Cell, Vol. 71, 97-105,


2. 1992, Copyright

0 1992 by Cell Press

The Translation Machinery and 70 kd Heat Shock Protein cooperate in Protein Synthegis FL John Neison,‘t Thomas Ziegelhoffer,’ Charles Nicoiet,** Margaret Werner-Washburne,*§ and Elizabeth A. Craig’t * Department of Biomolecular Chemistry t Program in Cellular and Molecular Biology University of Wisconsin-Madison Madison, Wisconsin 53706

Summary The function of the yeast SSB 70 kd heatshock proteins (hsplgs) was investigated by a variety of ap proaches. The SSB hsp70s (Ssbl12p) are associated with translating ribosomes. This association is disrupted by puromycin, suggesting that Ssbl/2p may bind directly to the nascent poiypeptide. Mutant ssbl ssb2 strains grow slowly, contain a low number of translating rlbosomes, and are hypersensitive to several lnhibltors of protein synthesfs. The slow growth phenotype of ssbf ssb2 mutants is suppressed by increased copy number of a gene encoding a novel translation elongation factor la (EF-la)-like protein. We suggest that cytosolic hsp70 aids in the passage of the nascent polypeptlde chain through the ribosome in a manner analogous to the role played by organella localized hsp70 In the transport of proteins across membranes. Introduction Molecular chaperones are a class of proteins that mediate protein-protein interactions. They function in the folding and oligomerization of newly synthesized polypeptides as well as in the assembly and dissassembly of multimeric structures. Hsp70 is a highly conserved, ubiquitous molecular chaperone important for cell growth even under optimal conditions. The eukatyotic cell utilizes several different species of hsp70 that are localized in the mitochondrion (Craig et al., 1989; Engman et al., 1989; Leustek et al., 1989; Mizzen et al., 1989), chloroplast (Marshall et al., 1990), endoplasmic reticulum (ER) (Rose et al., 1989; Normingtonet al., 1989) and thecytoplasm.Theprevalent hypothesis of hsp70 action holds that hsp70s bind to proteins via recognition of short amino acid sequences and, as a result, transiently impose local conformation and interaction constraints upon the target protein. The energy of ATP hydrolysis is required for the disruption of this interaction between hsp70 and the substrate protein. Investigation of the import of nuclear-encoded proteins into the mitochondria and ER indicates in both cases that the organelle-localized hsp70 binds to the protein as it iS $ Present address: Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin 53706. 5 Present address: Department of Biology, University of New Mexico, Alburquerque, New Mexico 87131.

being imported and plays a key role in its transport, folding, and assembly (Kang et al., 1990; Vogel et al., 1990; Scherer et al., 1990; Sanderset al., 1992; Boleet al., 1986; Gething et al., 1986). That the transport of proteins across two very different membranes utilizes hsp70 in the same way suggests that the general model for hsp70 function in protein biogenesis that has emerged from these studies is probably applicable to other systems, such as the biogenesis of cytosolic proteins. In support of this notion, Welch and coworkers have reported that Hsp72 and Hsp73, two mammalian cytosolic hsp70s, associate with proteins as they are being synthesized (Beckmann et al., 1990). Saccharomyces cerevisiae has proven particularly useful for the analysis of hsp70 function. Eight different yeast hsp70 genes have been isolated (Craig, 19%) and placed into four subfamilies based on structural and functional criteria. Of these subfamilies, KarPp is located in the ER lumen (Normington et al., 1989; Rose et al., 1989) Ssclp is mitochondrial (Craig et al., 1989), and the other two, SSA and SSB, are cytoplasmic (Ft. J. N. and E. A. C., unpublished data). This report focuses on the function of the SSB hsp70 subfamily, which has two members, SS67 and SS82, that are 99% identical (Boorstein et al., unpublished data). Because of this high degree of identity and the lack of any information indicating a functional difference between Ssblp and Ssb2p, these proteins are referred to collectively as Ssbl/2p in this work. Strains lacking SsbllPp are viable but grow slowly at all temperatures and are cold sensitive (Craig and Jacobsen, 1985). We find that Ssbll 2p is associated with translating ribosomes, and this association is disrupted by the drug puromycin, which causes the release of nascent polypeptides from the ribosome. Also, we find that ssbl s&2 mutant strains have a low number of translating ribosomes, are sensitive to certain inhibitors of protein synthesis, and that the ssbl ssb2 growth defect is suppressed by increased copy number of a gene encoding an EF-la-like protein. We conclude that hsp70 is important during protein synthesis, probably through interaction with the nascent polypeptide chain, and we suggest that the function of hsp70 in translation may be to aid in the passage of the nascent polypeptide through the ribosome channel into the cytosol. Results SsbllPp Are Associated with Translating Rlbosomes To aid in the determination of the subcellular location of Ssbl/2p, antibodies directed against Ssbl&p were raised. A chemically synthesized peptide correeponding to the 16 C-terminal amino acid residues of Sabl/2p, which is not present in any other yeast hsp70, was used for immunization. The resulting antiserum reacted only with Ssblnp as judged by immunoblotting a 2D gel of total yeast lysate (data not shown). Approximately 50% of SsbllPp was soluble, while the

