YEAST

VOL. 7: 699-716

(1991)

Functional Analysis of a Conserved Amino-Terminal Region of HSP70 by Site-Directed Mutagenesis CHARLES M. NICOLET* A N D ELIZABETH A. CRAIG+ Depurtment of Ph~siologiculChemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, U.S.A.

Received 23 January 1991, accepted I8 April I99 1

Hsp70 proteins have been highly conserved throughout evolution. As a first step in a structure-function analysis of hsp70, we constructed and analysed the consequences of mutations in a portion of the SSAI gene, a membcr of the Succharomvces cerevisiae HSP70 multigene family, that encodes a nearly invariant region near the amino terminus. Analysis of strains expressing SSAl proteins with alterations at positions 8, 11 and 15 showed that these conserved residues within this region are critical for normal functioning of the protein. SSAl protein containing either of two changes at position 15 was able to slightly complement the inviability of an ssalssaZssa4 strain, but was inactive in other complementation assays. The other mutant proteins tested were unable to complement any tested phenotype. Effective interallelic complementation of several phenotypes was observed when a mutant protein substituted at position 8 was expressed in the same cell with either of two proteins carrying substitutions at position 15, suggesting that hsp70 acts as a multimer. Evidence from previous studies suggests that hsp70 proteins engage in ATP-driven cycles of binding and release from peptides. The ability of the mutant proteins to bind ATP and a peptide was tested. The Ssalp carrying a substitution at position 8. which inhibits growth of cells carrying wild-type SSA proteins, showed a defect in release from a pcptide relative to wild type. Two mutations, one each at position 8 and 15, resulted in accumulation of phosphorylated isoforms which may be a normal, transient hsp70 intermediate. KEY WORDS - Saccharomyes

cerevisiae; heat shock; protein phosphorylation.

INTRODUCTION Cells from nearly every organism tested thus far respond to an increase in temperature by inducing synthesis of a stereotypical set of proteins-the heat shock proteins. The most abundant of the heat shock proteins found after temperature upshift is a 70 kDa protein, designated hsp70. hsp70s are among the most highly conserved proteins known, showing 60-70% identity among eukaryotes, and 4&50%0 identity between Escherichia coli hsp70, the dnaK gene product, and the corresponding eukaryotic proteins (reviewed in Craig, 1985). In most eukaryotic organisms, the HSP70 genes form a multigene family. Originally identified as inducible proteins, certain hsp70s are constitutively expressed as well; these constitutively expressed proteins are localized in a variety of cellular compartments, including the cytoplasm (Chirico et af., 1988). endoplasmic reticulum (ER) (Munro and *Current addrcss: Department of Medical Microbiology and Immunology. University of Wisconsin-Madison, Madison, Wisconsin 53706, U.S.A. Worresponding author OR9 I a X ! V I /070699-18 $09.00

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Pelham, 1986; Hendershot el al., 1988) and mitochondria (Craig et af., 1989; Leustek rt d.,1989). Geneticand biochemicaldata have shown that hsp70 proteins are crucial participants in normal cellular physiological functions (reviewed in Lindquist and Craig, 1988). Biochemical characterization of hsp70 proteins has uncovered a number of shared activities. All hsp70 proteins examined possess ATP binding and ATPase activity (Zylicz et al., 1983; Zylicz and Georgopoulos, 1984; Ungewickell, 1985; Chappell et al., 1986;Welch and Feramisco, 1985). hsp70 proteins have been shown to participate in a number of specific protein-protein interactions. For example, the DnaK protein of E. coli interacts specifically with bacteriophage lambda replication proteins (Liberek et af., 1988) as well as the cellular protein GrpE (Johnson et af., 1989). In mammalian systems, a member of the hsp70 family (designated hsc70) has been extensively studied as an uncoating ATPase, a protein involved in the disruption of clathrin cages surrounding coated vesicles (Schlossman et al., 1984; Ungewickell, 1985;

700 Chappell et al., 1986, 1987; Heuser and Steer, 1989). Disruption of clathrin cages is ATP-dependent, while continued high affinity binding of hsc70 protein to the triskelion is ATP-independent. grp78 (BiP), an hsp70 protein found in the lumen of the ER (Munro and Pelham, 1986; Hendershot et al., 1988), has been shown to bind to a number of proteins, particularly incompletely assembled, mutant and incompletely glycosylated proteins (Kassenbrock et al., 1988). Flynn et al. (1989) have studied binding of grp78 and hsc70 to peptides in vitro. A peptide from the vesicular stomatitis virus glycoprotein coupled to an agarose matrix behaved as an effective affinity resin, greatly enriching grp78 and hsc70 from crude lysates. While binding was not ATPdependent, elution of the hsp70s from the peptide columns required ATP. Since non-hydrolysable analogs were not effective in elution, ATP hydrolysis is most likely required for release. These results suggest that a major role for hsp70 proteins is mediating protein conformational changes in an ATP-dependent fashion. This general activity would allow hsp70 to potentially participate in a number of cellular processes, as suggested in many reports in the literature: hsp70s could bind to precursor proteins, facilitating their posttranslational import across membranes. hsp70s localized within the mitochondria and ER may participate in import, preventing a newly imported unfolded protein from inappropriate aggregation, as well as mediating the correct assembly of multimeric proteins which takes place within these compartments. It has also been suggested that hsp70s play a role in the normal folding process of a number of proteins, stabilizing the emerging newly synthesized peptide chain and mediating the correct folding pathway (Pelham, 1986; Rothman, 1989; Beckmann et al., 1990). In the yeast Saccharomyces cerevisiae, HSP70 genes can be grouped functionally into four subfamilies, SSA, SSB, SSC and KAR2. The gene products of the SSA group, comprising SSAI-4, are primarily cytosolic proteins (Chirico et al., 1988). Two of the members, S S A l and SSA2, are constitutively expressed at farily high levels, while the SSA3 and SSAI genes are expressed at high levels after heat shock. SSA gene products are partially functionally redundant-in rich glucose-based media ssal or ssa2 single mutants grow as well as wild-type (wt) cells, while ssalssa2 double mutants grow very slowly and are temperature sensitive for growth (Craig and Jacobsen, 1984). Expression of SSA3 and SSA4 is induced in ssalssa2 double

