Protein Localization

Peter TakizawaDepartment of Cell Biology

•Organelles

•Distribution of organelles by microtubules and motor proteins

•Signal sequences

•Examples of protein import

1. Role of signal sequences in targeting proteins.2. Describe how proteins get into ER, mitochondria, nucleus and peroxisomes.3. Why.

1. Illustrates important biochemical mechanisms that are common to many biological processes.2. Classical work in cell biology.3. Mutations in some pathways lead to disease.

Cells contain a variety of organelles that perform specific and unique functions.

1. Each organelle contains unique set of proteins and other components.2. Organelles perform special and unique set of tasks.3. Organelles increase efficiency by concentrating components of a common pathway in small environment.4. Protect cell from harmful components.5. Some organelles surrounded by membrane to prevent loss of material.6. Should be familiar with function of each organelle.7. Still image gives impression that organelles randomly distributed.

Cells use microtubules to position organelles within their cytoplasm.

1. Cells organize and localize organelles.1.1. Position where needed.1.2. Mitochondria at sites of greatest energy consumption.

2. Tether.3. Transport through cytoplasm.4. Microtubules play a critical role in organizing cytoplasm of cells.5. Golgi localize to central region, near nucleus.6. Disrupting microtubules leads to fragmentation of Golgi and distribution throughout cytoplasm.

Microtubules extend throughout the cytoplasm.

1. Microtubules effective for organizing cell because they are long filaments that extend throughout cytoplasm.2. Large enough to span most cells.3. Allows cells to position organelles in specific regions.

Microtubules are a hollow tube of tubulin dimers.

_-tubulin

`-tubulin +

-1. Heterodimer of alpha and beta tubulin.2. Both bind GTP.3. Assemble into filaments end on end to form protofilaments.

3.1. 13 protofilaments make a single microtubule.3.2. Hollow lumen or center.3.3. Extensive lateral contacts between subunits gives MT greater strength and allows it to grow longer than actin filaments.3.4. Similar to pvc pipe.

4. Note that microtubules are polarized.4.1. One end has alpha tubulin and the other end has beta tubulin.4.2. Grow from their plus ends as dimers associate with plus end.

Microtubules grow from their plus ends.

Video shows growth of microtubules in vitro. Microtubules grow from their plus ends as more dimers are added to the filament. Occasionally, microtubule will stop growing and then shrink as dimers fall off of plus end. Inside cells, the plus ends of microtubules are capped or stabilized so they don’t shrink. The repeated growing and shrinking allow microtubules to explore different areas of the cell and cells can reposition or reorient microtubules. Note that the minus ends of the microtubules emanate from a common center. This region is called the microtubule organizing center and stabilizes the minus ends of microtubules.

Proteins link organelles to microtubule network.

1. Cells stabilize minus ends through MTOC.1.1. Contains large number of proteins -> centrioles.1.2. Ring of gamma-tubulin that binds and stabilizes minus ends.

2.Location of MTOC determines orientation of MTs.2.1. In center, MTs radiate outward -> plus ends toward plasma membrane.2.2. Organelles contain proteins that bind MTs -> serve as tethers.2.3. Orientation allows cells to control distribution of organelles.2.4.

Kinesins and dynein mediate bidirectional transport of organelles along microtubules.

Dynein

1. Motor proteins move along MTs carrying organelles.2. Motor proteins move in either plus or minus direction. 3. Kinesins large family of motor proteins most of which move toward plus end.2. Dynein move toward minus end.3. Motors attach to organelles via receptors in organelle membrane.4. By controlling binding and activity of motors, cell can dictate movement along MTs and position.

Cells regulate activity of motor proteins to control distribution of organelles.

Organelles will often have both types of motors on their surface, allowing cells to adjust their position. For example, these melanocytes contain a pigmented organelle called a melanosome. In some organisms, melanocytes position melanosomes in response to the amount of light. In the presence of light. kinesin on the surface of melanosomes moves them to the periphery of cells. In the dark, dynein returns the melanosomes to the center of the cell.

Targeting proteins to specific organelles

Proteins synthesized in cytoplasm must get to correct organelle.

1. Each organelle contains unique set of proteins and other components.2. Organelles perform special and unique set of tasks.3. How do cells get certain proteins to the different organelles?