Figure 1. Ssbl/Zp Is Present in High Molecular Weight Form and Migrates to the Same Position as Do Ribosomes Extracts were prepared as described in Experimental Procedures from cells of wild-type strain JN228 grown at 30°C. Prior to lysis, cells were treated with 100 &ml cycloheximide to stabilize polysomes (Wettstein et al., 1964). The lysis buffer also contained 100 uglml cycloheximide. Samples were applied to 12 ml 20%-47% sucrose density gradients. After centrifugation, the gradients were run through a monitor reading 260 nm. All profiles are presented with the top of the gradient at the left, and the direction of migration is from the left to right as indicated by an arrow. Fractions from the region of the gradient containing ribosomes were collected and acetone precipitated; they were then run on an SDS-polyacrylamide gel and immunoblotted to detect SsbllPp and ribosomal protein L16. The immunoblots are placed to correspond to the profiles.

rest was particulate, as revealed by analysis of fractions of a yeast lysate (data not shown). Protease digestion of crude lysate showed that all the Ssbl/2p was sensitive to protease under conditions in which the KAR2 protein, which is localized in the ER, remained stable. The high sedimentation velocity of Ssbl/2p was not greatly affected by the addition of 0.1% Triton X-l 00. Taken together, the above results indicate that Ssbl/2p is a cytosolic protein associated with a nonmembraneous, large cell component. Since the ribosome is such a component, we investigated the possibility that SsblRp is ribosome associated.


To determine whether SsblRp was associated with ribosomes, crude cell extracts were prepared and centrifuged through a sucrose density gradient. During translation, more than one ribosome can be present on a given mRNA, resulting in the formation of “polysomes” whose sedimentation velocity increases in increments of 80s. Fractions of the sucrose gradients were run on an SDSpolyact-ylamide gel and immunoblotted using the Ssbl/ 2p antiserum. The migration of SsblRp coincided closely with the polysome profile and the migration of ribosomal protein L16 (Figure 1). This comigration was very reproducible, with up to 73% of the Ssbl/2p migrating into the gradient. We employed RNAase treatment of polysomes to degrade the mRNA that links the translating ribosomes together. When this was done, the ribosomes migrated with avelocityof 8OS, as indicated by both the profile and immunoblotting for ribosomal protein L16. Similarly, Ssbl/2p sedimented with a velocity no higher than 80s after RNAase treatment (Figure 28). These results indicate that Ssbl Rp is associated with a high molecular weight cellular component that contains RNA. The coincidence of migration of SsblRp with polysomes before RNAase treatment and with the 80s peak after RNAase treatment strongly suggests that SsbllSp are associated with ribosomes. To understand this association better, we tested the effect of the inhibition of translation on the association of Ssbl/2p with ribosomes. Treatment of cells with sodium azide results in the reduction of intracellular ATP levels. Initiation of translation requires ATP and thus is blocked by this treatment, but translation elongation, which is not ATP-dependent per se, continues. Thus, ribosomes translating at the time of the addition of sodium azide can complete protein synthesis, but reinitiation is blocked (Carter et al., 1980). As expected, sucrose density gradient analysis of lysate prepared from these cells shows that the ribosomes migrate primarily with a velocity of 80s (Figure 2C). Even though they are not actively translating, the ribosomal subunits are associated owing to their inherent affin-

Figure 2. Comigration of Ribosomes and Ssbll 2p Before and After Treatment with RNAase and the Translation Dependence of Association of Ssbl/2p with Ribosomes Extracts were prepared from cells of wild-type strain JN228 grown at 30°C as described in Experimental Procedures, except that 300 ug/ ml RNAase A was added to the lysis buffer for one-third of the cells; and, to allow translating ribosomes to run off and block reinitiation, onethird of the cells were treated with 10 mM sodium aside for 15 min prior to harvest. Centrifugation in sucrose density gradients was done as described in Figure 1. (A) Tracing of sucrose density gradients and immunodetection of Ssbl/2p and ribosomal protein L16 from mock-treated extract (A) and RNAase treated extract (B). (C) Extract from sodium axide treated cells. The immunoblotted fractions correspond to the profiles.

