Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins.

The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase. The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins. In contrast, the middle third of SIS1 is not similar to dnaJ proteins. This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD. The SIS1 gene is essential. Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus. About one-fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase. We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1.

T HREE DNA binding proteins, GCN4, BASI, and BAS2, are required for transcriptional activation of the Saccharomyces cerevisiae HIS4 gene (Arndt et al., 1987;Tice-Baldwin et al., 1989). Strains containing deletions of GCN4, BAST, and BAS2 require histidine for growth due to the lack of sufficient transcription of the HIS4 gene. The trans-acting sitl through sit4 mutations were isolated by their ability to restore sufficient transcription atthe wild-type HIS4 initiation site so that a strain containing deletions of GCN4, BAST, and BAS2 couldgrow in the absence ofhistidine  . All of the sit mutations alter the transcription of many diverse genes in addition to HIS4 and result in a slow growth phenotype . The sitl alleles are mutations in the gene encoding the largest subunit of RNA polymerase H and the sit2 alleles are mutations in the gene encoding the second largest subunit of RNA polymerase II  .
The sit4 alleles are mutations in a gene encoding a predicted protein that is 56% identical to mammalian type 2A and 43 % identical to mammalian type 1 serine/threonine protein phosphatases  . Subsequentgenetic analysis has shown that the SIT4 protein phosphatase is required for cell-cycle progression : SIT4 functions in late GI for progression into S phase (Sutton et al., 1991) . To gain insight into the role of SIT4 in transcription and cell cycle progression, we isolated wild-type genes that in high copy number suppress the slow growth phenotype of strains containing transcriptional suppressor mutations in SIP . One of these genes, termed SISI, encodes a protein that is similar to bacterial dnaJ proteins. The SISI gene is essential. Strains limited for the SISI protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SISI protein is localized throughout the cell but is more concentrated at the nucleus . About one-fourth of the SISI protein is released from a nuclear fraction upon treatment with RNase . We also show that overexpression of YDJI, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SISI.
The dnaJ gene of Escherichia coli was initially identified as a host mutation that results in the inability to replicate bacteriophage X DNA (Georgopoulos and Herskowitz, 1971). Subsequently, dnaJ was shown to be a member of the heat shock family of genes (reviewed in Lindquist and Craig, 1988) . Strains containing mutations in dnaJ have many phenotypes in common with strains containing mutations in dnaK, another heat shock gene in E. coli that is similar to HSP70 genes in eukaryotic organisms . Mutations in either dnaJ or dnaK result in slow growth at temperatures above 30°C, the inability to grow at temperatures of 43°C and higher, and the inability to replicate X phage DNA at any temperature (Sell et al., 1990) . Renaturation of denatured A phage c1857 protein is slower in dnaJ and dnaK mutants (Gaitanaris et al., 1990) . At the nonpermissive temperature, strains containing temperature-sensitive mutations in either dnaJ or dnaK have reduced phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase (Itikawa et al., 1989) and are severely inhibited forboth RNA and DNA synthesis (Wada et al., 1982) . The inhibition of RNA synthesis may be due to induction of the stringent response since high levels of ppGpp accumulate at the nonpermissive temperature and the inhibition of RNA synthesis (but not DNA synthesis) is relieved by reIA mutations (Itikawa et al., 1986) .
The common defects of dnaJ and dnaK mutants and the finding that dnaJ can stimulate the ATPase activity of dnaK in vitro (Sell et al., 1990) suggest that dnaJ may function in the same biochemical pathway as dnaK. However, dnaJ also functions independently of dnaK. dnaJmutants have a more severe defect in proteolysis than dnaKmutants (Straus et al., 1988). Also, mutation of dnaJ, but not dnaK, causes defects in the synthesis of a-galactosidase and certain membrane proteins at the nonpermissive temperature (Ohki et al ., 1987). For a-galactosidase, the defect in protein synthesis was shown to result from lowered levels of lac mRNA .
Despite these known defects due to mutations in dnaJ, the specific functions that dnaJ provides for bacterial cell growth are not currently known . The role of dnaJ in Pl and X phage DNA replication is better understood from studies using in vitro replication systems . For in vitro Pl phage DNA replication, addition of dnaJ and dnaK stimulates DNA replication (Wickner, 1990). The dnaJ protein binds directly to the P1 encoded repA protein (Wickner, 1990). In a reaction that is dependent on both dnaJ and dnaK, the repA protein is altered so that it is more active for binding to the Pl replication origin (Wickner et al., 1991) . For X phage DNA replication (Zylicz et al., 1989 ;Liberek et al ., 1990), dimers of XO protein bind to the X origin of replication and locally unwind the DNA. The dnaB helicase, complexed with XP protein, binds to the XO/oriX complex (at least partially via the XO-XP interaction) . However, in this complex, the activity of dnaB is inhibited by AP protein. The dnal protein (which specifically interacts with dnaB) and the dnaK protein (which interacts with XO) are essential for dissociation of this complex (releasing XP) and for activation of the dnaB helicase . Therefore, the results from the in vitro phage DNA replication systems suggest that the mechanism by which E. coli dnaJ functions is by dissociation of specific protein/protein complexes. The sequence similarity of the yeast SISI protein to bacterial dnaJ proteins suggests that SISI may perform its cellular functions by a similar mode of action. In this report, we present the isolation and initial characterization of the yeast SISI gene and its protein product .