C. M. NICOLET AND E. A. CRAIG

mutants. This expression is probably critical for the viability of ssalssa2 strains, since ssalssa2ssa4 triple mutants are inviable. Thus, the S S A genes comprise an essential subfamily (Werner-Wasburne et al., 1987). In vivo (Deshaies et al., 1988) and in vitro (Chirico et al., 1988) evidence has strongly implicated the Ssal and Ssa2 proteins (Ssal/2p) in the process of translocation into both the ER and mitochondria. The product of the essential KARZ gene is homologous to grp78 (BiP) of mammalian cells (Normington et al., 1989; Rose et al., 1989). Analysis of a temperature-sensitive (ts) KARZ mutant in yeast has implicated this gene product in the process of translocation of secreted protein precursors into the ER lumen (Vogel et al., 1990). The product of the essential SSCl gene is localized in the matrix of mitochondria. Analysis of a ts SSCl mutant indicates a role of Ssclp in both translocation and folding of protein imported into mitochondria (Kang et al., 1990). Analysis of the functional domains of hsp70 proteins has thus far relied on the analysis of deletion mutants and the study of proteolytic fragments (Chappell et al., 1987; Cegielska and Georgopoulos, 1989; Milarski and Morimoto, 1989). In order to begin a more precise analysis of structure-function relationships of the hsp70 protein, we introduced site-directed mutations in a highly conserved region of S S A I , a member of the S . cerevisiae HSP70 multigene family. This paper describes our examination of the phenotypic effects of these mutations, and a preliminary biochemical analysis of the mutant proteins. MATERIALS AND METHODS Yeast strains, culture media and general handling

The following strains were used: (1) DSlO (wild type): M A Ta Atrpl lysl lys2 ura3-52 leu2-3,112his311,15 ssal: DS10, with ssal::HIS3 (ssal-4); (2) ssalssa2: MATalpha Atrpl, lys2 leu2-3,112 ssal4 ssa2::LEU2; (3) ssalssa2ssa4 G A L 1 : S S A I : MA Talpha Atrpl ssal::HIS3 ssa2::LEU2 ssa4:: LYS2YCpGALI:SSAl. YCpGAL1:SSAl is acentromeric vector containing the wt SSAI gene under control of the regulable GAL1 promoter; it utilizes the URA3 gene as a selectable nutritional marker. Yeast culture media were made according to standard recipes as described by Sherman et al. (1986). Transformations were carried out by the lithium acetate procedure of Ito et al. (1983). Growth of

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FUNCTIONAL ANALYSIS OF A CONSERVED AMINO-TERMINAL REGION

15R C (Arg) 15s A (Ser) 8N A (Asn) 11A G (Ala) 15G G (Gly) I I I GTC CGT ATT GAT TTA GGT ACA ACA TAC TCG TGT GTT $SA1 (amino acids 5-16)

Val Gly Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val

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KAR2, maize, Dm hsp70

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Figure 1. Conserved amino-terminal region of hsp70. The amino acid sequence of Ssalp (amino acids 5-16) is compared to the sequence of a number of other hsp70s. An asterisk (*)denotes amino acid identity. Dm hsc70-4 is a heat shock cognate gene from Drosophilu melunogusfer;it is constitutively expressed and not appreciably induced after heat shock. KAR2 is the yeast grp78IBiP homolog. The other sequences all represent the major inducible hsp70 gene from the organism indicated. The wild-type SSAI nucleotide sequence and the changes introduced to generate the mutants utilized in this study are shown above the protein sequence. Thedenotations ofeach mutant used throughout the text are indicated in boldface type to the left of the change; the substituted amino acid is indicated in parentheses to the right. Sequences were obtained from the following sources: SSAl (Slater and Craig, 1989); Dm hsc70-4 (Craig et al., 1983); human (Hunt and Morimoto, 1985); KAR2 (Normington et ul., 1989; Rose ef ul., 1989); maize (Rochester et al., 1986); Dm hsp70 (Ingolia et al., 1980); E. coli(Bardwel1and Craig, 1984); B. subtilis (Hearne and Ellar, 1989).

yeast cultures was monitored by measuring the optical density at 600 nm (OD,) and assuming that 1 O D unit equals lo7 cells/ml. Except where otherwise noted, strains were maintained in minimal media missing the appropriate amino acid(s) to maintain plasmid selection. All cell growth was carried out at 30°C, except where otherwise noted. Bacterial strains, recombinant DNA manipulations and plasmid constructions MC1066A was the primary strain used for plasmid construction, propagation and maintenance. This strain is a recA derivative of MC1066 [E. coli K12 leuB6 A(1acIPOZ Y)X74 trpC9380 pyrF::Tn5 (Kan')StrA], and can be complemented by the yeast LEU2, URA3 or TRPl genes. All DNA enzymatic manipulations (restrictions, ligations, etc.) were carried out using conditions recommended by the manufacturers. Large and small scale plasmid DNA preparations were carried out by alkaline lysis, followed by EtBr-CsC1 gradients for large scale preparations (Maniatis et al., 1982). DNA fragments were purified using Gene-Clean (BiolOl, La Jolla, CA). Mutagenesis pYe(CEN3)30, a centromeric vector containing the TRPl gene as a selectable marker, was used for

construction of the mutant SSAI genes. The SSAl gene on a 3.9 kb BamHI-PvuII fragment, encompassing approximately 1.2 kb of 5' non-coding and 0.7 kb of 3' non-coding sequences in addition to the SSAl protein coding region, was inserted into BamHI-PvuII-restricted pYe(CEN3)30 to generate YCp-SSAI. For mutagenesis, a 1.7 kb PvuII-KpnI fragment containing the amino-terminal 0.5 kb of the S S A l 5' coding region was cloned into M13mpl9at theKpnIand HincII sites. Mutagenesis using the dut ung system of Kunkel(1985), as well as primer annealing, extension and mutant plaque screening, were carried out as described (Craik, 1985; Park and Craig, 1989). Oligonucleotides with the changes from the wt sequence indicated in Figure 1 were synthesized by the University of Wisconsin Biotechnology Center (Madison, WI). Mutations at position 15 were generated by using an oligonucleotide synthesized with mixed nucleotides at the position corresponding to the first base of the codon. The identity of each mutant was confirmed by dideoxy sequencing from an oligonucleotide primer which annealed within the coding region of the SSAl gene. Replicative form M13 DNA was prepared from the mutants, and the mutant fragment was excised from the M 13 vector using KpnI-HindIII. The Hind111 site was filled in with Klenow fragment, then this piece was ligated with the KpnI-PvuII cleaved gel-purified backbone

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from the original YCp-SSA1 vector. To insure that the actual mutated fragment was cloned into the vector, recombinants of the right size were further screened by digestion with PstI. Only those clones having mutant fragments obtained from the M 13 vector would have an additional M13-derived PstI site, 1.2 kb from the translation-initiating ATG. The YCp-SSA1 vectors utilizing TRPl as a selectable marker were changed into vectors utilizing LEU2 by the following protocol. A 2.2 kb XhoI-SalI LEU2 fragment was blunt-ended with Klenow fragment, then ligated to a partial XbaI digested, blunt-ended, YCp-SSA1 plasmid.