3.1. What targets proteins to these organelles.3.2. How do they cross the membranes surrounding the organelles?

Proteins are imported into organelles from cytosol or transported between organelles.

Cytosol

Golgi

Endoplasmic Reticulum

Late Endosome

Lysosome

Plasma Membrane

Secretory Vesicles

PeroxisomesMitochondriaNucleus

The synthesis of all proteins starts in the cytosol, but those proteins with signal sequences are delivered to their appropriate organelle. Many organelles receive proteins through the secretory pathway. Proteins are initially translated and inserted into the ER. Proteins are transported to the Golgi and then delivered to the different organelles. Some organelles receive proteins directly from the cytosol. The nucleus is a unique organelle as it allows bidirectional movement of proteins.

Proteins contain signal sequences that determine their final destination.

Function of Signal Sequence Example of Signal Sequence

Import into nucleus -Pro-Pro-Lys-Lys-Lys-Lys-Lys-Val-

Export from nucleus -Leu-Ala-Leu-Lys-Leu-Ala-Gly-Leu-Asp-Ile

Import into mitochondria+H3N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Glu-Arg-Asn-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu

Import into ER+H3N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln

Import into peroxisomes -Ser-Lys-Leu-COO-

1. Proteins contain short sequences that guide them to correct organelle or intracellular destination -> signal sequence.2. Signal sequence interacts with machinery on organelle that imports protein into organelle.

2.1. How does machinery get to organelle?3. Signal sequences often found at N-terminus but can be located internally or at C-terminus.4. Signal sequences often lack exact sequence conservation.

4.1. Can’t identify based on sequence.4.2. Properties of amino acids that function as signal -> hydrophobic, charged.

5. Glycosylation can also act as targeting signal.

Small changes in signal sequences have profound effects on location of proteins.

HumanNucleosideTransporter

PEXN

MouseNucleosideTransporter

PARS

Mitochondria

PlasmaMembrane

Slide illustrates how subtle changes in signal sequences can affect the localization of proteins. In humans, a certain nucleoside transporter contains a 4 amino acid motif that targets it to mitochondria. In mice, the same protein contains two changes in this motif and this targets the protein to the plasma membrane. If you experimentally change the motif in the human protein to the mouse version, the human transporter localizes to the plasma membrane. Is this medically relevant?

Fialuridine is a uridine analog that is a potent inhibitor of hepatitis B.

Human Mitochondrion

Mouse Mitochondrion

NucleosideTransporter

In the early 1990’s there was a clinical trial of a drug designed to treat hepatitis B in 15 patients. Hepatitis B is a virus that affects the liver, causing inflammation. One common treatment for this type of virus is nucleoside analogs. Nucleoside analogs are similar to the nucleosides that are normally used to synthesize DNA, so that they can incorporated into DNA, but they are altered so that they stop synthesis or affect the stability of the DNA. Fialuridine is a nucleoside analog that was found to be effective in reducing hepatitis B in animal models. It was subsequently used in human trials and initially it appeared to reduce the amount of virus. But then something went terribly wrong. Long after the clinical trials were concluded, researchers discovered something unique about the way fialuridine affected human cells. Because humans have a certain nucleoside transporter that localizes to mitochondria, fialuridine can enter mitochondria. Why is this bad? Fialuridine poisoned mitochondria, reducing their ability to generate ATP. Why wasn’t this discovered before the clinical trials? Fialuridine was tested on animal models for safety, but those animals were mice and rats whose nucleoside transporter lacks the mitochondrial signal sequence. Consequently, their mitochondria did not take up fialuridine and were not affected by the drug.

What happened in the clinical trials? By reducing the activity of mitochondria, fialuridine force energy-intensive cells to start using glycolysis to generate ATP. This lead to an increase in lactate in the blood and a condition called lactic acidosis. Because the liver processes lactate, the increase production of lactate lead to liver failure 7 patients,5 of whom died.

Protein import into the endoplasmic reticulum

ER signal sequences have a tripartite structure but little sequence similarity.

1. Most proteins that enter ER contains signal sequence at N-terminus.2. Signal sequence contains 3 domain.

2.1. Most important functionally is hydrophobic sequence.2.1.1. Will interact with hydrophobic tails of lipids.

2.2. On N-terminal side is series of charge residues of varying length.2.3. On C-terminal side is set of polar amino acids.