Yeast Strains
The yeast strains used in this study are shown in Table I . YPD, YNB, and SC media are as described  . The carbon source was 2 % glucose or 2 % galactose except as indicated .

Isolation ofSISI
Two sit4 strains (S/A225-23-5 = sit4-36 and S/A225-26-3 = sit4-37) were transformed with a high copy number library containing yeast genomic DNA inserts (Carlson and Botstein, 1982) . After 3 d at 30°C, the library plasmid was recovered from 84 fast growing sit4 colonies out of a total of 50,000 transformants. About 50 of these plasmids contained the wild-type S174 gene, but these plasmids were recovered from colonies that grew slightly better than the other fast growing colonies . In addition to SIT4, we obtained four different genes (SISI through SIS4) that dramatically increase the growth rate of either of the two starting sit4 strains. All four of these genes also increase the growth rate of strain S/A258 which contains the sit4-258 allele. Each of the four genes (SISI through SIS4) was independently isolated from at least three different fast growing transformants . Sequencing A 2-kb BstEII/BstEII restriction fragment containing the entire SISI gene was placed into pUC118 and sequenced completely on both strands as described (Tice-Baldwin et al ., 1989) . To demonstrate that the major open reading frame on this DNA fragment encodes the SISI protein, shifts in the reading frame were introduced at various positions by digestion with different restriction enzymes, treating with Klenow enzyme (Boehringer Mannheim Diagnostics, Inc., Houston, TX) or T4 DNA polymerase (New England Biolabs, Beverly, MA), ligating, and transforming into a sit4 strain The Journal of Cell Biology, Volume 114, 1991 to determine complementation . A frame shift either at nucleotide +209 (by cutting with Bg1II and treating with Klenow enzyme) or at nucleotide +682 (by cutting with KpnI and treating with T4 DNA polymerase) eliminates complementation .

Genetic Mapping ofSISI
The 2-kb BstEII/BstEII restriction fragment containing the SISI gene was cloned into the yeast URA3 integrating plasmid YIp5. This plasmid was digested with Xhol (cuts at nucleotide +205 of SISI) and transformed into strain W303. Integration was shown to occur at the SISI locus by Southern analysis . Meiotic mapping shows that the distance between SISI: : URA3 and pet8 is Q cM (180 parental ditypes, 0 nonparental ditypes, and 0 tetratypes in 180 tetrads) .
Preparation ofsisl-2 A 2-kb BstEII/BstEII restriction fragment containing the SISI gene was placed into the Xba I site of pUC118, regenerating the BstEII sites at each end . An oligonucleotide-directed deletion (Kunkel, 1985) removed DNA sequences encoding SIS1 amino acids 1-350 (of 352 total) and replaced them with a Bg1II site. Then, a 1 .8-kb BamHI/BamHI DNA fragment containing the HISS gene was placed into this BgIII site. The resulting plasmid was digested with BstEII and transformed into a W303 diploid, yielding strain CY406. Replacement of the SIS1 open reading frame with HISS at the SISI locus was confirmed by Southern analysis . This null allele of SISI is termed sisl-2 . Phenotypes identical to strains containing sisl-2 are obtained with strains containing a smaller deletion of SISI (termed sisl-1) . This smaller SISI deletion, which removes SISI amino acids 70-189, was prepared by replacing the sequences between the XbaI and Xhol sites with the HISS gene .

