase version 2.0 kit (United States Biochemicals) using oligo-nucleotides designed to make mutations just downstream as sequenc-ing primers.

Phenotypic analysis in S. pombe

Complementation in an srp54 disruption strain. Media and transfor-mation procedures were as described previously by Moreno et al.(1991) and Alfa et al. (1993). To test whether the mutant alleles couldcompensate for the absence of wild type Srp54p, we used a plasmidshuffle procedure in which pSP54-A plasmids carrying mutant al-leles were first transformed into an S. pombe haploid strain contain-

ing the srp54::LEU2 gene disruption in the chromosome and thepSP54-U (wild-type) plasmid (see text). After growth in liquid min-imal media suplemented with uracil for 24 h, cells were plated onminimal media with or without uracil and 5-fluororotic acid (5-FOA)to select against the plasmid carrying ura4. 5-FOA is converted to atoxic product by the ura4 gene product, thus killing cells carryingfunctional copies of this gene (Sikorski and Boeke 1991).

Growth rates of conditional mutants. Growth rates for haploid strainscarrying a heat- or cold-sensitive mutant allele as their sole sourceof Srp54p were determined by monitoring the increase of D600 in se-lective media using a Beckman DU-64 spectrophotometer. An over-night culture in minimal media was subcultured in 50 ml of fresh me-dia at 30°C and its D600 monitored until growth resumed. For tem-perature-shift experiments, cells in exponential phase were subcul-tured in 50 ml of fresh media pre-warmed to 30 or 37°C or pre-chilledto 18°C, placed in a shaking water bath at 18, 30 or 37°C, and theirgrowth monitored. The growth rate was measured by calculating theslope of the growth curve during the exponential phase.

Plate assays for conditional dominance. Viable alleles that showedconditional growth defects were tested for conditional dominance asdescribed previously (Althoff et al. 1994b). Briefly, growth of cellscontaining either a wild-type srp54 gene in the chromosome and themutant allele on a multicopy plasmid (pSP54-A) or both the wild-type and mutant alleles on multicopy plasmids was monitored at 18,30 and 37°C as follows. A single colony was re-streaked onto threeplates containing selective media, with the last plate streaked as acontrol (incubated at 30°C). Multiple isolates were examined in eachcase.

Microscopic examination. In addition to visual examination of con-ditional mutants, cells containing alleles that were phenotypicallyindistinguishable from the wild-type were scrutinized microscopi-cally in an effort to find anomalies other than reduced growth. Themorphology of cells in exponential phase growing at 18, 30 or 37°Cwas routinely monitored.

Phylogenetic analysis of Srp54p sequences. To construct a phylo-genetic tree, all known Srp54p sequences were first aligned usingthe PILEUP (Genetics Computer Group 1994) and CLUSTALW(Thompson et al. 1994) programs. The alignment was then opti-mized and used to generate a parsimony table using the DISTANC-ES program (Genetics Computer Group 1994) and to obtain an un-rooted tree with the GROWTREE program (Genetics ComputerGroup 1994). The tree was further corroborated using the programPROTPARS from the PHILIP package (Baum 1989) and employedfor phylogenetic analysis of the Srp54 protein. The programs PILE-UP, DISTANCES and GROWTREE are included in the GCG Wis-consin Package Version 8.0-OpenVMS (Genetics Computer Group1994). DRAWTREE and PROTPARS are included in the PHYLIP(Phylogeny Inference Package) software (Baum 1989). CLUS-TALW and the PHYLIP package were obtained from the IndianaUniversity server (ftp.bio.indiana.edu). Helical wheels were drawnwith the PLOT.A/HEL, which is part of the MacPROT software ob-tained from EMBL (netserv@embl-heidelberg.de). To analyze se-quence divergence, an evolutionary distance measured in PAM (-point mutations accepted per 100 aligned positions) units (Dayhoffet al. 1978) was assigned to each pair of protein sequences. The ev-olutionary distance separating two protein sequences can be de-scribed by a 1% PAM mutation matrix, which displays the probabil-ity of matching every amino acid with every other amino acid in analignment of two proteins diverged by one accepted point mutationper 100 residues (Dayhoff et al. 1978). The PAM distance betweentwo aligned protein sequences is the number of times the first pro-tein sequence must be transformed using the 1% PAM matrix toachieve the second sequence with highest probability. A family ofproteins can be divided into subfamilies defined by the maximumPAM width (MaxPW), the value assigned to the highest bridge con-necting proteins within the subfamily. The higher the PAM value ofthis bridge, the more sequence divergence the proteins in the sub-family display overall.

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Results

Evolutionary relationships among known Srp54 proteins

Because many new Srp54p sequences have recently be-come available, including the first archaeal representa-tives, we have updated the phylogenetic tree for this fam-ily of proteins as described in Materials and methods. Asshown in Fig. 1, this analysis indicates that, as expected,the archael Srp54p homologs cluster in a branch of the treedistinct from both the eukaryotic and eubacterial proteins.The degree of similarity among Srp54p homologs is ingood agreement with the evolutionary relationships estab-lished on the basis of 16S rRNA studies (Olsen et al. 1992).PAM calculations (data not shown), which provide a meas-ure of evolutionary divergence, indicate that the homologsin the eukaryotic branch of the Srp54p phylogenetic treeare nearly two-fold more conserved among themselvesthan are the prokaryotic homologs. Three subfamilies can

be distinguished among eukaryotic Srp54p sequences:mammalian, plant (cytoplasmic) and fungal. Within thefungal group, the S. pombe protein is more closely relatedto the Aspergillus niger homolog than to S. cerevisiaeSrp54p. The plant sequences display the most variability,with different cDNA sequences from the same species (ei-ther Lycopersicon esculentum or Arabidopsis thaliana)showing more divergence from each other than sequencesfrom different mammalian species (Can cannis, Mus mus-culus; see Fig. 1). Since these cytoplasmic forms were ob-tained from cDNA libraries, implying that they are ex-pressed, analysis of plant Srp54 proteins may identify re-gions of the protein that can accomodate amino acid sub-stitutions while remaining functional.

To generate a predicted secondary structure for theSrp54p family, the entire set of homologous sequencesreported to-date was aligned using the CLUSTALW pro-gram (Thompson et al. 1994) and sent to the “Predictpro-tein” EMBL electronic mail server, which uses the pro-gram PHD (Rost and Sander 1993, 1994 a, b; Rost et al.