2.3.1. Sequence specifies cleavage by peptidase.2.3.2. Removes signal sequence from protein.

3. Note lack of homology.

Most proteins imported co-translationally into the ER.

1. Most proteins that enter ER do so co-translationally.1.1. Inserted as they are made.1.2. Must coordinate translation with insertion.1.3. Recruit ribosomes to ER membrane.1.4. Energy consumed during protein translation drives protein through membrane.1.5. Protein passes across membrane unfolded -> smaller.

2. Co-translation prevents dangerous proteins from acting in cytosol.2.1. Lysosomal proteins digest macromolecules.2.2. Complete translation before insertion and protein may fold and become active.

3. How do cells ensure that ER proteins are translated on ER and not in cytoplasm.3.1. Conformational change in regulating activity of proteins.

Signal recognition particle binds to ER signal sequences and pauses translation.

Ribosome

mRNA

Protein

SRP

1. Coordination of translation and translocation are driven by SRP.1.1. Complex of 6 proteins and one RNA.1.2. SRP recognizes and binds to signal sequence.

1.2.1. Contains flexible, hydrophobic pocket.1.2.2. Binds hydrophobic region in signal sequence.1.2.3. Flexibility allows it to accommodate different hydrophobic sequences.

1.3. SRP wraps around ribosome and pauses translation elongation.

SRP receptor on ER binds SRP to recruit partially translated protein to ER membrane.

Cytosol

Lumen SRP receptor

Translocator

1. SRP bound to ribosome and signal sequence interacts with receptor in ER membrane -> SRP receptor.2. SRP receptor recruits nascent protein to ER membrane.3. How does receptor differentiate free SRP from SRP bound to signal sequence and ribosome?

3.1. SRP undergoes conformational change when bound to ribosome.3.2. Receptor likely recognizes conformation bound to ribosome.

Proteins threaded through translocator to cross the ER membrane.

Cytosol

Lumen

1. SRP receptor causes SRP to release from ribosome and signal sequence. SRP receptor induces conformational change to weaken interaction.

2. Proteins cannot cross membrane due to size and charge.3. Require protein channel -> translocator.

3.1. Transmembrane protein that forms aqueous pore.3.2. Crystal structure shows several transmembrane domains that surround pore.3.3. In absence of translation, pore contains helix that acts as plug, preventing small molecules from diffusing through pore.

4. SRP receptor brings ribosome to translocator.5. Translocator contains several ribosome binding sites.6. Translation continues through pore.7. Signal sequence binds to site on pore to open -> start transfer sequence.

Chaperones in ER help proteins fold after translocation.

1. Proteins inserted into ER as in unfolded state.2. Proteins must fold into correct conformation inside lumen.3. At low concentrations, most proteins fold successfully on their own.4. At high concentration, like in lumen where lots of proteins trying to fold, folding is less robust.5. Proteins contain hydrophobic domains that cluster in center of protein6. At low concentrations, these domains interact with each other.7. At high concentrations, hydrophobic domains from neighboring proteins interact.8. Chaperones such as BiP bind exposed hydrophobic domains in unfolded proteins and prevent aggregation with other proteins.

Integral membrane proteins contain stop transfer sequence.

N-terminus

C-terminus

Cytosol

Lumen

1. Many proteins are integral membrane proteins.2. These proteins contain another stretch of hydrophobic residues that function as stop transfer sequence.3. Stop transfer sequence will function as transmembrane domain.4. With this mechanism, N-terminus resides in lumen and C-terminus resides in cytosol.5. If transported to plasma membrane, N-terminus outside cell.

5.1. Receptors.6. Can you have opposite orientation?

Start and stop transfer sequences determine membrane topology of proteins.

Cytosol

Lumen

1. Many proteins span the membrane more than once.2. Proteins contain series of start and stop transfer sequences that determine membrane topology.3. For example, internal start transfer starts insertion with N-terminus facing outside.4. Stop transfer sequence ceases translocation.5. Translocator releases protein into membrane.6. Both N and C-termini face cytosol.7.

Sequential start and stop transfer sequences lead to multispan membrane proteins.

1. Proteins can span membrane several times.2. Series of start and stop transfer generate proteins that crosses membrane several times.3. Start and stop transfer sequences location dependent. Can switch function if positioned changed.

Protein import into peroxisomes

Peroxisomes detoxify chemicals, metabolize fatty acids and synthesize key lipid.