Epitope Tagging ofSISI
To epitope tag the SISI protein, an SpeI restriction site (5'-ACTAGT31 was inserted by oligonucleotide-directed mutagenesis (Kunkel, 1985) at positions corresponding to either the very amino terminus (between the codons for the first and second amino acids) or the very carboxyl terminus (between the codon for the last amino acid and the TAA stop codon) of SISI . A duplex oligonucleotide encoding the hemagglutinin epitope was then inserted into these SpeI sites . The sequence of SISI was changed from MVKET . . . to _MTSYPYDVPDYASSVKET . . . for the amino-terminal-tagged SISI protein . The sequence of SISI was changed from . . . DENF(so) to . . . DENF TSYPYDVPDYASS(LtoT) for the carboxyl-terminal-tagged SISI . Both epitope tagged SISI proteins were functional because expression of either one in cells containing a deletion of SISI resulted in a wild-type growth rate.

Conditional Expression ofSISI
A BamHI restriction site was created at position -13 of SIS1 (relative to the A of the SISI ATG) by inserting the self complementary oligonucleotide 5'-CTAGTCATTATTAGTTGGATCC AACTAATAA_TGA-3' (the BamHI site and SISI ATG are underlined) into the amino-tertninal Spel site of SISI that was created for insertion of the oligonucleotide encoding the epitope tag. The resulting plasmid was digested with BamHI and the fragment encoding SISI was inserted downstream of the pGAL promoter in YCp50. The resulting pGALSISI expression plasmid is pCB789. The SISI expressed from pCB789 has the sequence MSVKET . . . instead of MVKET . . . for wild-type SISI . On galactosemedium, strain CY719 (containing pCB789 and a chromosomal deletion of SISI) has a growth rate that is indistinguishable from that of an isogenic wild-type strain .

Immunoprecipitation ofSISI
Inununoprecipitation of SISI from extracts prepared from '5S-labeled cells grown on 3 % glucose YNB medium was performed as described in Sutton et al . (1991) except for the buffers used to prepare the extracts . For SISI, the cells were broken by vortexing for 1 min with glass beads in 0.3 ml of breaking buffer (100 mM Tris-HCI, pH 7.0 at 23°C, 200 mM NaCl, 1 mM EDTA, 5 % glycerol, 0.5 mM DTT, plus proteinase inhibitors). Then, 0.3 ml of RIPA buffer (50 mM Tris-HCI, pH 7.0 at 23°C, 200 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, plus proteinase inhibitors) was added and the cells were vortexed an additional 10 s . The liquid was removed from the glass beads and centrifuged for 10 min . The resulting 624 L, all strains in this series are isogenic to strain L3110 except as indicated . W, all strains in this series are isogenic to strain W303 MATa except as indicated . * Columbia University, New York, NY . # Camegie Mellon University, Pittsburgh, PA . § University of North Carolina, Chapel Hill, NC . supernatant was used for immunoprecipitation . The proteinase inhibitors used in the buffers are described in Sutton et al . (1991) .

Preparation ofMutations in SISI
Ollgonucleotide directed deletions in the SISI gene that removed 22 amino acid regions of SISI (see Fig. 3) were prepared by the method of Kunkel (1985) . Two separate deletion series were prepared : one series used the NH2 epitope tagged SISI and the other series used the COOH epitope tagged SISI as the starting gene . For each sisl mutation, the deletion junction and at least 100 bases on both sides were sequenced to confirm the desired mutation. The results from the NH2 epitope-tagged SISI series and the COOH epitope-tagged SISI series were identical except that strains containing NH2 epitope-tagged SISI-80 were inviable while strains containing COOH epitope-tagged SISI-80 were viable . To demonstrate that the inviability of NH2 epitope-tagged SISI-80 strains was not due to an unidentified mutation outside the region sequenced, a new NH2 epitope-tagged sisl-80 gene was prepared by subcloning a Xhol/Xba I restriction fragment containing the sisl-80 mutation from COOH epitope-tagged sisl-80 into the unmutagenized NH2 epitope-tagged SISI gene. A strain containing the new NH2 epitope-tagged sisl-80 mutation was also inviable.