SSAl strains of opposite mating type were transformed with the battery of plasmids, and restreaked onto synthetic galactose-based media to maintain selection for the plasmids. Freshly grown cells of each mating type in all possible plasmid combinations were then mixed together on galactose-based synthetic media to allow mating to proceed in the presence of wt Ssalp (from the GAL1 promoter) while still selecting for the mutant gene harboring plasmids. After at least 24 h to allow for some growth of diploids, portions of the mated patch were streaked onto glucose-based media to observe whether complementation could occur.

Assessment of complementation

Protein lysates, two-dimensional ( 2 0 ) gel electrophoresis and ATP column chromatography

Strains to be tested for complementation were transformed with the wt, mutant and vector plasmids, and selected under permissive conditions for growth. Four to five transformants from each plate were then streaked out to allow single colony formation, under both permissive and non-permissive conditions. A single transformant which showed ‘typical’growth from this group was then chosen for further analysis and/or photography. Since the strains were somewhat unstable, fresh transformants were periodically generated and taken through this regimen. Transformants were streaked out to observe single colony formation under the appropriate non-permissive condition-37°C for the ssalssa2 double mutant, and on glucose-based media for the ssalssa2ssa4 GAL1:SSAI mutant. The ability to form single colonies was scored as positive complementation, regardless of the length of time required. Cells transformed with the vector alone and the wt plasmid were included in all growth testsas a negative and positive control, respectively. Photographs were taken when the wt positive controls reached their maximal level of growth. Assessment of interallelic complementation in the ssalssa2double mutant was carried out essentially as described above. After transformation with the first set of plasmids, a transformant behaving as expected was selected, grown up in synthetic media without tryptophan or leucine to maintain plasmid selection, then retransformed with the second set of plasmids. Transformants were selected on synthetic media missing both tryptophan and leucine. Restreaking under both permissive and non-permissive conditions was also performed on synthetic media lacking tryptophan and leucine. To assess interallelic complementation in ssalssa2ssal strains, a different regimen wascarried out. Haploid ssalssa2ssa4 GAL:

Protein lysates were prepared by the following method: 1.5-3.0 x 10’ cells were suspended in 1 ml of ~ O ~ M - H E P E S pH , 7.5, 25mM-KC1, 5mM2-mercaptoethanol, 2 mM-MgOAc, 1 mM-PMSF, 1.4 pg/ml Pepstatin A and 100 pg/ml TLCK, in a 1.5 ml microfuge tube. Approximately 0.5 ml glass beads (45s600 micron diameter, Sigma, St Louis, MO) were added, then the cells were disrupted in a Bio-Spec (Bartlesville, OK) mini Bead beater by four 1-min pulses. Tubes were briefly spun, the supernatant was removed and re-centrifuged at 12,000 x g for 5 min. This cleared supernatant was quick-frozen in a dry-ice-ethanol bath and stored at - 70°C. This protocol generally yielded approximately 1 mg of protein per 1.5-3.0 x lo* cells used (mechanical disruption of cells grown in minimal media, as in these experiments, yields much less protein than cells grown in rich media). Two-dimensional gel electrophoresis was carried out in the first dimension (isoelectric focussing) by the method of O’Farrel(1979, using the exact composition of buffers and reagents described therein. Second dimensions were run in an SDS-discontinuous gel system as described by Laemmli (1970). The acry1amide:bis ratio was 29:1, utilizing a 7.5% resolving gel and 4% stacking gel. Gels were run at a constant voltage of 140 V. Proteins were visualized by staining in 0.25% Coomassie Brilliant Blue-R in H,O:methanol:HOAc (45:45: 10) and destaining in H,O:isopropanol:HOAc (80:10:10). Protein samples from the crude lysate to be run on 2D gels were subjected to the following regimen: approximately 100 micrograms of protein were treated with RNAaseA (final concentration 50 pg/ ml) and DNAaseI (final concentration 25 pg/ml) for 20min on ice. Protein was precipitated by the

FUNCTIONAL ANALYSIS OF A CONSERVED AMINO-TERMINAL REGION

addition of an equal volume of cold acetone, centrifuged. then the pellet was suspended in 60p1 of O'Farrel lysis buffer (O'Farrel, 1975). To analyse ATP column fractions, a similar protocol was utilized, except nuclease treatment was not carried out prior to acetone precipitation. Typically. the eluted fraction from the equivalent of 5-10 x lo8 cells was analysed by 2D electrophoresis. ATP-agarose column chromatography was carried out exactly as described in Craig ct ul. ( 1989). Peptide binding

Peptide columns were prepared and developed as described (Flynn et ul., 1989). Peptide A (KRQIYTDLEMNRLGK). as designated by Flynn er 01. (1989). was synthesized by the UW Biotechnology Center (Madison, WI). Coupling of the peptide to an agarose matrix was carried out by dissolving 8&100mg peptide in 2 ml l00mMHEPES. pH 7.5. then combining this solution with Zml (settled volume) of Afi-Gel 10 (Bio-Rad. S. Richmond. CA), prepared according to the manufacturer's instructions. A sample was withdrawn at this time to determine the absorbance at 280 nm of the solution prior to coupling. Coupling was carried out for 4 h at 4'C. At this time. another sample was withdrawn for an absorbance determination. The decrease in absorbance values typically indicated that 9-12mg;ml of peptide was coupled to the matrix. corresponding roughly to a concentration of 5 mM in the packed resin. After coupling. the reactive Affi-Gel groups were blocked by incubation with Tns, pH 7.5. for 1 h at 4 'C. Columns were poured to a final settled volume of 1.8 ml. then equilibrated with 50mM-HEPES. pH 7.5, 25mM-KCI (HK buffer) prior to loading. Lysates were prepared as described above. except the lysis buffer was on1 HK plus protease inhibitors. Typically 1-1.2 x 10' cells were lysrd to yield a final volume of 20 ml of 1.53 mgjml lysate. Prior to loading the peptidecolumn, lysates were adjusted to 10mM-EDTA and incubated for 10 min on ice. 0.5 -0.8 ml lysate was quickfroLen for a crude lysate sample, then the remainder was loaded onto the peptide column. The column was loaded at approximately 0.25 mljmin, then washed at a higher flow rate (0.5 ml/min) with five column volumes of HK-I0 mM-EDTA (HKE). five column volumes of HKE-I M-KCI. then three column volumes of HKE. Columns were then brought to room temperature for 10 min. Subsequent elutions consisted of three column volumes