Hsp7Cl Plays a Role in Protein 99


ity for each other (van Holde and Hill, 1974). Fractions from

ssbi ssbf Cells Are Sensitive to

this gradient were immunoblotted, showing that the vast majority of the SsblRp did not migrate into the gradient, but instead remained at the top (Figure 2C). For comparison see Figure 28, which charts results of an experiment performed at the same time with the same culture, and shows that a large amount of Ssbl/2p remains associated (even after collapse of the polysomes caused by RNAase treatment) during the fractionation and workup steps, which are identical in these two experiments.

Trsnsletion inhibitors

The Association of Ssbll2p with Ribosomes Is Sensitive to Puromycin The data presented thus far provide evidence that Ssbl I 2p is associated with translating ribosomes. A possible explanation for the association of Ssbl12p with translating ribosomes is that they bind to the nascent chain as it emerges into the cytosol. To test this possibility, we took advantage of the special properties of the drug puromycin. Puromycin, an analog of aminoacyl-tRNA, is covalently incorporated into the growing end of the polypeptide chain, blocking further amino acid incorporation and resulting in the release of the nascent chain from the ribosome (Smith et al., 1965). If SsblRp binds the nascent peptide chain, then puromycin treatment of yeast lysate should disrupt the association of SsblRp with ribosomes. We performed this experiment by treating yeast lysate with puromycin in the presence of 0.4 M KCI followed by fractionation in a sucrose density gradient. Importantly, there was no appreciable difference between the polysome profiles of the puromycin-treated and mock-treated lysate (Figures 3A and 3B), indicating that after treatment with puromycin the integrity of the ribosome remains largely intact. When fractions from these sucrose density gradients were immunoblotted to detect Ssbl/2p, it was apparent that puromytin treatment resulted in a dramatic reduction in the amount of the Ssbl/2p migrating with the polysomes (compare Figures 3A and 3B). This result is consistent with the possibility that SsblRp is associated with translating ribosomes via direct interaction with the nascent polypeptide.

To determine the importance of Ssbl/2p in translation, we analyzed the phenotype of strains containing “knock out” alleles of both SS87 and SS02. Protein lysate from ssbl-7 ssb2-7 cells contain no Ssblnp as judged by immunodetection utilizing serum that reacts with either the conserved amino terminus or the SsblRp-specific carboxyl terminus (data not shown). Wild-type yeast cells are sensitive to a variety of compounds that inhibit translation by different mechanisms. If a mutant strain is more sensitive than the wild type to these drugs, i.e., if it is hypersensitive, this suggests that the mutation borne by this strain results in a defect in translation (Bayliss and Vinopal, 1971; Masurekar et al., 1981). With this in mind, we determined the sensitivity of ssbl ssb2 strains to several such drugs, including paromomycin, hygromycin B, 6418, cycloheximide, anisomycin, and verrucarin A. The ssbl ssb2 strains were hypersensitive to paromomycin (Figure 4) hygromycin B, and G418 (data not shown), all of which are aminoglycosides and act on the 40s ribosomal subunit to inhibit polypeptide chain elongation (Gonzales et al., 1978; Gale et al., 1981). The ssbl ssb2 mutant strain was also sensitive to verrucarin A (data not shown), a member of the trichothecene family of antibiotics, that blocks peptide bond formation and act primarily on the 60s ribosomal subunit (Fried and Warner, 1981; Gale et al., 1981). However, ssbl ssb2 strains did not exhibit increased sensitivity to cycloheximide or anisomycin (data not shown), both of which are also known to act on the 60s subunit to inhibit peptide bond formation (Stocklein et al., 1981; Fried and Warner, 1981). The increased sensitivity of ssbl ssb2strains to asubset of translation inhibitors argues against the possibility that ssbl ssb2 strains are generally more pemmabM or susceptible to drugs and suggests that a specific defect in translation results from the absence of SsbllPp.