Luke et al . Characterization of SISI
Immunofluorescence Microscopy Subcellular Fractionation 625 DAPI staining and immunological staining of formaldehyde fixed cells was carried out as described (Kilmartin and Adams, 1984) . For tubulin staining, the primary antibody was anti-tubulin antibody YOL/34 (Sera Lab Limited, Crawley Down, Sussex, England) and the secondary antibody was goat antirat IgG-FITC (Boehringer Mannheim Diagnostics, Inc.) . For SISI localization, the primary antibody was ascites of 12CA5 monoclonal antibody (Field et al., 1988) and the secondary antibody was goat anti-mouse IgG-FITC (Jackson Immunoresearch, Westgrove, PA) . For SISI localization, the cells were blocked with 1% BSA and 2% normal goat serum to reduce nonspecific background staining. Cells were viewed with a Zeiss Axiophot microscope using a 100x objective . Kodak Tri-X pan 400 film was used for photography.
Yeast nuclear fractions were prepared according to the method of Kalinich and Douglas (1989) . The yeast nuclear fractions were treated with DNase I (Worthington Biochemical Corp., Freehold, NJ), RNase A (Sigma Chemical Co., St . Louis, MO), NaCl, or Triton X-100 (Bio-Rad Laboratories, Richmond, CA) according to the method of Allen and Douglas (1989

Isolation ofSISI
Transcriptional suppressor mutations in the SIP protein phosphatase alter the transcription of many diverse genes and cause a slow growth defect (doubling times in YPD medium of ti 300 min compared to 90 min for an isogenic wild-type SIP strain) . To identify genes whose products : (a) regulate SIT4 activity ; (b) can partially substitute, directly or indirectly, for SIT4 ; or (c) are substrates of SIT4 (or act downstream of SIT4 in the SIT4 pathway), we isolated wildtype genes that, when present in high copy number, can suppress the slow growth phenotype of sit4 strains . From this screen (see Materials and Methods), we obtained four genes that, when present on a high copy number 2p. plasmid, increase the growth rate of sit4 strains . We term these four suppressor genes SISI through SIS4, for sit4 suppressor. The suppression of the sit4 growth defect is not specific to a particular sit4 allele because each of the SIS genes in high copy number can increase the growth rate of three different sit4 mutants . The suppression of the growth defect of the sit4-37 strain by high copy number SISI is shown in Fig . 1 A .
The SISI through SIS4 genes in high copy number are not able to suppress the temperature sensitive phenotype of sit4 strains . In addition, SISI through SIS4 in high copy number are not able to suppress the lethality (Sutton et al ., 1991) due to deletion of the SIP gene . Therefore, none of the SIS genes in high copy number can substitute for SIT4 . Suppression by The Journal of Cell Biology, Volume 114, 1991 SISI, SIS2, or SIS4 is specific for sit4 strains because none of these genes in high copy number can suppress the growth defect of sitl, sit2, or sit3 strains. In addition, the SIS genes in high copy number do no increase the growth rate of wildtype strains (data not shown) .
The suppression of the growth defect of sit4 strains is greater with high copy number SISI or SIS2 than with high copy number SIS3 or SIS4 (data not shown) . The SIS4 gene (renamed PPH2a) encodes a predicted protein that is 80% identical to mammalian type 2A protein phosphatases (Sutton et al ., 1991) . Therefore, overexpression of a type 2A protein phosphatase can partially compensate for one or more of the defects due to mutation of the SIP protein phosphatase. The analysis of SIS2 and SIS3 will be presented elsewhere . In this report, we present the initial characterization of the SISI gene .