1. Several vital functions.1.1. Metabolize many harmful chemical.

1.1.1. Phenols, formaldehyde, ethanol1.1.2. Enzyme catalase oxidizes these substances.1.1.3. Prominent in liver.

1.2. Metabolize fatty acids.1.2.1. Oxidize carbon chains 2 at a time to form acetyl CoA -> important for metabolism.

1.3. Catalyze first step in formation of plasmalogens.1.3.1. Lipids specifically found in myelin that wrap axons of neurons.1.3.2. Increase conduction velocity of neurons.

Proteins inserted post-translationally into peroxisomes.

© 2002 Nature Publishing Group384 | MAY 2002 | VOLUME 3 www.nature.com/reviews/molcellbio

P E R S P E C T I V E S

nsPME–PEX5 complexes shortly after theirsynthesis but before their import. We referto these pre-import complexes of nsPMEsas ‘preimplexes’.

We see preimplex formation as a stochasticprocess that is controlled primarily by the con-centrations of the relevant proteins in thecytoplasm and their affinities for one another(FIG. 2). In such a system, there are many waysfor nsPMEs to interact with preimplexes. Forexample, peroxisomal enzymes that oligomer-ize rapidly and/or have a low affinity for PEX5are more likely to oligomerize before theyenter preimplexes, whereas proteins thatoligomerize more slowly and/or have a rela-tively high affinity for PEX5 might enter thepreimplex as monomers. Even monomericproteins should enter preimplexes, providedthey contain a PTS1. An intrinsic part of thepreimplex hypothesis is that some nsPMEsmust oligomerize before, or during, preim-plex formation, otherwise the nsPME–PEX5interactions would not be mutually multiva-lent. However, various factors (enzyme con-centration, degree of enzyme oligomerizationand affinity of the PTS1 of each enzyme forPEX5) make it difficult to predict exactly whatproportion of peroxisomal enzymes has tooligomerize before, or during, preimplex for-mation in order for it to proceed.

Another prediction of the preimplexhypothesis is that other PEX5-binding pro-teins might have a significant effect on preim-plex dynamics. For example, PEX14 is partic-ularly suited to having such a role. PEX14 isthe primary PEX5-docking site in the peroxi-some membrane, and it is a dimeric proteinwith at least one high-affinity PEX5-bindingsite per monomer29,34. Each PEX5 monomerhas several PEX14-binding sites, and soPEX5–PEX14 interactions are also mutuallymultivalent. In addition to helping tetherpreimplexes to the peroxisome surface,PEX5–PEX14 interactions might actually pro-mote preimplex expansion by linking other-wise distinct nsPME–PEX5 complexes atPEX14 dimers (FIG. 2).

Although the biochemical properties ofperoxisomal enzymes, PEX5 and PEX14 indi-cate that preimplex assembly is probable, whatpossible benefit is derived from assemblinglarge nsPME–PEX5 complexes on the peroxi-some surface before nsPME translocation?One possibility is that the multivalent natureof nsPME–PEX5–PEX14 interactions allowsthe rapid and specific delivery of nsPMEs tothe peroxisome using only diffusion and thebinding energy of these protein–protein inter-actions. Such a mechanism might explain whyPEX5-docking factors are the only proteinsthat have been implicated in delivering PEX5

and X-ray-crystallography structure datahave shown that PEX5 binds one PTS1 perPEX5 monomer, and that PEX5 behaves asan oligomer — most probably a tetramer —with several PTS1-binding sites10,29. On itsown, the fact that PEX5 is multivalent withrespect to its PTS1 ligands is not remark-able. However, the fact that PEX5 can bindseveral nsPMEs becomes more intriguingwhen we consider that peroxisomes canimport oligomeric peroxisomal enzymes2–4,and most, if not all, peroxisomal enzymesare oligomers30–33. So, the high-affinity inter-actions between PEX5 and nsPMEs (Kd =~100 nM)10 are likely to be mutually multi-valent. On the basis of these considerations,we propose that nsPMEs form large

nsPME import, but less is known about theirpoint of action11.