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N domain:R8A/R9A 5′-CAGATTTAGGGGCCGCTTTGAACTCTGC-3′E25A/E26A 5′-ACTTCAGTGAATGCTGCTCTTGTCGATAC-3′K58A/K59A 5′-CAAATATTAAGGCCGCCATCAATGTATC-3′K72A/R73A 5′-GGTATAAATGGGGCCGCCATCGTCCAGAAG-3′D81A/E82A 5′-GCTGTCTTTGCTGCCCTTTGCAGT-3′K97A/K98A 5′-TTTACTCCTGCCGCCGGGAGACCT-3′G domain:R127A/R128A 5′-CACTACCAAGCCGCCGGTTTGAAATC-3′K173A/E174A 5′-CGTTATTGCCGCCGCCGGTGTTGATAAG-3′D177A/K178A 5′-GAAGGTGTTGCCGCCTTCAAAAACGATAG-3′D182A/R183A 5′-GTTCAAAAACGCTGCCTTTGACGTT-3′R194A/H195A 5′-GATACATCTGGTGCCGCTCAGCAGGAGCAG-3′K237A/E238A 5′-CAAAGCGTTTGCCGCCACTGCTGACTTTG-3′E277A/H278A 5′-GGTACCGGTGCCGCTATTAATGATTTG-3′E283A/R284A 5′-AATGATTTGGCCGCCTTTTCTCCAC-3′Mc domain:E305A/H306A 5′-GGTCTGATGGCCGCCGTTCAGTCTTTAG-3′K314A/K315A 5′-GATTTTGATGCCGCCAATATGG-3′R329A/D330A 5′-GTTTACGGTGGCCGCCTTTCGAGATCAAC-3′R332A/D333A 5′-CGAGACTTTGCCGCCCAACTCGG-3′D363A/E364A 5′-CGGTATGAATGCCGCCGAGGGATCTTTGCG-3′K371A/R372A 5′-TC TTTGCGTATGGCCGCTATGCTCTACATC-3′E414A/E415A 5′-GTTTTAGAGGTGGCCGCTACCATTTCTCAG-3′K428A/K429A 5′-CAAATGGCGGCCGCCATAGGAGG-3′Mv domain:K433A/D434A 5′-GATAGGAGGGGCCGCCGGAATTTTGGG-3′K449A/K450A 5′-GCAGCTCTGGCCGCTCCTCGTCAAC-3′K460A/R461A 5′-GCTATGCAGGCCGCCATGCAAGCC-3′PGB:T275N 5′-GTTCATTGGTAACGGTGAACATA-3′E277Q 5′-TGGTACCGGTCAACATATTAATG-3′PRBM:R398A 5′-GAACAACCCTCTGCTGTTTTACGTGTTGC-3′R401A 5′-CTCTCGTGTTTTAGCTGTTGCAAGGGGTAG-3R398A/R401A 5′-GAACAACCTTCTGCTGTTTTAGCTGTTGCAAGG-3′R404A 5′-CGTGTTGCAGCCGGTAGTGG-3′R404K 5′-CGTGTTGCAAAGGGTAGTGG-3′G408P 5′-GGGGTAGTCCTACAAGCG-3′G408I 5′-GGGGTAGTATTACAAGCG-3′

Table 1 Mutagenic oligonu-cleotides

1994). Figure 2 shows the predicted positions of α-heli-ces and β-sheets superimposed on the amino-acid se-quence of Srp54p from S. pombe. For the most part, thecomputer-based predictions coincide with the three-di-mensional models that recently became available for theFfh (fifty four homolog) protein from a thermophilic bac-terium (Freymann et al. 1997; Keenan et al. 1998; see be-low). Also indicated in Fig. 2 are the domain boundariesinferred from the sequence comparison (data not shown).

In agreement with earlier analyses based on more limiteddata (e.g., Bernstein et al. 1989; Althoff et al. 1994a),three distinct regions can be identified: the N domain atthe amino-terminus followed by the G domain which in-cludes the GTPase consensus motifs and the methionine-rich M domain at the carboxyl-terminus. In addition, thepresent analysis allowed the M domain to be divided intotwo subdomains: Mc, which is structurally conservedamong all organisms, and Mv, which is variable (con-served only among homologs from specific evolutionarygroups). Of the predicted structural domains, the G do-main alone is also extensively conserved at the primarysequence level.

The consensus distribution of hydrophilic and hydro-phobic residues in the α-helices of the N and Mc domainswas analyzed using the PLOT.A/HEL program, with theresults shown in Figs. 3 and 4. Three of the α-helicespresent in the N domain (NH1, NH2 and NH3) display aconserved hydrophobic face, whereas helix NH4 shows aconserved pattern of alternating hydrophobic/hydrophilicresidues not organized into a defined pattern (Fig. 3). OnlyNH2 and NH3 display a significant amphipathic character.Within the Mc domain, the α-helices McH3, McH5 andMcH6 are highly amphipathic, while McH1 is markedlyhydrophobic and McH2 is hydrophilic (Fig. 4). Biochem-ical data demonstrate that the methionine-rich domain isresponsible for the interaction of Srp54p with both SRPRNA and the signal sequence (Römisch et al. 1990; Zopfet al. 1990; High and Dobberstein 1991). The special char-acteristics of this amino acid led to the proposal that me-thionine residues form a flexible “brush” potentially ca-pable of recognizing a diverse array of ER targeting sig-nals (Bernstein et al. 1989). Notably, the number of methi-onine residues in the Mc domain varies widely, from nine(Mycoplasma mycoides) to 18 (S. cerevisiae). Among theeukaryotic sequences, only four methionine residues areconserved, and not a single conserved methionine is foundamong the prokaryotic sequences (Fig. 4; data not shown).S. pombe Srp54p contains 13 methionines in the Mc do-main, only one of which is shared by all eukaryotic homo-logs (Figs. 2 and 4). The analogous position for five othermethionines can be occupied by any hydrophobic residue,while three other methionines can vary to any non-hydro-philic amino acid and four positions can be occupied byany residue. In aggregate, analysis from an evolutionaryperspective suggests that the hydrophobic character of me-thionine may be just as important as the flexibility of itsside chain for interactions with signal peptides.