ssbl ssb2 Cedls Contsln a Low Number of Trsnslating Ribosomes Further evidence supporting the idea that translation is compromised in ssbl ssb2 cells was gained by analysis of

Figure 3. The Association of Ssbl/2p with Ribosomes Is Sensitive to Treatment with Puromycin Wild-type strain JN225 was grown at 30% in YPD and Iysed as described in Experimental Procedures except that the lysis buffer contained 400 mM KCI and for one-half of the cells 5 mM puromycin was added. Both the puromy&and mock-treated tysates were incubatedat21°Cfor 15mtn, thenincubatedfor 15 min on ice and quick frozen in a dry-ice-ethanol bath. After fractionation on sucrose density gradients 88 described in Ftture 1, the conespondtng fractions were immunobbbed using Ssbl/2p antteerum. (A) mock-treated lysate; (B) puromycin-treated lysate.

Cell 100


YPD + 150pglml paromomycin

JN212 Figure 4. Disruption of Both SSB Genes Confers Paromomycin Sensitivity Wild-type strains JN54 and the ssbl-7 ssb2-7 mutant strain JN212 were grown in YPD medium to a cell density of approximately 1 x lo6 viable cells per milliliter. Serial 1:20 dilutions of the cultures were prepared, and 10 ul of each dilution was spotted onto plates of YPD and YPD containing 150 us/ml paromomycin. The plates were incubated for 3 days at 30%. Similar levels of inhibition were seen using YPD plates containing the drugs 6413 or hygromycin B, both at a concentration of 100 pglml.

ssbl ssb2

the polysomesof ssbl ssb2strains. Lysates prepared from ssbl ssb2 strain JN208 and wild-type strain JN54 were centrifuged through a sucrose density gradient and analyzed as described above (Figure 5). Comparison of the

polysome profiles of these two strains, as well 3 other ssbl ssb2 and wild-type strains, consistently rev&fed that lysate from ssbl ssb2 strains contained a smaller amount of polysomes and that the average number of ribosomes comprising the polysomes was lower. The lysate from the mutants also contained higher relative levels of free 80s subunits. We observed the 40s subunit peak in ssbl ssb2 polysome profiles other than the one shown, however, because of poor resolution, it was difficult to get an estimate of the quantity of material. The ssbl ssb2 Mutant Phenotype Can Be Suppressed by Increased Copy Number of a Previously Unidentified Gene, HBS7, Which Encodes an EF-la-llke Protein We carried out a selection to isolate genes whose increased expression could suppress the slow growth phenotype of ssbl ssb2 strains. Characterization of such genes could be helpful in gaining further insight into the function of Ssblnp. Strain JN208 (ssbl ssb2) was transformed with DNA of both a YCp59-based (Rose et al., 1987) and a YEpPCbased (Carlson and Botstein, 1982) yeast genomic library. The plates containing the transformants were incubated at 23%, a semipermissive temperature for growth. Transformants that formed large colonies were picked for further analysis. Protein lysates of JN208 isolates bearing putative suppressor clones were screened by immunoblotting with the Ssblnp antiserum to eliminate clones bearing either the SSfl7 or SSBP genes. SSB7 and SS62 were were isolated multiple times from both libraries. Four clones that conferred a suppressor phenotype and did not contain either SSB7 or SSB2 were recovered from the YCpBO-based library and retained for further analysis. Analysis of restriction enzyme fragments of these clones indicated that they had a 4 kb

/ Figure 5. ssbl ssb2 Mutants Have a Reduced Amount of Polysomes Extracts were prepared as described in Experimental Procedures from wild-type strain JN54 and the ssbl ssb2 mutant strain JN208 and centrifuged through sucrose density gradients as described in Figure 1. Cultures of both strains were grown at 30°C, treated with 100 ug/ ml cycloheximide prior to harvest, and lysed in CSB containing 100 pg./ml cycloheximide.