This general model of peroxisomal-matrix-enzyme import has been useful forassigning general functions to several of theknown peroxins, but suffers from a lack ofmechanistic detail. For example, peroxisomal-matrix-enzyme import requires ATP27,28, andthis model makes no predictions about whereATP is consumed or how ATP hydrolysis pro-motes enzyme translocation. This model alsofails to explain how peroxisomes importfolded, oligomeric enzymes across a sealedmembrane2–4. In light of these limitations, wehave re-examined the existing data on peroxi-somal-matrix-protein import from a mecha-nistic perspective. The remainder of this arti-cle presents a more detailed description ofwhat has arisen from our reconsideration ofthese data.

The ‘preimplex’ hypothesisTo gain insight into the molecular mecha-nisms of matrix-protein import, we concen-trated our attention on those aspects of per-oxisomal-matrix-protein import for whichthe biochemical data are most reliable. Webegan with the recognition of PTS1-con-taining enzymes by PEX5, the step inimport that has been subjected to the mostrigorous biochemical analysis. Biochemical

Figure 1 | A general model of peroxisomal-matrix-enzyme import. The dynamic distribution of PEX5indicates that matrix-enzyme import involves: a | binding of enzymes (red circles) by the import receptors(shown here as PEX5 in green); b | transport of receptor–enzyme complexes to the peroxisome surface; c | docking of receptor–enzyme complexes through protein–protein interactions with peroxisomalmembrane proteins, such as PEX14 (purple) and, perhaps, PEX13 (grey); d | dissociation ofreceptor–enzyme complexes and enzyme translocation through a proteinaceous pore (perhaps containingPEX8, PEX10 and PEX12, all shown in blue); and e | receptor recycling, which might involve PEX4 andPEX22 (both shown in orange). PEX, peroxin.

PEX5

PEX14 PEX13PEX8

PEX12 PEX10

PEX4

PEX22

a Binding

b Transport

c Docking

d Translocation

e Receptor recycling

Cytosol

Peroxisome

“We see preimplexformation as a stochasticprocess that is controlledprimarily by theconcentrations of therelevant proteins in thecytoplasm and their affinitiesfor one another.”

1. Protein import is critical for proper functioning of peroxisomes.2. Similar mechanism to mitochondria.

2.1. Peroxisomal proteins contain signal sequence.2.2. Sequence binds receptor that targets protein to peroxisome.2.3. Protein transits across membrane through channel.

2.3.1. Passes through as folded protein.2.4. Proteins involved in protein import called Pex.

Mutations in Pex proteins lead to loss of peroxisomes.

41

Fig. 1A–H PEX1 expressionrestores peroxisome biogenesisin CG1 patient cell lines. Cul-tured cells were transfectedwith either control pcDNA3plasmid (left panels: A, C, E,G) or the pcDNA3-PEX1 plas-mid pBER81 (right panels: B,D, F, H) and analysed by indi-rect immunofluorescence toperoxisomal matrix proteins byusing an anti-SKL antibodyand a Texas-Red-labelled goatanti-rabbit secondary antibody.A, B Patient 4065, C, D pa-tient 2775, E, F patient 3488,G, H patient 1742. PEX1 celltransfection efficiency was11%–18%

Fig.2A, B PEX1 exon 13base insertion in CG1 patients.A Automated sequence chro-matogram of a region of exon13 amplified by PCR from ge-nomic DNA of patient 4756, aheterozygote for the exon 13insT mutation. Left Subclonedwild-type allele, right sub-cloned exon 13 insT allele.B PEX1 protein sequence en-coded by the wild-type (WT)and exon 13 insT (Ex13insT)alleles. Histidine 690 is num-bered. Tyrosine 700 of the mu-tant sequence is underlined toindicate the position of theframeshift from the exon 13insT allele. Asterisk Termina-tion codon

pex1 Normal

Cells that contain mutations in the Pex genes can’t import porteins into peroxisomes. Consequently, the protein content of peroxisomes can’t be maintained and the cell loses its peroxisomes.

Pex mutations cause Zelleweger Syndrome and demyelination of nerves.

1. Several diseases associated with defects in genes that encode peroxisomal proteins.2. Set of diseases that result from mutations in genes that encode proteins involved in import into peroxisomes.

2.1. These mutations result in an absence of peroxisomes at birth.2.2. Results in build up of toxic materials including copper and iron-> enlarged liver.2.3. Lack myelination around axons.

2.3.1. Loss of muscle tone, inability to move and suckle.2.3.2. Can’t synthesize lipid for myelin.

3. Poor prognosis.3.1. Death by 6 months.