Systematic mutagenesis of the srp54 gene

Design of mutations

In an effort to avoid folding problems, which occurred withcertain previously analyzed Srp54p deletion mutants (Römisch et al. 1990), we adopted clustered charged-to-alanine mutagenesis as our principal approach to alteringthe S. pombe homolog. This strategy has been employed

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Fig. 1 Unrooted phylogenetic tree of the Srp54p family of proteins.All available sequences of Srp54p homologs were aligned and usedto generate the figure as described in Materials and methods. Thedistances along the branches are proportional to the PAM distanceas indicated by the bar. Abbreviations used are as follows: Aa Aci-dianus ambivalens (Moll and Schaefer 1996); Af Archaeoglobus ful-gidus (Klenk et al. 1997); An Aspergillus niger (Thompson et al.1995); Atcp A. thaliana (chloroplast protein; Franklin and Hoffman1993); Atct Arabidopsis thaliana (cytoplasmic protein; Chu et al.1994; Lindstrom et al. 1994); Bb Borrelia burgdorferi (Fraser et al.1997); Bs Bacillus subtilis (Honda et al. 1993); Ca Candida albicans(Hosking et al. 1997). Cc Can cannis (Bernstein et al. 1989); Ce Cae-norhabditis elegans (Wilson et al. 1994); Ec Escherichia coli(Römisch et al. 1989; Bernstein et al. 1989); Eh Entamoeba histo-lytica (Ramos et al. 1997); Hp Helicobacter pylori (Tomb et al. 1997);Hs Homo sapiens (Römisch et al. 1989); Hi Haemophilus influen-zae (Fleischmann et al. 1995); Hv Hordeum vulgare (Chu et al. 1997);Le Lycopersicon esculentum (Krolkiewicz et al. 1994); Met Methan-obacterium thermoautotrophicum (Smith et al. 1997); Mj Methano-coccus jannaschii (Bult et al. 1996); Mm Mus musculus (Römisch etal. 1989); Ml Mycobacterium leprae (Seeger and Harris 1997); MtMycobacterium tuberculosis (Seeger and Harris 1997); Myg Myco-plasma genitalium (Fraser et al. 1995); Mym Mycoplasma mycoides(Samuelsson 1992); Myp Mycoplasma pneumoniae (Himmelreich et al. 1996); Sa Sulfolobus acidocaldarius (Moll et al. 1997); Sc Sac-charomyces cerevisiae (Hann et al. 1989); Sm Streptococcus mutans(Gutierrez et al. 1997); Sp Schizosaccharomyces pombe (Hann et al.1989); So Synechococcus sp. (Packer and Howe 1996); Sy Synecho-cystis sp. (Kaneko et al. 1996); Ta Thermus aquaticus (Freymann etal. 1997); Yl Yarrowia lipolytica. (Lee and Ogrydziak 1997)

to systematically analyze several proteins (e.g., Wertmanet al. 1992; Diamond and Kirkegaard 1994; Hasset andCondit 1994; Reijo et al. 1994), revealing new insights intotheir structures and functions. The method is based on theobservation that clusters of charged amino acids are likelyto occupy exposed positions on the surface of a protein,since they cannot be readily accommodated in the hydro-phobic core. Moreover, changing these charged residues toalanine (a small amino acid) does not usually perturb thestructure of the polypeptide (Bennett et al. 1991; Heinz etal. 1992; De Filippis et al. 1994). Thus, this mutagenesisstrategy should minimize the number of mutations thathave drastic effects on protein folding and maximize thenumber that disrupt protein-ligand interactions. In additionto providing information about regions involved in inter-molecular contacts, this approach has produced a largefraction of heat- and cold-sensitive alleles in previous stud-ies. The conditional mutants can then be used in geneticstrategies to identify interacting proteins.

A variety of charged-to-alanine mutagenesis schemesranging from single mutations to changes in five residuesin a seven amino-acid window have been previously re-ported (Wertman et al. 1992; Diamond and Kirkegaard1994; Hasset and Condit 1994; Reijo et al. 1994). An eval-uation of the phenotypes that emerged indicates that thehighest frequency of heat-sensitive (ts) and cold-sensitive(cs) mutants (21% and 13%, respectively) was obtained bychanging both contiguous charged residues in a two amino-acid window. To systematically analyze the S. pombe Srp54

protein, we therefore mutated each pair of adjacent chargedresidues to alanine as described in Materials and methods.The locations of the 25 charged-to-alanine mutations areindicated above the amino-acid sequence of the fissionyeast Srp54 protein shown in Fig. 2. Because the two ala-nine codons preferentially used in fission yeast are GCTand GCC (Russell 1989), we incorporated these into themutagenic oligonucleotides to minimize effects on expres-sion levels of the mutant proteins.

In addition to charged-to-alanine scanning of the Srp54protein, we performed directed mutagenesis of two con-served motifs identified in our laboratory via detailed se-quence comparisons (Althoff et al. 1994a). First, near theC-terminus of the G domain is a highly conserved non-apeptide which, based upon its location and conservationin both Srp54p and SRα, was proposed to serve as a bind-ing site for a putative common guanine nucleotide-disso-ciation factor; this element was thus designated the PGB(Althoff et al. 1994a); two single substitutions were gen-erated in this sequence. Second, between the third andfourth amphipathic segments of the M domain is a con-served heptapeptide which, based on its resemblance toRNA-binding motifs found in HIV TAR and related pro-teins, was proposed to function in the recognition of SRPRNA and hence designated the PRBM (Althoff et al.1994a); six single point mutations and one double substi-tution were introduced into this element.

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Fig. 2 Amino-acid sequenceand secondary structure predic-tion for the S. pombe Srp54protein. Pairs of charged resi-dues that were changed to ala-nine are indicated by asterisks.The regions predicted to foldinto α-helices by the secondarystructure algorithm PHD areshown as hatched boxes; β-sheets are indicated by solidboxes. Also indicated on thefigure are the different structu-ral domains, as well as the posi-tions of the GTPase consensusmotifs (G-1, G-2, G-3 and G-4), the putative guanine nu-cleotide dissociation factorbinding site (PGB), and the pu-tative SRP RNA recognitionmotif (PRBM)

Complementation analysis and phenotypic characterization of the mutants

The phenotypes of the mutant alleles analyzed in thepresent work are shown in Tables 2 and 3. The viability ofhaploid cells containing each mutant allele was first as-sessed using a plasmid shuffle assay (Sikorski and Boeke1991). For these experiments, the transformation recipientwas a haploid that contains an srp54::LEU2 disruption inthe chromosome and the pSP54-U plasmid (a ura4 plas-mid carrying the wild-type srp54 gene); this strain requiresadenine for growth (Althoff et al. 1994b). The recipientwas transformed with various derivatives of the plasmidpSP54-A (same as pSP54-U but containing an ade6marker; Szankasi et al. 1988) carrying the mutant srp54 al-leles. After transformation, cells were plated on minimalmedium in the presence or absence of uracil + 5-FOA andincubated at the standard growth temperature (30°C). Noneof the mutant alleles interfered with growth in minimal me-dium at 30°C in the absence of any supplements. Since bothplasmids must be maintained to meet the nutritional needsof the cell, we conclude that the corresponding mutant pro-teins do not result in dominant lethality in combination

with wild-type Srp54p under these conditions (but see be-low). In the presence of uracil + 5-FOA, only the plasmidcontaining the mutant allele can be maintained. Mutant al-leles that failed to produce colonies at the standard growthtemperature under these conditions were considered lethal.Mutations that were compatible with growth at 30°C weretested for conditional phenotypes by replica-plating thehaploids at high and low temperature.