Hindlll fragment in common. No suppressor plasmids were isolated from the high copy number YEp24 library; this


be partially


by our




4 kb Hindlll suppressor fragment was deleterious to yeast growth when borne by a high copy number yeast vector (data not shown). In an attempt to identify the gene responsible for suppression, a mutation was constructed by inserting the LEU2 gene into the unique EcoRl site of the 4 kb Hindlll fragment. The effect of the LEU2 insertion on the ability of the 4 kb Hindlll fragment to suppress the slow growth phenotype of ssbl ssb2 was tested (Figure 8). The clone bearing the insertion mutation was not able to confer suppression, suggesting that the EcoRl site lay within the coding sequence of the suppressor gene. Molecular Analysis of HBSl Sequencing of the region flanking the EcoRl site revealed that it lies in a single large open reading frame (as indicated by underlining, Figure 7A). The putative start codon of this reading frame is preceded by stop codons in all three reading frames, and the putative stop codon is followed by several in-frame stop codons as well as stop codons in all three reading frames. This gene was designated HBS7 for Hsp70 subfamily B suppressor. The deduced amino acid

Hsp70 101



a Role in Protein



thought to be key residues for GTP binding and hydrolysis activity. The spacing between these sequences is also conserved (Dever et al., 1987). Sequence blocks closely resembling the EF-la-GTP binding and hydrolysis consensus sequence are found in Hbslp, as indicated by shading (Figure 7C), and their spacing is exactly the same as in EF-la.

ssb1 ssb2 [SSS]

SSbl ssb2 [YCpSO)

Discussion ssbl ssb2 [Hk?S7] Through avariety of approaches, we have investigated the function of the yeast SSB hsp70 subfamily. Analysis of the ssbl ss62 mutant together with the determination of the cellular characteristics of Ssbll2p indicates that hsp70 plays a hitherto unproposed role in translation.

SSbl ssb2 [htw:LEUZ]

Figure 6. The ssbl ssb2 Slow Growth Phenotype Is Suppressed Increased Dosage of a Gene for an EF-la-like Protein, Hbslp


Growth rate comparisons of ssbl ssb2 mutant JN206 with and without the HB.9 gene on the centromeric plasmid YCp60. Also shown is the growth character of JN206 carrying a YCp50 borne copy of the mutant /-KM allele hbsl-I. Cell cultures were adjusted to the same optical density and spotted on uracil omission media at the indicated temperature.

sequence of Hbslp shows that it is a protein of 520 aa (Figure 7A). The predicted molecular size of Hbslp is 58,300 kd. During sequencing of the 4 kb Hind81 suppressor fragment, we found that the MRP-LPO gene was adjacent to Hi3S7 (Figure 76). AMP-L20 encodes mitochondrial ribosomal protein L20 and has been mapped to chromosome 11 (Kitakawa et al., 1990). Thus, we tentatively assign HBS7 to chromosome 11. Searching GenBank with the HBS7 nucleotide sequence revealed that HBS7 is a previously unidentified gene but has significant sequence similarity to the EF-1 a family of translation factors (Figure 7C). This search also revealed a similarity between HBS7 and the SUF72 gene (also known as SUP2, SUP35, and GST7), which encodes an EF-la-like protein, first isolated as a translational suppressor (Hawthorne and Leopold, 1974; Culbertson et al., 1982; Surguchov et al., 1984). Further sequence comparisons revealed no significant similarity between Hbslp and other translation initiation, elongation, or termination factors. EF-1 a is a highly conserved protein found in all eukaryotes. Because EF-1 a is so highly conserved, for simplicity only the comparison of yeast EF-1 a (Nagata et al., 1984) and Hbslp is shown (Figure 7C). The similarity between the amino acid sequences of Hbslp and EF-la extends throughout their entire lengths, and overall they are 33% identical. The similarity between Hbsl p and EF-1 a is 570% as calculated based on the values of relatedness between amino acids normalized by Gribskov and Burgess (1986). This alignment also indicates that Hbsl p has an additional 69 aa at the amino terminus and that EF-1 a has an additional 5 aa at the carboxyl terminus (Figure 7C). EF-1 a and Hbsl p are the most similar at their amino termini. Three sequence blocks are found in the amino terminus of EF-1 a that are highly conserved in EF-1 a from all species and are