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of HK-3 mwMgC1, (HKM), then HKM with varying amounts of ATP (at the slower flow rate of 0.25ml/min). A final wash was carried out with three column volumes of 8 M-urea. Columns were re-equilibrated with at least ten column volumes of HK. In accordance with the results of Flynn et al. (1989), weobservedsomeelutionofhsp70swith HK plus no added nucleotide. However, a much greater amount of hsp70 was recovered in the presence of ATP. Elution of wt Ssal/2p was complete with 20pi-ATP-i.e., no more of these proteins were recovered with subsequent elutions using higher concentrations up to 5 mM-ATP. The 8 M-urea wash contained very low levels of numerous cellular proteins, but not a specificenrichment ofany hsp70s. To visualize the eluted fractions, the dilute fractions were precipitated with 1.5 volumes acetone. Generally, one-halfof the recovered fraction was subjected to 2D gel electrophoresis in order to obtain an easily visualized pattern. Yields were somewhat variable, although in every case the amount of Ssa2p recovered provided a reliable internal control.

In vivo pliospholahelling Cells were grown in selective synthetic media to early log phase, then passed into low phosphate Y PD media (Schweingruber and Schweingruber, 1981)at a concentration of 7-9 x lo5cells,'ml. Cultures were allowed to attain a cell density of 4-6 x lo6 cellsjml (2-3 generations). At this point, 2 x lo7 cells were removed, centrifuged, and resuspended in 1 ml low phosphate YPD. 150 pCi of j2Porthophosphate was added (85W9200 Ci/mmol; Dupont-New England Nuclear, Boston, MA), and incubation continued for an additional 2 h (approximately one generation). Cells were pelleted. the supernatant was removed, and the pellets were quick-frozen in a dryice+thanol bath. The remainder of the culture, not receiving label, was also grown for an additional 2 h, and harvested. These unlabelled cells, grown in the same low phosphate media. served as a source of cold protein which could be mixed with the labelled protein, in order to align a Coomassie-stained total cell protein pattern with a phospholabelled protein pattern. To prepare lysates for visualization by 2D gel electrophoresis, an amount of protein lysate corresponding to 7--8x lo7 unlabelled cells plus I -2 x lo7labelled cells was nuclease treated, acetone precipitated, and run as described in a previous section. Gels were stained, then dried and subjected to autoradiography at - 70°C with an intensifying screen to visualize the labelled spots.

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preparations (data not shown) levels were indistinguishable from that of wt Ssalp. In contrast, 15R Construction of mutant SSAl genes containing lysates (Figure 2F) consistently conUsing site-directed mutagenesis, we introduced tained a much greater amount of Ssalp. In accordfive mutations into a highly conserved region of the ance with the protein data, the mRNA levels for the S S A l gene spanning amino acids 5-17 (Figure 1). wt, 8N, 11A, 15s and 15G genes were indistinguishThe 12 amino acid region is completely conserved able, but the mRNA level of 15R was elevated two between SSAI and human hsp70, and shows only a to three-fold (data not shown). The increase in 15R single conservative change in maize and Drosophila. mRNA level may result from an alteration in autoChanges that we judged most likely to have an regulatory properties of the 15R mutant protein effect, based on their chemical characteristics, were (Stone and Craig, 1990). Since the 15R and 8N mutations result in an made at positions within this conserved stretch. As shown in figure 1, the aspartate at amino acid 8 was additional net positive charge on the polypeptide changed to an asparagine, substituting an chain, a shift toward the basic pole relative to wt uncharged residue of similar geometry for the only Ssalp was expected. The expected shift was negatively charged position over a 21 amino acid observed, but the pattern for both these proteins region. This altered gene (and protein was desig- was unexpectedly complex in that modified forms of nated 8N. A possible site of modification, threonine the proteins were apparent. To address the nature of at position 1 1 , was changed to alanine (designated the modification of 8N and 15R, we performed an 1 IA). Three mutations were generated that altered in vivo phospholabelling. Phosphorylation would be the cysteine at position 15, an amino acid that might expected to shift the protein towards the acidic pole, participate in intra-or inter-molecular interactions. without necessarily changing the apparent molecuIn the altered proteins, this cysteine was changed to lar weight. After ssal strains harboring the battery either the positively charged residue arginine (15R), of plasmids were labelled with phosphate, protein or two uncharged polar residues, serine (15s) or lysates were prepared and analysed by 2D gel electrophoresis. Unlabelled proteins from cells grown glycine (1 5G). simultaneously in the same media were prepared and combined with labelled proteins, in order to Expression and modijication of mutants visualize the spots by Coomassie staining prior to Before assessing the complementing ability of the autoradiography. The putative modified isoform of mutant proteins, it was important to determine that 8N and 15R (migrating between the more basic they were present at levels comparable to the wt. We form and Ssa2p) was strongly phospholabelled compared the levels of accumulation of the mutant (Figure 3). Under these growth and labelling conand wt proteins in an ssal background, using 2D gel ditions, radioactive phosphate incorporation was electrophoresis to resolve Ssalp from other hsp70s not detected in the wt or other mutant Ssal proteins, which co-migrate on a one-dimensional gel. or in other members of the hsp70 family. Although Ssalp and Ssa2p were not always completely resolved as distinct spots, their appearance Complementation ability of the mutants as a broad nearly resolvable doublet in extracts from The S S A subfamily is essential; ssalssaZssa4 cells ssal cells carrying the wt SSAI gene on a centromeric plasmid (Figure 2B) was clearly distinguish- are inviable but can be maintained by growing cells able from the sharper cone-shaped spot consisting harboring a GAL1 promoter-SSA 1 fusion plasmid only of Ssa2p observed in extracts from cells harbor- (designated GAL1:SSAl) on galactose-based media ing the parent vector (Figure 2C). In wt cells, Ssalp (Werner-Washburne et al., 1987). After a shift to and Ssa2p were present in approximately equimolar glucose-based media, the level of Ssalp falls and the amounts (data not shown). As judged by the Ssalp: cells die. We used the phenotype of inviability of SSa2p ratio (Figure 2B), the level of expression of these strains on glucose-based media to ask if the S S A l from the centromeric vector was similar to mutant proteins were functional in vivo. Results of a that from the chromosome. In lysates from cells typical experiment are shown in Figure 4A. Three of containing 8N (Figure 2D), 1 IA (Figure 2E), 15s the mutant proteins, 8N, 11A and 15R, were non(Figure 2G), and 15G (Figure 2H), the mutant pro- functional at all temperatures tested, as indicated by teins accumulated to a slightly higher level than in their inability to support single-colony formation cells carrying the wt gene. However, in other protein on glucose-based media. At 15°C (not shown) and RESULTS