Out of 25 charged-to-alanine mutants analyzed, eightwere recessive lethal, three were unable to grow at 37°C,one was unable to grow at 18°C, and the remaining 13 dis-played growth indistinguishable from an isogenic wild-type strain (Table 2, lines 1–25). The two point mutationsgenerated in the PGB region, T275 N and E277Q, showeda lethal and a wild-type phenotype, respectively (Table 2,lines 26 and 27). Of the six single-point mutations ana-lyzed in the putative RNA-binding motif, three were le-thal, two were heat-sensitive, and one was cold-sensitive

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Fig. 3 Helical wheel representations of α-helices NH1, NH2, NH3and NH4 (A, B, C and D respectively) from the N domain of S. pombeSrp54p (Fig. 2). The evolutionary conservation at each position isindicated by both symbols and letters. Internal symbols designate thecharacter of the consensus amino acid for each position in the helixas follows: open circle, hydrophobic residue; hatched circle, hydro-phobic or neutral residue (non-hydrophilic); hatched triangle, neu-tral residue; hatched square, neutral or hydrophilic residue (non-hy-drophobic); filled square, hydrophilic residue. Positions lacking asymbol are variable. Specific amino acids conserved in all knownSrp54p sequences appear inside the corresponding symbol. The res-idue present at each position in the S. pombe protein is indicated out-side the wheel. Amino acids mutagenized in the present work areunderlined

Fig. 4 Helical wheel representations of α-helices McH1, McH2,McH3, McH5 and McH6 (A, B, C, D and E respectively) from theMc domain of S. pombe Srp54p (Fig. 2). Amino-acid character andconservation are indicated as in Fig. 3. McH4 is not shown due to itssmall size (five amino acids)

(Table 2, lines 28–29 and 31–34). An allele containing bothof the heat-sensitive PRBM mutations was unable to sup-port growth (Table 2, line 30). Finally, a deletion withinthe Mv domain was lethal (Table 2, line 35) even thoughthe missing segment is not conserved through evolution.Microscopic examination of cells containing mutant alleles(data not shown) indicated that all strains appeared normalexcept for the R194A/H195A mutant, which showed an

aberrant morphology at 18°C (enlarged, highly vacuolatedcells which often aggregated); this phenotype is similar tothat previously observed in cells containing single substi-tutions at position 194 (Althoff et al. 1994b).

The proportion of our charged-to-alanine mutants foundto be lethal, 8 out of 25, is similar to what was observed inprevious similar studies (Wertman et al. 1992; Diamondand Kirkegaard 1994; Hasset and Condit 1994; Reijo et al.1994). However, the number of conditional mutants, only4 out of 25 alleles, was lower than expected. The low fre-quency of heat- or cold-sensitive mutants may be due tothe existence of an extensive series of interactions betweenSrp54p and its ligands, as proposed in one model for therecognition of SRP RNA and signal peptides by this pro-tein (e.g., High and Dobberstein 1991; Zopf et al. 1993).An alternative possibility (which we favor) is that the con-tacts between Srp54p and other components of the SRP cy-cle involve small specific regions, but there are simplyfewer of these than in other proteins studied by charged-to-alanine mutagenesis. Actin in particular is known tointeract with a vast number of other polypeptides (see Wert-man et al. 1992 for a discussion).

The conditional mutants derived from charged-to-ala-nine mutagenesis were examined in greater detail as fol-lows. First, the relative growth rate of haploid cells har-boring each heat- or cold-sensitive mutant as its sole sourceof Srp54 protein was determined at 18, 30 and 37°C, withthe results shown at the top of Table 3. When present asthe sole source of the protein, the R194A,H195A allele wasunable to support growth at either high or low temperature,whereas the remaining mutants were heat-sensitive only.

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Table 2 Locations and haploid phenotypes of mutations introducedinto the Schizosaccharomyces pombe Srp54 protein. The table is di-vided into sections, with the first 25 lines devoted to charged- to-al-anine mutations, lines 26 and 27 to mutations in the putative guaninenucleotide exchange factor binding site (PGB), lines 28–34 to mu-tations in a putative RNA binding motif (PRBM) and line 35 to a de-letion within the Mv domain.

Line Mutation(s)a Locationb Detectionc Pheno-# typed

1 R8A, R9A N/H1 AhaII (–) Lethal2 E25A, E26A N/H2 BsmI (+) WT3 K58A, K59A N/H3 HaeIII (+) Lethal4 K72A, R73A N/H5 HinfI (–) WT5 D81A, E82A N/H5 DNA seq. Lethal6 K97A, K98A N/L6 DNA seq. WT7 R127A, R128A G/H1 DNA seq. WT8 K173A, E174A G/H3 StyI (–) Lethal9 D177A, K178A G/H3 DNA seq. Lethal

10 D182A, R183A G/L5 DNA seq. WT11 R194A, H195A G/L6 DNA seq. CS12 K237A, E238A G/L9 DraI (–) Lethal13 E277A, H278A G/L11 DNA seq. TS14 E283A, R284A G/L11 HaeIII (+) WT15 E305A, H306A Mc/H1 HaeIII (+) Lethal16 K314A, K315A Mc/H2 DNA seq. TS17 R329A, D330A Mc/H3 AvaII (–) WT18 R332A, D333A Mc/H3 DNA seq. WT19 D363A, E364A Mc/H5 BsmI (+) WT20 K371A, R372A Mc/H5 HaeIII (+) Lethal21 E414A, E415A Mc/H6 HaeIII (+) WT22 K428A, K429A Mc/H6 HaeIII (+) TS23 K433A, D434A Mv/L1 HaeIII (+) WT24 K449A, K450A Mv/L1 HaeIII (+) WT25 K460A, R461A Mv/L1 HaeIII (+) WT