Genetic Mapping of SIS1
The minimal region of the high copy number SISI plasmid necessary for suppression was determined by restriction mapping the yeast insert, followed by deletion analysis and/or subcloning and assaying for suppression of the growth defect of sit4 strains . Full suppression of sit4 strains for faster growth is obtained by using a 2 .  TCCCCGATGACCCTCAAAGGATGCTACTGGATTGGGATTTGTCGTCCGAATGTCATAAAGCCGTACGTTTT  GATGGCCTGCTGTTGCCAACAACTTTTTATCGTTGGTGATTTCTAAGCGATTAACCTGTGAGTCGGAATGC  TGAATTGTTCTTGAACAAACCCCAGTAAGAGCCTCCCAAAATCTTATTGTGTGGTCATAGCCAGCAGATAC  TAAGATAACAGACATCAATCAGAACTCCTTTTTTATATGGATGACAAGACTGCCACGTTGATCGCTGCGCT  AATAATATATGGTTGTCGCACTCTTCTATAACATGATCAGTAATAGCTAGTCGTTACTTTTCTGAATCTTA  ACGTATTGTGTTACCCTCTTTGGTTTATATGAACGTTCCAGAAACTTCTGGAAAAAGAATGGGATATCCTT  TTACGGGTTTTTCAACCACTGTCAGCACAAACTCCTTGCCAAGGTAGTTCGAGATTTAATATATATTGTAT  AGATTTCTCGTCCCTTTACCGTATTGTTAATCCCTTTTTAAACTTATGCGCTTTCTTGTCAAAAATTCTAA  TTAATATTATTTAAGCTCTTACACGAATTCTTTTAACGACTTAGAATTGTATAACCATCCAGAAACTCTTC  AGAGGATAAAGGATAAGTTGTTTGCATTTTAAGATTTTTTTTTTAATACATTCACATCAATATAATAGAGT  ATAGTATACAGAACTAATA ATG GTC AAG GAG ACA AAA CTT TAT GAT TTA CTT GGA

SISI Encodes a dnaJ Homologue
The DNA sequence ofthe SISI gene predicts a protein of352 amino acids with a molecular mass of 37,592 D (Fig. 2) . Searches of the databases reveal that SISI is similar (ti28 identical overall) to E. coli andM. tuberculosis dnaJ proteins (Fig. 3) . SISI is most similar to bacterial dnaJ proteins in the amino-terminal third of the proteins (50 % identity for SISI and E. coli dnaJ for the first 104 amino acids terial dnaJ proteins. The carboxyl-terminal third of SIS1 is also similar to dnaJ proteins. However, the middle third of SISI (residues 100-220) has very little similarity to the dnaJ proteins. The middle third of dnaJ proteins contains four cysteine-rich direct repeats whose common feature is Cxx CxGxG (indicated by arrows in Fig . 3). SISI does not contain these cysteine-rich repeats. In place of two of the cysteine-rich repeats, the SISI protein has a striking glycine/methionine rich sequence; GMGGMPGG MGGMHGGMG-GMPGG , which can be viewed either as a (GGMGGMP) . motif or as containing two direct repeats of the sequences GMGGMPGG (Fig. 3) . Within this region of SISI, the six methionines are positioned M .M_ and would lie along the same surface if this region formed an a helix . However, due to the high glycine content and the two proline residues, this region is predicted not to be a helical . The SISI glycine/methionine-rich region is reminiscent of the glycine/methionine-rich carboxyl terminus of the E. coli groEL protein and eukaryotic HSP60 proteins . The carboxyl terminus of the groEL protein is (GGM)4M (Hemmingsen et al ., 1988) while that of the S. cerevisiae HSP60 protein is (GGMP) ZGMP-GMM (Johnson et al., 1989) . The function of the carboxylterminal regions of these proteins is not known . SISI is also similar to dnaJ homologues identified in yeast . The SEC63 protein, which is required for import of proteins into the endoplasmic reticulum, contains a 70-amino acid region that is similar to the amino-terminal 70 amino acids of SISI or bacterial dnaJ proteins Sadler et al ., 1989 ; Fig . 3) . SEC63 has no similarity to dnaJ proteins outside this 70-amino acid region . SISI is also similar to the yeast YDJI protein, identified by Caplan and Douglas (1991) by using antibodies directed against matrix lamina pore complexes, and to the SCJI protein, identified by its ability, when overexpressed, to possibly cause alterations in protein sorting (Blumberg and Silver, 1991) . The The Journal of Cell Biology, Volume 114, 1991 Figure 3. SISI is similar to dnaJ proteins. The predicted SISI protein is compared to E. coli dnaJ (Bardwell et al ., 1986), M. tub. dnaJ (Lathigra et al ., 1988), the yeast YDJI protein (Caplan and Douglas, 1991), and a region of the SEC63 protein (Sadler et al ., 1989) . Only identical amino acids shared between SISI and the other proteins are boxed and shaded . The asterisk for the SEC63 sequence indicates an insertion of the amino acids SVMEETY The first amino acid shown for SEC63 is at position 120 of 663 total amino acids . The four arrows indicate the four cysteine rich repeats that are absent in SISI. The solid bars above the SISI sequence indicate the 22-amino acid region deleted for each altered SISI protein . Two attempts to prepare a SISI-83 mutation did not give the desired mutation . The dots under the SISI-82 bar indicate the regularly spaced methionines in this region of SISI.
amino-terminal third and carboxyl-terminal third of SISI are both similar to the corresponding regions of YDJI (Fig . 3) and SCJI. However, the middle third of SISI has very little similarity to the corresponding region of YDJI or SCJI . The central third region of both YDJI and SCJI has four cysteinerich repeats that are very similar to the four cysteine-rich repeats in the two bacterial dnaJ proteins .