FUNCTIONAL ANALYSIS OF A CONSERVED AMINO-TERMINAL REGION

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Figure 2. Expression of wild-type (wt) and mutant Ssa I proteins. Protein lysates were prepared from ssa1 cells containing the wt, mutant and vector plasmids, then resolved by two-dimensional gel electrophoresis. The arrow indicates the position of the Ssalp (in the case of 8N and ISR, the arrow designates the most basic isoform). (A) Schematic of the gel region shown, with several members of the hsp70 family identified; (B) wt; (C) vector alone; (D) 8N; (E) 1 IA; (F) 15R; ( G ) 15s; (H) 15G. In this and all other figures of gels, the acidic pole is oriented towards the right.

23°C (Figure 4A), 15s and 15G allowed singlecolony formation, though at a much slower rate than wt. This partial complementation is temperature sensitive: 15s allowed no growth at 30°C or 37"C,while 15G supported growth at 30°C but not 37°C. The very large, well-growing colonies visible in this figure are revertants, probably resulting from mitotic recombination or gene conversion events.

However, true complementation can be distinguished from revertant growth, since revertant colonies are much fewer in number compared to the more slowly growing mutant cells. A biochemical consequence of depletion of SSA proteins is the accumulation ofprecursors ofsecreted proteins, including preproalpha factor (Deshaies et af.,1988). We monitored precursor accumulation

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Figure 3. Phosphorylation of mutant Ssal proteins. Phospholabelled proteins were prepared and analysedby two-dimensionalgel electrophoresisas describedin the text. The arrow indicates the position of the phosphorylated isoform in the Coomassie-stainedgel (A and C) and in the corresponding autoradiograph (B and D). (A and B) 8N; (C and D) 15R.In the Coomassie-stainedgel, the non-phosphorylated isoform and the Ssa2 protein are visible to the left and right of the arrow, respectively.

inanssalssa2ssa4 GAL1:SSAI strain in the presence of the mutant SSAI proteins, after a shift to glucosebased media. Cells containing the mutant SSAI genes, as well as cells carrying the parent vectors, showed a substantial accumulation of precursor (data not shown). Even 15G, which does allow growth of the cells on plates, has no apparent ability to rescue the translocation defect of the ssalssa2ssa4 triple mutant. However, since complementation of the growth defect is poor, differences in transport efficiency may be too small to detect by the assay used. ssalssa2 strains grow very slowly at 23°C and 30°C, and are strongly temperature sensitive, unable to form single colonies at 37°C (Craig and Jacobsen, 1984). These phenotypes are not necessarily due to

loss of the same cellular functions that result in inviability of ssalssa2ssa4 strains, so it was of interest to examine the complementing ability of the mutant Ssal proteins in the ssalssa2 background. The mutant proteins did not rescue the slow growth phenotype nor the temperature sensitivity of ssalssa2 cells, and in fact had an inhibitory effect on growth (Figure 4B). In particular, the 8$J mutant protein is strongly inhibitory. Repeated analyses showed that the order of inhibition caused by the mutant proteins was 8N > 1 1 A > 1 5s > 15G = 15R. The complementation experiments described above were carried out in the absence of Ssalp or the very closely related Ssa2p. To determine if any of the mutant defects were dominant over the wt, we examined wt, ssal and ssalssa2ssa4 GAL1:SSAI

FUNCTIONAL ANALYSIS OF A CONSERVED AMINO-TERMINAL REGION

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Figure 4. Complementation test of SSAl mutant proteins. Wild-type (wt), and vector transformants ofeither an .~.~ul.s.~u2.~.~a4 CALI: SSAl strain (A) or an ssalssa2 strain (B) were streaked out on glucosebased media to observe single colony formation, at the indicated temperatures. The plasmid utilized is indicated next to the streak (V, vector alone).

strains expressing the mutant Ssal proteins. 8N mutant protein inhibited growth in all strains while the other mutant plasmids had little if any effect (Figure 5). Thus 8N has a dominant negative effect, suggesting that 8N is not simply an inactive protein, but that it interferes in the normal functioning of the SSA subfamily of proteins. Interallelic complementation We investigated whether any of the mutant proteins exhibited interallelic complementation by examining the growth properties of the ssulssa2 strains containing different pairwise combinations of the mutant SSAI genes. To permit selection for

two centromeric plasmids, a set ofvectors harboring the mutant genes was generated by cloning the LEU2 gene into the resident TRPl gene. Certain combinations of the mutant plasmids allowed better growth than either plasmid alone. The 8N plus 15G or 15s proteins in an ssulssu2 strain obviated the inhibitory effect of any of these plasmids transformed singly, and in fact these combinations allowed nearly wt growth rates at 30°C. The 8N plus 15G combination also showed the unique ability to rescue the ts phenotype of the ssa/.wa2 strains (Figure 6). However, certain combinations had a synergistic deleterious effect. For example, the 8N and 1 1A plasmids individually strongly inhibit growth of an ssalssu2 mutant (Figure 4B), and our

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Figure 5. Inhibition of growth by the 8N mutant protein. The growth rate of wild-type (wt) cells, ssal mutants and ssalssa2ssa4 GALI: SSAl mutants, was compared in transformants carrying the wt, mutant and vector plasmids wt and .ssa/, glucose-based media; ssalssaZssa4 CALI: SSAl galactose-based media.

Figure 6. Interallelic complementation ofssalssa2strains. ssalssa2 strains were transformed with various combinations of the wildtype (wt), mutant and vector plasmids. Growth ability was observed at the temperatures indicated. The numbers refer to the particular combination of plasmids in the transformant streaked (V,vector). I , wt/V; 2,8N/V;3, 15R/8N;4, 15G/8N;5, 15S/8N;6,15G/V;7, 15S/V;8,15G/llA;9, 15G/15R,10,15G/15S; 11,15S/llA; 12,15S/15R; 13, 15R/V;14,IIA/V;15, 15R/llA.