26 T275N G/L11 KpnI (–) Lethal27 E277Q G/L11 DNA seq. WT

28 R398A Mc/B2 DNA seq. TS29 R401A Mc/B2 DNA seq. TS30 R398A/R401A Mc/B2 DNA seq. Lethal31 R404A Mc/B2 DNA seq. Lethal32 R404K Mc/B2 DNA seq. WT33 G408P Mc/L6 DNA seq. Lethal34 G408I Mc/L6 DNA seq. CS

35 ∆445–472 Mv/L+H1 DNA seq. Lethal

a Indicated are the position numbers of the amino acids (see Fig.2)affected by the various mutationsb The location of each mutation is indicated according to the affect-ed domain (N, G, Mc or Mv) and secondary structure element (H = α-helix; B = β-sheet; L = loop), designated as in Fig. 2c The method used for initial identification of each mutant is indi-cated as follows: (+) and (–) refer to creation or destruction, respec-tively, of a restriction site in the srp54 gene by the mutagenesis. Theseand the remaining mutants were verified by sequence analysisd The effects of the mutations on growth are indicated as follows:WT (indistinguishable from wild-type growth), CS (cold-sensitive),TS (heat-sensitive) or lethal

Table 3 Detailed phenotypic characterization of heat- and cold-sen-sitive fission yeast Srp54p mutants. The top one-third of the tableshows the generation times of cells containing a mutant allele as theonly source of Srp54p relative to an isogenic wild-type haploid, whilethe remainder shows the results of plate assays to determine wheth-er the conditional alleles display dominance over the wild-type at ex-treme temperatures as described previously (Althoff et al. 1994 b;see text). The middle three lines show relative growth when the mu-tant allele is present at the same copy number as the wild-type allele,while the bottom three lines show results when the mutant allele ispresent in excess (see text). The data are the average of four differ-ent experiments with a standard deviation between 5 and 10 minutes

Temp. WT R194A, E277A, K314A, K428A, H195A H278A K315A K429A

18°C 1a >8 1.2 1.1 130°C 1a 1 1 1 137°C 1a >8 7.3 6.8 6.5

18°C ++ + ++ ++ ++30°C ++ ++ ++ ++ ++37°C ++ – + + +

18°C ++ – ++ ++ ++30°C ++ ++ ++ ++ ++37°C ++ – – – –

a Generation time of the srp54 haploid strain carrying a null allelein the chromosome and the wild-type allele on a multicopy plasmid(pSP54-A) growing in minimal medium supplemented with uracil(EMM2 + ura): 18°C, 22 h 40 min; 30°C, 6 h 12 min; 37°C, 5 h 55 min

Growth curves for all of the conditional mtants (data notshown) indicate that cell death ensues quite rapidly afterthe temperature shift, since the optical densities of the cul-tures plateau in less than one generation. This observationsuggests that the mutations are likely to affect the functionrather than the synthesis or stability of the fission yeastSrp54 protein. If the mutations altered only the amount ofprotein present, a protracted lag during which pre-existingprotein pools were depleted would be expected prior tomanifestation of the growth defect.

A second genetic assay used to characterize the new mu-tants involved testing for conditional dominance, since ourearlier work showed that certain GTPase consensus motifmutants interfere with growth at extreme temperatures instrains also carrying a wild-type allele (Althoff et al.1994b). As documented in the lower two-thirds of Table 3,the proteins encoded by the newly isolated conditional al-leles interfere with growth at one or both extreme temper-atures as compared to an isogenic strain containing onlywild-type Srp54p. Because the pSP54-A and pSP54-Uplasmids contain the same origin of replication, they areexpected to be present at a similar copy number. In this sit-uation, only the R194A,H194A mutant completely elimi-nates growth at 37°C, although a marked effect is observedfor the remaining alleles tested (Table 3). To analyze con-ditional dominance when these mutant genes were presentin excess over the wild-type allele, we introduced them intoSpAS1, a diploid strain containing one wild-type chromo-somal copy of the srp54 gene and an srp54::LEU2 genedisruption on the other chromosome (Althoff et al. 1994b).In such strains, growth is completely blocked at 37°C byall of the conditional alleles (Table 3). Thus, the severityof conditional dominance in diploid strains mirrors the hap-loid phenotypes conferred by the mutations when presentin the sole copy of srp54 (cf. Table 2, lines 11, 13, 16 and22). Although further work will be required to interpretthese results in detail, the incomplete dominance of the mu-tants over wild-type srp54 implies that complexes contain-ing the altered proteins do not stoichiometrically block theaction of signal recognition particles containing the nativeprotein in vivo. Some of the semi-dominant phenotypescould be due to delayed release of complexes containingmutant proteins from the SRP receptor in the ER mem-brane, thereby blocking access by wild-type complexes; asimilar scenario was previously suggested for mutants car-rying substitutions in the GTPase motifs (Althoff et al.1994b). For other alleles described here, the partial domi-nance may be due to titration of another component of theSRP cycle. In any case, the ability of the mutant proteinsto interfere with the function of wild-type Srp54p providesfurther evidence that they are not only produced but arestable at the restrictive temperature.

The mutations that confer lethal or conditional pheno-types are distributed throughout the protein. Those thatmap to the N+G domain are shown on the three-dimen-sional structure depicted in Fig. 5; these include: (1)R8A/R9A, K58A/K59A, D81A/E82A (Table 2, lines 1, 3and 5), which map to three distinct α-helices within the Ndomain; (2) K173A/E174A and D177A/K178A (Table 2,

lines 8 and 9), which lie within an α-helix unique to theG domains of the Srp54p subfamily of GTPases (Frey-mann et al. 1997); (3) R194A/H195A (Table 2, line 11),which lies within the G-3 GTPase motif believed to playa direct role in catalysis (Pai et al. 1989); (4)K237A/E238A (Table 2, line 12), which maps to a shortα-helix within the G domain; and (5) E277A/H278A andT275N (Table 2, lines 13 and 27), which lie within a loopcontaining the PGB motif. One lethal and one conditionalmutation (E305A/H306A, K314A/K315A; Table 2, lines15 and 16) lie in the region for which crystallographic dataare unavailable due to lack of order in the peptide con-necting the G and M domains (Keenan et al. 1998). Theremainder of the M-domain mutations that affect growthare shown on the three-dimensional structure depicted in Fig. 6; these include: (1) K371A/R372A andK428A/K429A (Table 2, lines 20 and 22), which lie withinthe terminal amphipathic α-helices in the conserved re-gion of the M domain implicated in the recognition of signal peptides (Bernstein et al. 1989); and (2) R398A,