Phenotypic Analysis of the ssbl ssb2 Mutant We observed that ssbl ssb2 strains have a lower percentage of their ribosomes in polysomes and that the average number of ribosomes simultaneously translating any given message is lower than in the wild type. Hypothetically, this difference could be due to the slow growth rate of ssbl ssb2 cells, however detailed study of the characteristics of polysomes in Escherichia coli (Forchhammer and Lindahl, 1971) shows that the distribution of ribosomal material between polysomes, 70s ribosomes, and free 30s and 50s particles is independent of the growth rate. As far as we know, detailed studies on polysome characteristics of this nature have not been performed in yeast, but it is likely that in yeast also, the fraction of actively translating ribosomes is independent of the growth rate. Therefore, our observation of a reduction in the fraction of translating ribosomes represents an aberrant situation. This could be due to a decrease in the rate of initiation, or aberrations of elongation such as premature termination, or, less likely, an increased elongation rate. While this result does not allow us to distinguish between these possibilities, it indicates that that there is a defect in translation caused by the absence of Ssbll2p. The notion that SsbllPp is important for translation is further supported by our observations of the drug hypersensitivity of ssbl ssb2 mutants. All of the drugs we tested inhibit translation elongation, but their modesof action are different. Thus, the observation that s&7 ssb2 strains are sensitive only to a subset of these drugs suggests that a specific step or steps in translation elongation is defective. The most compelling piece of evidence supporting the interpretation that translation elongation is defective in ssbl ssb2 cells is the finding that the protein encoded by the increased dosage suppressor gene HBS7 is an EF-1 alike protein. EF-1 a plays a key role in translation elongation by bringing aminoacyl-tRNAs to the ribosome and facilitating codon-anticodon recognition. Based on the similarity between Hbslp and EF-la, it is likely that increased dosage of HBS7 affects translation elongation in a way that reduces the defect resulting from the absence of Ssbl/2p. Localization of Ssbll2p As expected for a protein involved in translation, we found that Ssbll2p iscytosolic and, to a largedegree, associated

Cell 102


B 2000







C lblp





I 0.1



200 I:


II 131 250


: I: 181 300



332 I 263 302



::.,I 305 432



I 213



31 150




I EF-la

:,:I 353 477 I


I:I 401 516 451



7. Molecular


of HBS7

(A) The sequence of the HBSl gene and flanking region and the deduced amino acid sequence of Hbslp. The EcoRl site into which the LEU2 gene was inserted between codons 213 and 214 to create the mutant allele hbsl-7 is indicated by underlining. (Et) Restriction map of HBSI region including the flanking MRP-LPO gene. The EcoRl site into which the LEUZ gene was inserted to create the mutant allele hbsl-1 is indicated by an asterisk. (C)Comparison of the deduced amino acidsequenceof Hbslp with the amino acid sequence of yeast EF-la. The deduced amino acid sequence of Hbsl p was aligned with EF-1 a using the default values for the Genetics Computer Group program, GAP. The top sequence is Hbslp, the lower EF-1 a. The regions of the EF-la tripartite GTP binding and hydrolysis consensus sequence are indicated by the shaded boxes. The location of insertion of the LEUZ gene is between codons 213 and 214 and fails between two of these consensus elements as indicated by the arrowhead.

Hsp70 103


a Role in Protein


with translating ribosomes. This finding corroborates our analysis of the ssbl s&2 mutant that indicates that Ssbll 2p plays an important role in translation. Ssbl/2pcould be associated with translating ribosomes either because it is binding directly to the nascent peptide chain or because it is involved in the assembly or dissassembly of a component of the translation machinery. The resultsof our experiments utilizing puromycin support the notion that Ssbll2p is associated with translating ribosomes by virtue of its association with the nascent polypeptide chain. Puromycin acts as an analog of aminoacyl-tRNA and is incorporated into the growing polypeptide chain resulting in termination of protein synthesis and release of the peptidyl-puromycin fragment (Smith et al., 1965). Treatment of polysomes with puromycin under conditions of high ionic strength results in the release of the nascent chain. If the puromycin-treated polysomes are maintained at low temperature, they remain intact after release of the nascent chain (Blobel and Sabatini, 1971). We found that the association of Ssbl/2p with ribosomes is disrupted by treatment with puromycin. Importantly, under the conditions we employed there was no destruction of the polysome profile because of the addition of puromycin. This makes it likely that only the nascent chain and any proteins associated with it were released by this treatment. Thus, this result suggests that Ssbll2p is released from polysomes upon treatment with puromycin because it is associated with the nascent polypeptide chain. This interpretation is consistent with the results of Welch and coworkers who have reported results suggesting that in the mammalian cell, cytosolic hsp70 associates transiently with nascent polypeptides (Beckmann et al., 1990). Implications for Protein Synthesis and Protein Folding During protein synthesis, the newly synthesized polypeptide chain passes through a channel in the large ribosomal subunit. Approximately 40 aa of polypeptide in an extended conformation are contained within the channel (Milligan and Unwin, 1966; Malkin and Rich, 1967; Blobel and Sabatini, 1970). The nascent polypeptide chain enters into the cytosol from an opening in the channel termed the exit site. An analogy can be drawn between the emergence of the polypeptide chain from the ribosome channel into the cytosol and the transport of polypeptide chains across a lipid bilayer. In both cases, the polypeptide is passed through a tunnel or channel in an extended conformation. The hsp70 subfamilies that reside in the mitochondrial matrix or the lumen of the ER have been shown to bind transiently to polypeptides as they are being transported into the organelle from the cytosol (Kang et al., 1990; Scherer et al., 1990; Sanders et al., 1992). Yeast strains bearing mutations in their mitochondrial or ER-localized hsp70s fail to transport proteins across the membrane (Kang et al., 1990; Vogel et al., 1990; Nguyen et al., 1990). These results suggest that organelle-localized hsp70 is necessary for polypeptide transport and that it acts by interacting directly with the polypeptide being transported. We propose a very similar role for cytosolic hsp70 in pro-