FUNCTIONAL ANALYSIS OF A CONSERVED AMINO-TERMINAL REGION

709

Figure 7. lnterallelic complementation of ssalssaZ.~ssdstrains. Haploid ssalssaZsso4 GALI: SSAl strains of opposite mating types were transformed with the wild-type (wt), mutant and parent vector plasmids. then mated in various combinations (on galactosebased media). Diploid-enriched patches were streaked out to observe single colony formation on dextrose-based media at the temperatures indicated. The numbers indicate the mating combinations tested. I , wt x V: 2, 15s x V: 3. 15G x V; 4. 8N x V; 5. I S S x t " 6 , 15GxXN:7. 1 5 R x 8 N ; 8 , I l A x 8 N ; 9 , I I A x l S R ; 10. I I A x l S S ; I I , I I A x l S G : 12, 1 I A x V ; 13. 15Rx15S: 14, ISR x 15G; 15. 15R x V: 16. 15Sx 15G. At 37"C.only thosecombinationsallowinggrowthareshown.

inability to recover .s.sal.sso2strains harboring both plasmids suggests this combination is lethal. We wanted to assess interallelic complementation in the ssulssa2ssal background, as well as in s.salssu2, since some differences in complementation were observed in the two strain backgrounds. Because of a limitation in the number of auxotrophic markers, interallelic complementation after mating of strains carrying different plasmids was tested. As expected from the results of the complementation assays, the strains containing 15G with any other plasmid allowed slow growth. However, strains containing the pairwise combination of 15G plus 8 N showed much more robust growth. Furthermore, the combination of 15s plus 8 N allowed growth, even though at 30°C neither 15s nor 8N alone is able to complement an ssaIssa2.s.sal strain. While none of these mutant proteins permit even very slow growth at 37°C in an ssalssa2s.sul strain (Figure 4B), these two combinations also allowed growth at 37°C (Figure 7). Therefore, we conclude that interallelic complementation occurs

efficiently in the ssa Issa2ssa4 triple mutant, as well as s.saIssa2 with the combinations 8N plus 15G and 8 N plus 15s alleles. A TP binding

The ability to bind ATP is a universal property of hsp70 proteins. We utilized ATP-agarose chromatography to examine the ATP-binding properties of the mutant hsp70 proteins. Protein lysates were prepared from .ssaI cells harboring each of the plasmids. These lysates were applied directly to ATP-agarose columns and eluted with ATP. Eluants were resolved by 2D gel electrophoresis to identify the different hsp70s recovered (Figure 8). The protocol utilized consistently resulted in the recovery of the major yeast hsp70 proteins- Ssalp, Ssa2p, Ssclp, Ssb1/2pand Kar2p. We used the ratio of Ssalp to Ssa2p in the ATP eluate as a relative measure of binding affinity. The ratio of mutant Ssa 1 p to Ssa2p recovered in the ATP-eluted fraction of8N (both isoforms), 1 IA, 15Gand 15s-containing

710

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M. NICOLET AND E. A. CRAIG

Figure 8. ATP binding of mutant Ssal proteins. Protein lysates from ssal strains transformed with the battery of plasmids were prepared and analysed by ATP affinity chromatography. Eluted fractions were resolved by two-dimensional gel electrophoresis. The arrow indicates the position of the Ssal protein (in the case of the 8N and 15R crude lysates the arrow designates the most basic isoform); to the immediate right is the Ssa2 protein. For the wild-type (wt) (A), 11A (B), 15s ( C ) and 15G (D) proteins, the ratio of Ssal:SsaZ was the same in the crude lysate and eluted fractions, so only the ATP-eluted fractions are shown. (E) 8N crude lysate; (F) 8N ATP-eluted fraction; (G) 15R crude lysate; (H) 15R ATP-eluted fraction.

lysates was the same as the wt (Figure 8) and equivalent to that found in the crude lysates (crude lysate only shown for 8N). Step gradient elution with varying ATP concentrations did not preferentially elute the mutant proteins, nor did GTP or a high salt wash (data not shown), indicating that the binding affinities of these mutant proteins are approximately the same as that of the wt. In contrast, the 15R phosphorylated isoform is not recovered from the ATP column (compare Figure 8G and 8H). Since ATP column flow-through fractions show an enrichment of the phosphorylated isoform relative to the other hsp70s (data not shown), and since this isoform was not eluted even with 25 mM-ATP (data not shown), it is likely that the phosphorylated isoform is defective in its ability to bind ATP, rather than unable to be eluted from the column. The 15R unmodified isoform was able to bind and be eluted with ATP, but the Ssalp:Ssa2p ratio was higher in the crude lysate than in the eluted fraction. The fact that mutant protein produced in E. coli can be enriched by ATP-agarose chromatography as effectively as wt E. coli-produced protein (data not shown) indicates that 15R is able to bind ATP efficiently, and in the yeast lysates was not

simply complexing with other hsp70s already bound on the column.

Peptide binding A newly described biochemical activity of hsp70 proteins which directly relates to their in vivo function is their ability to bind to oligopeptides, as described by Flynn et al. (1989). We analysed the ability of S S A l wt and mutant proteins to bind to the peptide A used in their studies, KRQIYTDLEMNRLGK, since this peptide effectively bound hsp70-related proteins. Protein extracts derived from ssal strains expressing the wt and mutant genes were chromatographed on columns containing the oligopeptide coupled to an Affi-Gel matrix. Figure 9 shows the ~ O ~ M - A T P eluted fractions of wt and mutant-containing lysates, resolved by 2D gel electrophoresis. The mutant proteins fall into three classes. The first class, consisting of 15S, 15G and the basic isoform of 15R, behaved like wt S s a l p t h e y quantitatively bound to the peptide column and were completely eluted by 20 p ~ - A T PThat . is, the ratio of Ssalp:Ssa2pwas the same in the crude and eluted

FUNCTIONAL ANALYSIS OF A CONSERVED AMINO-TERMMAL REGION

71 1

Figure 9. Peptide binding of mutant Ssal proteins. Protein lysates from ssol strains transformed with wild-type (wt), mutant and parent vector plasmids were prepared and analysed by peptide affinity chromatography. Only the ATP-eluted fractions are shown from those mutants in which the crude lysates and eluted fractions had equal proportions of Ssa1:SsaZ. The arrow indicates the position of Ssal protein (in the case of 8N and 15R the arrow designates the most basic isoform); to the immediate right is the Ssa2 protein. (A) wt; (B) 15s; (C) 1%; (D) 8Ncrudelysate; (E) 8N ATP-eluted fraction; (F) I IAcrudelysate; ( G )I IAATP-eluted fraction; (H) ISR crude lysate; (I) 15R ATP-eluted fraction.

fractions for this group. The 8N and 11A proteins represent the second class of mutant proteins. Both proteins showed a reduced ratio of Ssalp:Ssa2p in the 20 pv-ATP-eluted fractions relative to the crude lysate. This is most likely due to a defect in release, since elution with higher amounts of ATP (1 mM) increases the yield of these mutant proteins (data not shown). This suggests that these proteins have a defect in ATP hydrolysis, although purified protein is needed to address this question directly. The sole representative of the third class is the phosphorylated isoform of 15R,which was not recovered from the peptide columns. For technical reasons, it is not possible to determine unambiguously whether the phosphorylated isoform is enriched in the flowthrough (non-binding) or urea-eluted (binding com-

petent, but release defective) fractions. However, the small amount of 15R observed in the urea fraction by one-dimensional SDS-PAGE is insufficient to account for phosphorylated isoform, suggesting that the phosphorylated isoform of 15R has a defect in binding, not release.