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Fig. 5 Three-dimensional ribbon diagram of the N+G domainshowing the locations of lethal and conditional mutants. The imageshown was prepared using the coordinates supplied by Freymann etal. (1997). The amino- and carboxyl-termini of the fragment are in-dicated as [N] and [C], respectively. The amino acids correspondingto those substituted in our charged-to-alanine mutants are shown inblack

Fig. 6 Three-dimensional ribbon diagram of the M domain show-ing the locations of lethal and conditional mutants. The image shownwas prepared using the coordinates supplied by Keenan et al. (1998).The amino- and carboxyl-termini of the fragment are indicated as[N] and [C], respectively. The amino acids corresponding to thosesubstituted in our charged-to-alanine mutants are shown in white

R401A, R404A, G408P and G408I (Table 2, lines 28, 29and 31–34), which lie within an α-helix/turn structure inthe Mc domain implicated in SRP RNA-binding (Althoffet al. 1994a; Keenan et al. 1998).

Discussion

The possible molecular consequences underlying the phe-notypes observed for our Srp54p mutants are discussed indetail in the following sections, making reference when-ever possible to the recently determined X-ray crystalstructures for either the N+G or the M domain (Freymannet al. 1997; Keenan et al. 1998).

N-domain mutants

Sequence analysis of Srp54p and SRα revealed that theseproteins contain similar N and G domains, which was pro-posed to reflect homotypic interactions (Bernstein et al.1989). Recent crystallographic data indicate that theshared region adopts a very similar structure in both pro-teins, with the N domain tightly packed against the G do-main (Freymann et al. 1997; Montoya et al. 1997). A com-pact structural relationship between the N and G domainswas previously proposed based on partial proteolysis ofmammalian Srp54p, which revealed that only the boun-dary between the G and M domains is sensitive to diges-tion (Römisch et al. 1990; Zopf et al. 1990). The N do-main of S. pombe Srp54p is 101 amino-acids long and ispredicted to contain four α-helices designated NH1, NH2,NH3 and NH4, separated by three loops designated NL1,NL2 and NL3 (see Fig. 2); the helical boundaries in thecomputer-generated structure closely correspond to thosein the recently determined crystallographic model for eu-bacterial Ffh (Freymann et al. 1997). A fourth loop (NL4)which connects the N and G domains is quite hydrophilic,and the corresponding segment in the crystal structurestretches across the surface of the G domain (Freymannet al. 1997).

Because the N domain is highly conserved in structurebut quite variable in sequence, it is notable that two of thethree lethal mutants obtained in this region, K58A/K59Aand D81A/E82A (Table 2, lines 3 and 5; Fig. 5), affect atleast one amino acid that is moderately conserved through-out evolution (see Fig. 3 above; Fig. 5 of Althoff et al.1994a). Two of the lethal mutants (R8A/R9A andD81A/E82A) lie near the interface with the G domain(Fig. 5), consistent with the idea that the α-helical bundleformed by the N domain might serve to sense or controlnucleotide occupancy (Freymann et al. 1997; Montoya etal. 1997). Such a function would be analogous to that ofthe “extra” helical domain present in the Gα subunits ofhetero-trimeric GTPases, which has been implicated inswitching on (via GTP binding) and off (via GTP hydrol-ysis) members of this subfamily (Noel et al. 1993; Lamb-right et al. 1994). Notably, Gα proteins display a kcat for

GTP hydrolysis 100-fold higher than other GTPases suchas p21ras or EF-Tu (reviewed in Bourne et al. 1991). Thekcat for both Srp54p and SRα is nearly 10-fold higher thanin p21ras (Connolly and Gilmore 1993; Samuelsson andOlsson 1993; Miller et al. 1994), suggesting that the pres-ence of the N domain, or another feature unique to theseproteins (see below), may facilitate catalysis.

G-domain mutants

As anticipated based on the high degree of structural con-servation between other members of the GTPase superfam-ily despite limited sequence identity (reviewed by Bourneet al. 1991; Bourne 1995), the G domain of Srp54p adoptsan overall folding pattern similar to that of distantly relatedGTPases including EF-G and α-transducin (Noel et al.1993; Aevarsson et al. 1994; Czworkowski et al. 1994;Lambright et al. 1994). In S. pombe Srp54p, this domainis 194 residues long (Fig. 2) and is predicted by the com-puter algorithm to contain eight α-helices (GH1–8), 6 β-sheets (GB1–6) and 13 loops (GL 1–13). A comparisonwith the recently determined structure for eubacterial Ffh(Freymann et al. 1997) indicates two minor differences:GH7 and GH8 are actually part of a longer continuous he-lix, and a short segment of β-sheet between helices GH2and GH3 was not picked up by the computer. The structu-ral similarity of Srp54p and previously characterized mem-bers of the GTPase superfamily is compatible with our ear-lier observation that the G-1 through G-4 consensus mo-tifs in the S. pombe protein show similar mutational sen-sitivity to the corresponding regions of p21ras and EF-Tu(Althoff et al. 1994b). The double mutation R194A/H195Adescribed here (Table 2, line 11; Fig. 5), which modifiesone of the residues in the G-3 motif implicated in GTP hy-drolysis, displays a phenotype similar to that conferred bysingle mutations at position 194 (Althoff et al. 1994b),namely cold sensitivity and conditional dominance at ex-treme temperatures.

In other GTPases, the regions outside the four consen-sus motifs, which are unique to different subfamilies, areknown to contain the binding sites for specific modulatoryfactors such as guanine nucleotide-dissociation stimula-tors (GDSs) and GTPase activating proteins (GAPs; re-viewed in Boguski and McCormick 1993). One notable ex-ception to the structural similarity between Srp54p homo-logs and other members of the GTPase superfamily is thepresence of an insertion of approximately 30 amino acidsbetween the G-2 and G-3 motifs (Freymann et al. 1997 andreferences therein); SRα contains an even larger insertionat this site relative to other GTPases (see the alignment inFig. 5 of Althoff et al. 1994a). In the three-dimensionalmodels, the “extra” residues emerge from the G domain atthe surface opposite the interface with the N domain andare largely α-helical (Fig. 5; Freymann et al. 1997; Mon-toya et al. 1997). Modeling based on a low-resolution struc-ture of the intact protein suggested that this segment liesin proximity to the M domain (Czarnota et al. 1994) andmay thus increase the protein’s affinity for GTP in the pres-

96

ence of a ribosome-nascent chain complex (Millman andAndrews 1997). Alternatively, since the position of the an-cillary loop corresponds to the “effector” region of p21ras-related GTPases, it may be involved in GAP response(Freymann et al. 1997; Montoya et al. 1997). BecauseSrp54p and SRα mutually stimulate each others’ GTPaseactivity (Powers and Walter 1995; Rapiejko and Gilmore1997), the ancillary loops may contain the points of con-tact between these two proteins (Freymann et al. 1997).Regardless of its precise function, the fact that we haveisolated two lethal mutations (K173A/E174A andD177A/K178A; Table 2, lines 8 and 9; Fig. 5) in this seg-ment strongly implies that it plays an important role in thefunction of Srp54p despite showing only limited evolu-tionary conservation.