SISI Is an Essential Gene
A deletion allele of SISI, termed sisl-2, was prepared that replaces DNA sequences encoding amino acids 1-350 of SISI (of 352 total amino acids) with a 1 .8-kb DNA fragment that contains the HIS3 gene (see Materials and Methods). When a sisl-2/SISI diploid strain (CY406) is sporulated and the tetrads dissected, each tetrad gives rise to two normalsized His SISI colonies and two very small colonies, Fig .  1 B. After 6 d of incubation at 23 or 30°C, each small colony contains -500 inviable cells, about one-half of which are very large in size. This inviability does not result from a defect acquired only during spore germination . When a sisl-2/SISI diploid containing the wild-type SISI gene on the URA3 centromere plasmid YCp50 is sporulated and the tetrads dissected, haploid sisl-2 (His') progeny containing the wild-type SIS1 gene on YCp50 are obtained . When this strain is grown on 5-fluoro-ortic acid medium, which al- Figure 4. Levels of SISI protein after conditional expression or heat shock . (A) An exponentially growing culture of strain CY719 in SC galactose medium was centrifuged, washed once in SC glucose medium, resuspended in SC glucose medium, and grown at 30°C . At various times cells were collected from a portion of the culture, frozen in dry ice/ethanol, and stored at -70°C. A parallel culture was also grown for 30 h on SC raffinose medium (indicated by R) . At all times, the ODD of the cultures was maintained between 0.1 and 0.5 by appropriate dilution. Extracts were prepared from the cells and 75 hg of total protein for each extract was loaded onto a SDS/10% polyacrylamide gel . The separated proteins were transferred to nitrocellulose membrane and probed with anti-SISI antiserum No. 252 which was raised against a trpE-SISI fusion protein containing amino acids 74-191 of SISI . Undiluted (a), 1 :10 dilution (b), and a 1 :100 dilution (c) of an extract prepared from cells grown on SC galactose medium (Gal) were loaded as standards . (B) Levels of SISI protein after heat shock. An exponentially growing culture of strain W303 in YPD medium at 23°C was divided into two portions and centrifuged . One of the cell pellets was resuspended in 23°C YPD medium and grown at 23°C. The other cell pellet was resuspended in 39°C YPD medium and grown at 39°C . These cells were harvested at 13 or 26 min after transfer to 39°C. Extracts were prepared and 40 ug of protein for each extract was loaded onto a SDS/10% polyacrylamide gel . The levels of SISI were determined by Western analysis using No . 252 anti-SIS1 antiserum . For both A and B, the asterisk indicates a nonspecific band that cross-reacts with the No. 252 antiserum and can be used as control for loading similar amounts of cell extract .
Therefore, S. cerevisiae cells grow, on the average, for about nine generations (^500 cells total) in the absence of the SISI gene . There are two possibilities for such a delayed lethality. One possibility is that the cells cannot divide at all in the absence of the SISI protein and that the cells take nine generations to dilute the SISI protein below some critical level, at which point the cells are inviable. The second possibility is that the cells run out of SISI very rapidly but can divide in the absence of SISI . Here, each division in the absence of sufficient SIS1 protein either results in some probability of cell death or can result in a specific cellular defect . If enough defects are accumulated for a given cell, the cell becomes inviable .
Two lines of evidence support the second model for the delayed lethality. First, pedigree analysis of haploid sis]-2 progeny resulting from a sisl-2/SISI diploid shows that not every sisl-2 cell goes through nine generations . Some individual cells are inviable after only three generations from the original sisl-2 spore while other cells (only a few lineages) are viable after 13 generations from the original sisl-2 spore. The second line of evidence comes from the analysis of strain CY719 that conditionally expresses SISI protein . Strain CY719 contains a deletion of SISI at the chromosomal SISI locus and a low copy number URA3/centromere plasmid containing the SISI coding sequences under control of the GALJ promoter. CY719 is viable when grown on galactose medium (GALL promoter induced, SISI expressed) but inviable on glucose medium (GALL promoter repressed, SISI not expressed) . When CY719 is transferred from galactose to glucose medium, the levels of SISI protein decrease ( After 8 h, the levels of SISI have decreased to about one-tenth the starting levels . At this time, the culture grows with a wild-type doubling time (75 min) and >95% of the cells are viable. After 22 h, the levels of SISI have decreased to NI/100 of the starting levels. At this time, the culture continues to grow (but with a doubling time of 180 min) and about 50% of the cells are viable. When strain CY719 is grown on raffinose medium for 30 h (raffinose gives low level induction of the GALL promoter), the cells have about the same amount of SISI as do cells after 8 h on glucose medium (Fig . 4 A) . In contrast to strain CY719 grown on glucose for 8 h where >95 % of the cells are viable, strain CY719 grown on raffinose for 30 h has <20% viable cells . Continued growth on raffinose medium results in complete loss of viability. These results suggest that yeast cells can grow and divide when the levels of SISI are below some critical level but that cell division under these conditions eventually results in inviability.