DISCUSSION Examination of the effect of three alterations in a 12 amino acid highly conserved amino-terminal region of an hsp70 protein of S. cerevisiue, Ssa I p, demonstrates the essential nature of this region for function (summarized in Table 1). Substitutions of an asparagine for an aspartate at position 8, an alanine for a threonine at position 1 I , or an arginine for a

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C. M. NICOLET AND E. A. CRAIG

Table 1. Properties of wild-type (wt) and mutant HSP70 proteins Complementation ssalssa2 wt

8N 11A 15R

15s 15G

+

-

ssalssa2ssal

+

-

__

-

-

-

-

Interallelic Phosphorylated complementation isoform NA

15S, 15G

(+>* (+I*

-

8N 8N

-

+ +

-

-

ATP binding

+ + + +t + +

Peptide binding/release

+/+ +/(+IS

+A+)$ +ti+

+/+

+/+

*Complementationis not to wt levels. TPhosphorylated isoform does not bind. $Complete release requires more ATP than wt protein. NA, not applicable.

cysteine at position 15 results in a non-functional protein, as assayed by in vivo complementation. However, substitution of a serine or glycine at position 15 results in a partially functional protein that allows slow growth of ssalssa2ssa4 cells. The fact that certain substitutions for the cysteine at position 15 do not completely eliminate function is consistent with the observation that the ability of Ssa 1p to facilitate protein translocation across microsomal and mitochondria1 membranes is unaffected by NEM, which alkylates sulfhydryl groups of cysteine (Chirico et al., 1988). The mutant proteins were not able to complement the slow growth or ts phenotypes of the ssalssa2 double mutant. In fact, the slow growth of ssaZssa2 strains was exacerbated by the presence of the mutant SSAI proteins. The 8N protein in particular was inhibitory in an ssalssa2 background. Additionally, and unlike the other mutant proteins, the inhibition by 8N was seen in all strain backgrounds examined. One explanation for this dominant negative effect is that the hsp70s act in vivo as dimers (or higher order structures), and that the presence of the mutant monomers interferes with the ability to form functional multimers. This would be analogous to the situation with particular dominant mutations in the l a d gene, which result in the inability to form functional tetramers even when wt protein is present (reviewed in Miller, 1980). Further suggestiveevidence for in vivo multimer formation is afforded by the interallelic complementation results discussed below. Interallelic complementation We have observed efficient interallelic complementation in strains carrying the combination of

either 8N plus 15G or 8N plus 15s. Two types of explanations for this interallelic complementation are possible. Particular combinations of mutant alleles showing low or no activity individually could combine to produce a functional hsp70 activity. Alternatively, the amounts of slightly active protein could be increased to some threshold level by gene dosage, thereby allowing complementation. Gene dosage is an unlikely explanation. While there is a slightly higher level of SSAl proteins in the double transformants, the fact that only two out of 15 possible combinations work indicates a specificity in the interactions. In addition, strains transformed with two copies of 15G, 15s or 8N show no greater complementation ability than single transformed strains. What are the possible mechanisms that could allow interallelic complementation? Often interallelic complementation occurs when two mutations change amino acids in different domains of a protein; by acting together, the two mutant proteins can generate a functional activity. This may explain interallelic complementation which has been observed with another member of the hsp70 family, KAR2. In the case of KAR2, two widely separated mutations each showed the ability to complement in the presence of the original unmapped kar2-2 allele (Rose et al., 1989). However, in our case the mutations are very close together; it would be surprising if residues only seven amino acids apart were important components of two different domains. The results suggest the possibility that multimerization of hsp70 molecules is involved. hsc70 from mammalian cells is known to oligomerize in the absence of ATP or in the presence of nonhydrolysable analogues (Schlossman et al., 1984;

FUNCTIONAL ANALYSIS OF A CONSERVED AMINO-TERMINAL REGION

Heuser and Steer, 1989). Homomultimers of 8N, 15s and 15G may be unable to form, or form in an inactive conformation, whereas heteromultimers of 8N and 15s or 15G are more stable and/or more active. Perhaps in multimers the amino-terminal regions are adjacent, thus allowing cooperative interactions amongst the mutated regions. Biochemical properties of mutant SSAI proteins

Our data are consistent with the idea that this conserved amino-terminal domain is not critical for ATP binding. Except for the phosphorylated isoform of 15R, all of the mutant proteins were able to bind ATP, as shown by their retention on ATPagarose columns. The efficiency of binding of the mutant proteins from yeast lysates was similar to that of wt by a number of criteria. Indeed, a preliminary analysis of the wt, 8N, 1 lA, 15R and 15s proteins expressed in E. coli having a deletion of the HSP70-related dnaK gene has shown that all these proteins can be recovered in similar amounts using ATP-agarose chromatography (data not shown). Our analysis is consistent with the observation that amino acids 5-122 in mammalian hsp70 can be deleted without affecting ATP binding (Milarski and Morimoto, 1989). However, in our assay, the ability of the mutant proteins to bind to and be released from peptide columns was differentially affected. Similar levels of wt and the 15S, 1% and 15R basic isoform mutant proteins were quantitatively recovered from the peptide column by elution with 20 PM-ATP, as shown by the similar ratios of Ssalp:Ssa2p in the crude and eluted fractions. The experiments of Flynn et al. (1989) suggest that the release of hsp70 from peptide is most likely coupled to ATP hydrolysis. Therefore, the fact that these mutant and wt proteins were quantitatively released with low amounts of ATP (20 PM) suggests that the ATPase activity of the mutants substituted at position 15 is not drastically reduced. The behavior of both 8N isoforms and the 11A protein suggests that these proteins may be defective in ATP hydrolysis, since quantitative elution from the peptide column is only achieved with higher amounts of ATP. However, since we have only used one peptide in these experiments, we cannot rule out the possibility that the mutant proteins are affected in their ability to recognize other peptides. Recently, the three-dimensional structure of the amino-terminal 44 kDa fragment of bovine hsc70 was reported (Flaherty et al., 1990). Based on the