The region of p21ras containing amino acids corre-sponding to one of our lethal mutations (K237A/E238ATable 2, line 12; Fig. 5) has been implicated in nucleo-tide exchange (Willumsen et al. 1991). Thus, the inabil-ity of the mutant Srp54 protein to support growth may bedue to a disruption in this process. Finally, as noted pre-viously (Althoff et al. 1994a), the binding site for theknown GDS of EF-Tu (EF-Ts) lies downstream from theG-4 motif, in a position analogous to the extended regionof similarity (PGB) that we previously noted betweenSrp54 proteins (as well as SRα homologs). In the presentstudy, we have obtained a heat-sensitive and a lethal mu-tant (E277A/H278A and T275N, respectively; Table 2,lines 13 and 27; Fig. 5) in the vicinity of this motif, pro-viding clear evidence that the region is functionally im-portant. Based on sequence similarity between Srp68pand guanine nucleotide-exchange factors, we proposedthat this protein might participate in GDP release fromboth Srp54p and SRα (Althoff et al. 1994a). A more re-cent study suggests that the ribosome serves as a nucle-otide-exchange factor for Srp54p (Bacher et al. 1996).Suppressor or synergistic lethal analysis with our condi-tional mutant should help to distinguish between thesemodels.

M-domain mutants

Mc subdomain

As noted above, the M domain of Srp54p can be dividedinto two subdomains, of which only the first is present inall organisms. The Mc domain of fission yeast Srp54p ispredicted by the computer algorithm to contain six α-hel-ices (McH1, McH2, McH3, McH4, McH5, and McH6),one β-sheet (McB1) and seven loops (McL1, McL2,McL3, McL4, McL5, McL6 and McL7; see Fig. 2). Thisstructure correlates quite well with the newly determinedthree-dimensional model for the Thermus aquaticus pro-tein (Keenan et al. 1998) with one minor and one more-important difference (compare Fig. 2 with Fig. 5 of Kee-nan et al. 1997). At the amino terminus of the Mc domainare three helices which have been strongly implicated insignal sequence recognition by the structural data as well

as previous computer-based modeling (Bernstein et al.1989); the helices predicted by PHD overlap, but are notprecisely coincident, with those in the crystallographicmodel. Because our mutagenesis targeted charged aminoacids, the hydrophobic residues proposed to form the sig-nal-sequence binding groove (Keenan et al. 1998) re-mained untouched. Notably, however, despite the empha-sis on hydrophobicity in models for the function of thisregion, we find that it contains four pairs of polar aminoacids where alanine substitutions produce conditional orabsolute lethality (Table 2, lines 15, 16, 20 and 22). Twoof the mutagenized amino-acid pairs (E305/H306 andK314/K315) lie outside the region for which structuraldata are available (the polypeptide chain linking the G andM domains is disordered; Keenan et al. 1998). The thirdpair (K371/R372) is located near the middle of α-helixM2 and the fourth (K428/K429) lies near the C-terminusof α-helix M4 (Fig. 6). Although we do not yet know theprecise role played by these amino acids, it is notable thatthe presence of at least one basic residue at the site of themutations has been quite conserved throughout evolution(see Fig. 4 above; Fig. 7, Althoff et al. 1994a; Fig. 5, Kee-nan et al. 1998).

A feature revealed by the crystallographic data, but un-anticipated from previous molecular modeling (Fig. 2;Bernstein et al. 1989) is the presence of a helix-turn-helix structure in the region implicated in SRP RNA rec-ognition (Althoff et al. 1994a; Kurita et al. 1996; Keenanet al. 1998). This segment lies between two of the helicesproposed to contact signal sequences and contains a dis-crete, highly conserved element (consensus ROX-ROA(R/K)GSG; X=any amino acid; O = hydrophobic)similar to the RNA recognition motif found in the HIVTAR RNA-binding protein and the DEAD-box family ofRNA helicases (Althoff et al. 1994a). We have isolatedboth lethal and heat-sensitive mutants that map to the pu-tative RNA-binding motif, which spans part of helix M3and the following loop (Fig. 6). A positive charge at thethird basic residue of the S. pombe Srp54p element is absolutely critical based on the lethality of the R404A al-lele; however, this need not be supplied by an arginine,since R404K is viable (Table 2, lines 31 and 32; Fig. 6).The first and second arginines appear to be less crucial,since alanine substitutions at these positions produce con-ditional rather than absolute lethality (R398A and R401A;Table 2, lines 28 and 29). Notably, however, synthetic le-thality is observed when both arginines are mutated simul-taneously, consistent with their participation in the sameinteraction. In the DEAD-box proteins, the related motifalso consists of three arginines alternating with other res-idues (HRIGRXXR), and mutating any one of the invari-ant arginine residues is lethal (Pause et al. 1993). In addi-tion, our data further indicate that a conserved glycine justdownstream from the basic region is critical for the func-tion of Srp54p, since a proline substitution is lethal andreplacement with isoleucine produces cold-sensitivity (Table 2, lines 33 and 34; Fig. 6). Additional evidence forthe importance of the putative RNA-binding motif comesfrom a study by Kurita et al. (1996), who performed a more

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limited mutagenesis of the analogous region of Bacillussubtilis Srp54p (ffh) and obtained results similar to ours.