tein synthesis; as the nascent polypeptide chain emerges into the cytosol it interacts with hsp70, and this interaction is crucial for continuous transport of the polypeptide through the ribosome channel into the cytosol. According to our hypothesis, emergence of the peptide from the ribosome may be slower in the ssbl ssb2 mutant and thuscausethepolypeptide to”backup”in thechannel. This in turn would perturb protein synthesis because of a slowdown of the rate at which the nascent chain could be fed into the channel. During the translocation step of protein synthesis, the ribosome advances acodon and the site for binding of the EF-1 a-aminoacyl-tRNA complex is revealed. It is this step that would be most likely effected by a polypeptide “back up.” A defect in the translocation step would reduce the accessibility of the EF-la-aminoacyl-tRNA binding site. In this context, it is interesting to speculate on the function of Hbslp. Increased dosage of this EF-la-like protein could suppress the slow growth phenotype of ssbl ssb2 strains because it is more efficient than the normal EF-la at bringing aminoacyl-tRNA to the ribosome. This could compensate for a less accessible EF-la-aminoacyl-tRNA binding site. Future experiments utilizing an in vitro translation system and the SSB and HBS mutants as well as translation inhibitors should prove a powerful approach to investigate further the role of hsp70 in translation. Hsp70 has been shown to associate with nascent polypeptides (Beckmann et al., 1990) and there is ample evidence indicating that hsp70 binds to unfolded proteins. Thus, it appears likely that hsp70 associates with nascent cytosolic proteins to aid in their folding and prevent aberrant protein-protein interactions, as has been a topic of discussion for some years (Pelham, 1986; Rothman, 1969). Our analysis of hsp70 mutants has led us to conclude that there is additional significance to the association of hsp70 with nascent polypeptides. We suggest that hsp70 may also be needed to prevent the nascent polypeptide from interfering with translation by clogging the ribosome channel. At the present time it is not clear to what degree these two hsp70functions are connected. It is possible that there are two distinct hsp70 functions in translation, one to aid in the passage of the polypeptide through the ribosome channel and another to act in subsequent folding and assembly events. In the eukaryotic cell there are at least two species of cytosolic hsp70. One of these species could act very early as the nascent polypeptide enters the cytosol to aid in its passage through the ribosome channel, while another species could act in subsequent folding and assembly events. This study suggests that the influence of hsp70 on protein folding may extend to earlier events than previously thought by influencing the rate of passage of the nascent chain into the cytosol. Experimental Translation

Procedures lnhlbitor



Antibiotic stock solutions were prepared in water except for those of verrucarin A and anisomycin, which were prepared in 70% ethanol. For the water-soluble antibiotics, YPD plates containing various amounts of the drugs were prepared. Cultures were serially diluted and 10 ~1 aliquots of each were spotted onto the plates. Verrucarin A and anisomycin were applied to filter disks that had been placed on

Cell 104

lawns of the appropriate strains on YPD plates (30 ttg per disk for verrucarin A, 60 pg per disk for anisomycin). Equal volumes of 70% ethanol were added to control disks. All plates were incubated at 30DC.