Phenotypic Defects Due to Limiting Levels ofSISI
When a sisl-2 strain expressing SISI under control of the GALL promoter (CY719) is transferred from galactose to glucose medium, the levels of SISI decrease over time (Fig .  4) . After 31 h in glucose medium, the culture has an overall doubling time of 200 min. At this time, many of the CY719 cells have become very large (50% of cells) and many of the cells appear blocked for migration of the nucleus from the mother cell into the daughter cell (30% of cells) (Fig. 5) . In some of the cells that appear blocked for nuclear migration, the nucleus partially migrates into the neck between the two cells . Much of the volume of most of the large cells is filled with a large structure that is seen as a rim just inside the cell The panels show either phase contrast, staining for a-tubulin by indirect immunofluorescence, or DAN staining of DNAby fluorescence. Exponentially growing cultures of strain CY719 (pGAL:SISI on YCp50) on SC-ura galactose medium or W303 (wild-type SISI) on SC galactose medium were centrifuged and resuspended in SC-ura or SC glucose media. The cells were collected after 31 h in glucose medium (maintaining the cultures between OD6N of 0.1 and 0.75 by dilution). The cells were fixed with formaldehyde and treated with zymolyase to remove the cell wall before incubation with the and-a-tubulin antibody. (B) Nomarski optics and DAPI straining of DNA for CY719 cells after 31 h in glucose medium as in A. The cells were collected and fixed in 70% ethanol before staining of DNA with DAPI . Bars, 10 pm . surface ( Fig . 5 B) (Nomarski) . This structure was shown to be a vacuole as determined by vital staining (Pringle et al ., 1989) for the red dye that accumulates in the vacuole in ade2 strains grown on limiting adenine (data not shown) . In most of the cells with a large vacuole, the nucleus (located by DAPI staining) appears pressed against the side of the cell (Fig . 5 B) . The phenotypic defects shown for strain CY719 grown on glucose medium for 31 h are also visible after 22 h on glucose medium but in a smaller percentage of the cells. These phenotypes are seldom seen for wild-type cells grown under identical conditions (Fig. 5) .