Mv subdomain

The C-terminal region, which we have designated the Mvdomain, ranges from 19 residues in eubacteria (B. subtilis)to 104 residues in a dimorphic yeast (Yarrowia lipolytica).This domain is the major source of variability when Srp54psequences are compared between evolutionary groups. Forexample, use of this region of the S. pombe protein as aprobe in FASTA (homology) searches identified only eu-karyotic Srp54p homologs (data not shown). In all cases,the Mv subdomain is basic (pI 9–10) with an especiallyhigh concentration of lysine residues; it also contains thehighest frequency of methionine (10–29%) and glycine(12–24%) within the M domain. As shown in Fig. 7A, thesecondary structure program predicts a terminal α-he-

lix/loop/α-helix in all eukaryotic Srp54p homologs. Theinternal loop sequence seems to be somewhat conservedbetween animals and plants, while the fungal homologshave diverged dramatically, with a high content of glycineand methionine as the only common denominator(Fig. 7A). Deletions in this region affect binding of the pro-tein to both SRP RNA and signal sequences in vitro (Lütckeet al. 1992), which may account for the lethality associatedwith the ∆445–472 allele of fission yeast Srp54p in vivo(Table 2, line 35). The Mv deletion ends at two prolineswhich presumably terminate an α-helix, and thus its lethal-ity is unlikely to reflect a gross structural perturbation. Incontrast to the deletion, none of the three point mutationsin this subdomain noticably affect growth (Table 2, lines23–25). Interestingly, long Mv regions are present in thecyanobacterial, Mycobacterium, and Arabidopsis thalianachloroplast Srp54 proteins, but these do not seem to be re-lated to each other nor to the corresponding region of anyeukaryotic homolog (data not shown). Thus, the picturewhich emerges from these observations is reminiscent ofthe evolutionary relationships among various SRP RNAs,in which the most dramatic divergences are observedamong eubacteria and their organellar relatives (Althoff et al. 1994a).

The secondary structure of SRP RNA from eukaryotesand archaea can be divided into four different domains,each of which interacts with a specific protein of the SRPcomplex: Srp9/14p binds to Domain I, Srp68/72p to Do-main II, Srp19p principally to Domain III and Srp54p toDomain IV (Siegel and Walter 1988a, c). Eubacterial SRPRNAs lack one or more domains; for example, while SRPRNA from B. subtilis contains Domains I, II and IV, thosefrom E. coli and M. mycoides have only Domain IV. It hasbeen proposed that SRP RNA has undergone several re-ductions in size and complexity such that certain eubacte-rial homologs may be capable of directly binding only the

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Fig. 7 A Comparison of Mv domains from known eukaryoticSrp54p homologs. Shown are the carboxy terminal amino acid se-quences and predicted secondary structures (depicted as in Fig. 2)for the homologs indicated. Sp Schizosaccharomyces pombe; An As-pergillus niger; Sc Saccharomyces cerevisiae; Yl Yarrowia lypolyti-ca. For animals and plants, the consensus sequences are annotatedas follows: absolutely conserved residues are underlined, residuesconserved in 90% of the sequences are shown in normal type, hy-drophobic residues are designated “O” and hydrophilic residues “J”.For references, see the legend to Fig. 1. B structural alignments ofSrp19p homologs from diverse groups. Mj Methanococcus jannas-chii (Bult et al. 1996) Hs Homo sapiens (Lingelbäch et al. 1988), DmDrosophila melanogaster (Lai and Langley 1995); Os Oryza sativa(CW Zwieb and SD Black, unpublished data cited in Chittenden et al. 1994); Sc Saccharomyces cerevisiae (Hann et al. 1992) Yl Yar-rowia lypolytica (Sanchez et al. 1997) Secondary structure elementsare depicted as in Fig. 2. The experimentally determined SRP RNA-binding region (Chittenden et al. 1994) is boxed

Srp54 protein (Althoff et al. 1994a and references therein).Upon publication of the complete genomic sequences fortwo eubacteria, Haemophilus influenzae and Mycoplasmagenitalium (Fleischmann et al. 1995; Fraser et al. 1995),we searched for open reading frames related to the remain-ing SRP polypeptides. Notably, no significant homologieswere uncovered (E. M.-F. and J. A. W., unpublished). Al-though the lack of a signal could be due to poor primarysequence conservation, we favor the idea that Srp19p, atleast, is truly absent from eubacteria, since all homologsof SRP RNA from this phylogenetic group lack DomainIII. Interestingly, a protein with quite extensive similarityto mammalian Srp19p was found in the the first archaealgenome sequenced (Methanococcus jannaschii; Bult et al.1996), consistent with the presence of Domain III in eachof the many sequenced SRP RNAs from this phylogeneticgroup (reviewed in Althoff et al. 1994a).

The dramatic differences among eukaryotic groups inthe Mv domain suggest that this region of Srp54p mayinteract with an SRP component whose sequence alsoshows extensive divergence. In searching for such an en-tity, we noticed that the pattern of conservation within theMv domain parallels that of the Srp19 protein. To-date, ho-mologs of Srp19p have been found in a variety of eukar-yotes, including animals (Lingelbäch et al. 1988), plants(C. W. Zwieb and S. D. Black, unpublished data cited inChittenden et al. 1994), and yeasts (Stirling and Hewitt1992; Sanchez et al. 1997). Figure 7B shows predicted sec-ondary structures for the available Srp19p homologs, de-rived by the method described above for Srp54p. The sim-ilarity between animal and plant Srp19p revealed by thesequence comparison correlates with biochemical data in-dicating that complexes can be formed between Srp54p andSRP RNA from L. esculentum in the presence of Srp19pfrom C. cannis (Krolkiewicz et al. 1994). Based on thesedata, we propose that the Mv domain is the segment ofSrp54p that interacts with the Srp19p/SRP RNA complexduring formation of the particle in vivo.

Conclusion

In the present work, we have analyzed the consequencesin vivo of a large number of mutations distributed through-out the Srp54 protein from the fission yeast S. pombe. Inaddition to confirming the importance of a number of ev-olutionarily conserved regions outside the GTPase consen-sus motifs, the mutagenesis data, combined with structu-ral analyses (Freymann et al. 1997; Keenan et al. 1998),suggest several potentially fruitful avenues for future dis-section of the role of Srp54p in signal recognition particlefunction. The seven mutations identified here that produceconditional growth defects are likely to be particularly use-ful for deciphering the interactions of this polypeptide withits RNA and protein ligands.

Acknowledgements The authors thank Steve Althoff and Dave Se-linger for helpful discussions during the early stages of this project,and Wim van Heeckeren for critical comments on the manuscript.

This work was supported by NSF Grant #BCS 92–13895, awardedto J. A. W. and K. Dane Wittrup (University of Illinois at Urbana-Champaign). E. M.-F. was supported in part by a fellowship from theSpanish Ministry of Education.

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