and Treatment

of Yeast


Ail cell lysis was performed by bead beating for 3 min using a mini bead beater (Biospec Products). One hundred ODmo units of cells were typically lysed into complex stabilization buffer (CSB), which contains 300 mM sorbitol, 20 mM HEPES (pH 7.5) 1 mM EGTA, 5 mM MgCI,, 10 mM KCI, 10% glycerol, and 2 mM dithiothreitol with specific modifications as described below. After lysis, the cell extract was clarified by centrifugation for 5 min at full speed in a microfuge. To freeze ribosomes during translation, cycloheximide was added to a concentration of 100 @ml to cells growing at 30°C. The culture was then quickly cooled, and the cells were isolated by centrifugation and lysed into CSB containing 100 jrg/ml cycloheximide. To allow ribosomes to run off and block reinitiation, 10 mM sodium azide wasadded to the culture, which was then incubated for 15 min at 30°C, cooled on ice, and lysed into CSB containing 10 mM sodium azide. RNAase treatment of cell lysate was performed by lysing cycloheximide treated cells into CSB containing 300 &ml RNAase A and 100 uglml cycloheximide. Puromycin treatment of lysate was accomplished by lysing cells not treated with cycloheximide into CSB containing 400 mM KCI with 5 mM puromycin. The puromycinand mock-treated lysates were incubated at 21 OC for 15 min, then incubated for 15 min on ice, and quick frozen in a dry-ice-ethanol bath. All lysates were centrifuged through 20%47% sucrose density gradients containing 20 mM HEPES (pH 7.5) 1 mM EGTA, 5 mM MgCb, and 10 mM KCI. Either 12 ml or 5 ml gradients were employed and were centrifuged for 3.75 hr or 70 min. respectively, at 40,000 rpm in an SW 41 rotor at 4OC. Gradients were run through an ultraviolet monitor reading 260 nm. Fractions were collected, and acetone was precipitated and subjected to SDS-polyacrylamide gel electrophoresis.

DNA Sequencing

and Analysis

DNA sequencing was carried out by the dideoxychain termination method (Sanger et al., 1977) utilizing alkali-denatured DNA and the Sequenase enzyme system (US Biochemical Corporation) according to the instructions of the manufacturers. As the sequence was determined, primers were designed that annealed to the 3’ end, and sequencing proceeded successively in this manner. Oligonucleotide primers were purchased from Operon Technologies, Inc. Assembly of sequence fragments was performed utilizing the SEQMAN program of DNA Star Inc. Both strands were determined for all sequences presented.



A peptide of sequence NH2-CAEVGLKRWTKAMSSR-COOH was synthesized at the University of Wisconsin Biotechnology Center and injected directly into rabbits. This peptide corresponds to the 16 C-terminal amino acids of SsbllPp and has an additional N-terminal cysteine added for chemical coupling purposes. This serum was shown to react with both Ssblp and SsbPp. lmmunoblotting was performed as described previously (Craig et al., 1969). Visualization of antigen was done with incubation with ‘251-protein A Bolton-Hunter reagent (Amersham).


DNA Procedures

E. coli MC1066A was the primary strain used for molecular cloning procedures. This strain is a recA derivative of [E. coli K-l 2 /e&6 A(/ac/PGZY)X74 bpC936OpyrF::Tn5 (Kan’) s&A]. All cloning steps were car-


1. Yeast


and Miscellaneous

All of the yeast strains utilized construction of the ssbl-1 and viously (Craig and Jacobsen, method of Ito (Ito et al., 1963). cal Corp.

in this study are listed in Table 1. The ssbbl alleles has been described pre1965). Yeast transformation was by the All chemicals were from Sigma Chemi-

Acknowledgments We thank John Woolford for providing the L16 antiserum, Carol Gross for comments on the manuscript, and Irv Edelman for help on sequence analysis. This work was supported by Public Health Service grants and a molecular and cellular biology training grant from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 USC Section 1734 solely to indicate this fact. Received

June 6, 1992; revised


JN226 are from this study except


ssbl-1 ssb2-1 his3-11,515 his3-11,515 leu2-3,2-112 his3-11.3-15 leu2-3.2-172 ssbl-1 ssbbl his3-11,515 pep4-3 ade6


et al., 1961).

July 24, 1992.

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The accession M98437.


Number for the sequence


in this paper


The translation machinery and 70 kd heat shock protein cooperate in protein synthesis.

The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches. The SSB hsp70s (Ssb1/2p) are associated w...
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