713

position of amino acids relative to bound ATP and the similarities in structure between hsc70 and hexokinase, it was proposed that either aspartic acid at position 10 or glutamine at position 175 serves as a catalytic proton acceptor anticipated to be involved in the ATPase reaction (Flaherty et al., 1990). Alterations in the proton acceptor would be expected to result in a decrease in ATPase activity, consistent with the characteristics of the 8N protein. Therefore, this aspartic acid remains a strong candidate for the proton acceptor. It is interesting that 8N and 1IA, the two mutant proteins analysed that inhibit the growth ofssalssa2 the most, required higher levels of ATP to effect quantitative release from the peptide column, suggesting a defect in ATP hydrolysis. A correlation between a defect in peptide release and a dominant negative effect would be consistent with predictions from current models of hsp70 action, whether hsp70 acts as a monomer or multimer. If the rate of ATPdriven cycles of protein binding and release by mutant hsp70 is reduced due to defective ATP hydrolysis, prolonged interactions with protein substrates would result in an inhibition of a variety of cellular processes. Phosphorylation of Ssalp

It is intriguing that mutations in two positions of the conserved sequence, 8N and 15R, result in the accumulation of a phosphorylated form. Though at different amino acid positions, both changes result in a relative increase in charge in this region. Although in the labelling conditions used in the experiments shown here we did not detect labelling of the other hsp70s, under other conditions we have observed phosphorylation of a small proportion of SSAI and SSA2 proteins (unpublished observations). It is possible that phosphorylation of the protein is a rapidly cycling modification, resulting in only a small percentage of the protein being modified at any one time. The 8N and 15R mutations may result in the inhibition of turnover of the phosphate moiety to the extent that a phosphorylated intermediate accumulates to much higher levels than is normally seen. It has been shown that the major hsp70 of Dictyostelium is phosphorylated under non-heat shock conditions, and that rapid turnover of the phosphate group occurs (Loomis et al., 1982).Phosphorylated forms of DnaK (Zylicz et al., 1983), Ssclp (unpublished observations) and grp78 (Hendershot et al., 1988) have also been observed in vivo. At least two explanations (which

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C. M. NICOLET AND E. A. CRAIG

are not mutally exclusive) of the mechanism of Cegielska, A. and Georgopoulos, C. (1989). Functional domains of the Escherichia colidnaK heat shock protein phosphorylation are possible. The phosphorylation as revealed by mutational analysis. J . Biol. chem 264, may represent a normal transient intermediate in 21122-21130. the ATPase reaction such as occurs in the P-type ATPases (reviewed in Pedersen and Carafoli, 1987). Chappell, T. G., Konforti, B. B., Schmid, S. L. and Rothman, J. E. (1987). The ATPase core of a clathrin Alternatively, the phosphorylation may occur uncoating protein. J. Biol. Chem. 262,746751. independently of the ATPase activity. In either case, Chappell, T. G., Welch, W. J., Schlossman, D. M., Palter, the phosphorylation may play a regulatory role, K. B., Schlesinger, M. J. and Rothman, J. E. (1986). modulating the interactions with substrates, either Uncoating ATPase is a member of the 70 kilodalton by regulating the ATPase activity required for profamily of stress proteins. Cell 45,3-13. tein release or the interaction with protein sub- Chirico, W. J., Waters, M. G. and Blobel, G. (1988). strates. Interestingly, the phosphorylation of grp78 70K heat shock related proteins stimulate protein translocation into microsomes. Nature 332,805-8 10. was negatively correlated with function-in contrast to the unphosphorylated form, the phosphory- Craig, E. A. (1985). The heat shock response. CRC Crit. Revs. in Biochem. 18,239-280. lated form was not found associated with other Craig, E. A., Ingolia, T. D. and Manseau, L. J. (1983). proteins in the ER (Hendershot et al., 1988). Expression of Drosophila heat-shock cognate genes In conclusion, the wide range of effects caused by during heat shock and development. Dev. Biol. 99, alterations in the amino-terminal region indicates 418426. that this highly conserved region plays an important E. A. and Jacobsen, K. (1984). Mutations of the role in hsp70 function. Although this region is not Craig, heat inducible 70 kilodalton genes of yeast confer directly involved in ATP binding, alterations result temperature sensitive growth. Cell 38,841-849. in changes in phosphorylation and peptide release. Craig, E. A., Kramer, J., Shilling, J., Werner-Washburne, The results reported here suggest that the aminoM., Holmes, S., Kosic-Smithers, J. and Nicolet, C. M. terminal region of hsp70 may be of importance in (1989). SSCI, an essential member of the yeast hsp70 the regulation of ATpase activity, and therefore in multigene family, encodes a mitochondrial protein. Mol. Cell. Biol. 9,3000-3008. the interaction with peptide substrates, perhaps, in part, by affecting the phosphorylation state of the Craik, C. S. (1985). Use of oligonucleotides for sitespecific mutagenesis. BioTechniques 3, 12-1 9. protein. By purifying the mutant proteins, we will be able to examine more precisely the activities of the Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A. and Schekman, R. (1988). A subfamily of mutant proteins, and thereby gain more insight into stress proteins facilitates translocation of secretory and the function(s) of the conserved amino terminus. ACKNOWLEDGEMENTS We thank Hay-Oak Park for technical advice on site-specific mutagenesis, Carol Gross, Peggy Farnham and John Nelson for critical comments on the manuscript, and members of the Craig laboratory for advice and discussion over the course of this work. This work was supported by Public Health Service grants R 0 1 G M 31 107 (to E.A.C.) and GM 12381 (to C.M.N.) from theNational Institutes of Health. REFERENCES Bardwell, J. C. A. and Craig, E. A. (1984). Major heat shock gene of Drosophila and the Escherichia coli heat inducible dnaKgene are homologous. Proc. Natl. Acad. Sci ( U S A ) 81,848-852. Beckmann, R. P., Mizzen, L. A. and Welch, W. J. (1990). Interaction of hsp70 with newly synthesized proteins: implications for protein folding and assembly. Science 248,850-854.

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Zylicz, M. and Georgopoulos, C. (1984). Purification and properties of the Escherchia coli dnaK replication protein. J. Biol. Chem. 259,8820-8825. Zylicz, M., LeBowitz, J. H., McMacken, R. and Georgopoulos, C. (1983).The dnaK gene of Escherichiu coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system. Proc. Natl. Acad. Sci. (USA) 80,6431435.