The Levels of SISI Increase About 7Wo-Fold After Heat Shock
The E. coli dnaJ protein is a typical heat shock protein : the rate of synthesis of E. coli dnaJ protein increases 10-fold af-Luke et al . Characterization of SISI Figure 6. SISI associates with p40 and p88. (A) The epitopetagged SISI in extracts (100 ul ; all 1.6 mg protein/ml) prepared from 35S-labeled cells was immunoprecipitated using the indicated amounts ofascites of 12CA5 monoclonal antibody. The strains used were : CY455 (wild-type SISI) for the None lanes, CY461 (COOH epitope-tagged SISI) for the COON lanes, CY661 (high copy number NHZ epitope-tagged SISI ) for the High copy # NHZ lanes, or CY457 (NHZ epitope-tagged SISI) for the NHZ lanes . Where indicated (+), the ascites was preincubated for 30 min with 10 gg of competing hemagglutinin epitope peptide (YPYDVPDYA) before addition to the extracts. The asterisks indicate SISI degradation products as determined by Western analysis. (B) Immunoprecipitation of SISI tagged at the amino-terminal end with one (lane 1, strain CY457) or two (lane 2, strain CY678) copies of the hemagglutinin epitope . (C) Longer exposure of a region of the gel shown in A. Western analysis of a parallel gel shows that the band labeled HSP70 reacts with antiserum raised using a peptide corresponding to a conserved amino-terminal region of yeast HSP70 proteins (gift from E. Craig) . The a bracket indicates two bands that increasein intensity when SISI is overexpressed .
ter a heat shock from 30 to 43°C (Bardwell et al ., 1986) . As determined by Western analysis, the overall level of the SISI protein increases about twofold after a heat shock from 23 to 39°C (Fig. 4 B). Under these conditions of heat shock, the overall levels of HSP70 proteins also increase about twofold as determined by probing the same Western blot with antiserum (gift from E. Craig) directed against a peptide representing a highly conserved amino-terminal region of yeast HSP70 proteins (data not shown) .
The finding that the level of the SISI protein increases upon heat shock is consistent with the presence of the sequence GAA CGTTCCAGAAAC_TTCTG GAA (the first G is at -344 relative to the A of the SISI ATG ; Fig . 2) in the SISI promoter. This sequence is similar to the heat shock consensus element, which is composed of 5 by sequences of xGAAx alternating with xTTCX (Slater and Craig, 1987;Amin et al ., 1988;Xiao and Lis, 1988

SISI Associates with p40 and p88
Immunoprecipitation of SIS1 was used to determine if SISI associates with other proteins . The SISI protein was tagged with a 9-amino acid epitope for which a high affinity monoclonal antibody is available (12CA5; Field et al., 1988) . The 12CA5 monoclonal antibody has low cross-reactivity to endogenous yeast proteins. To epitope tag the SISI protein, a DNA sequence encoding a 9-amino acid influenza hemagglutinin epitope was inserted into the SISI gene at a position corresponding to either the very amino terminus or the very carboxyl terminus of the protein (see Materials and Methods) . An isogenic set of three strains was prepared differing only in the source of the SISI protein : strain CY455 contains wild-type SISI without the epitope tag, strain CY457 contains only amino-terminal-tagged SISI, and strain CY461 contains only carboxyl-terminal-tagged SISI . The epitope tagged versions of SISI are functional since the strains containing only the epitope-tagged forms of SISI have phenotypes and doubling times indistinguishable from those of isogenic wild-type strains .
When the epitope-tagged SISI protein is immunoprecipitated from extracts prepared from "S-labeled cells, SISI specifically coinununoprecipitates with a protein of apparent The Journal of Cell Biology, Volume 114, 1991 Figure 7. p40/SISI elutes as a very large complex . 0 .5 ml of an extract (5 mg total protein/ ml ; in 50% RIPA/50% breaking buffer) prepared from 31Slabeled cells containing NHZtagged SISI (strain CY457) was loaded onto a 1 .0 x 40cm Sepharose CL-6B column equilibrated in 50 % RIPA/50 % breaking buffer. Immunoprecipitates of alternate fractions were loaded onto a SDS/10% polyacrylamide gel . For this figure the gel was overexposed to show that the SISI protein is present in the high molecular mass p40/SISI complex . The asterisks indicate SISI degradation products. The column standards were blue dextran (2,000K), thyroglobulin (660K), ferritin (440K), and bovine serum albumin (67K) . molecular mass of 40 kD, which we term p40 (Fig . 6 A) . The p40 protein is not a slower migrating form of SISI by two criteria . First, p40 is not detected by Western analysis of SISI immunoprecipitates using either of two antisera directed against different regions of SISI (data not shown) . These sera readily detect SISI in this analysis . The second criterion that p40 is nova form of SISI is that altering the mobility of SISI by addition of a second nine-amino acid hemagglutinin epitope does not change the apparent mobility of p40 (Fig . 6  B) . This experiment also shows that SISI does not coimmunoprecipitate with a protein of the same mobility as the single epitope-tagged SISI protein .
Overexpression of SISI increases the amount of SISI in the immunoprecipitates (Fig. 6 A) . In contrast, the amount of p40 in these immunoprecipitates does not increase. Therefore, there may be a limited amount of p40 that can associate with SISI . In addition, the amount of p40 present in the immunoprecipitates depends on the buffer used to prepare the extract. RIPA buffer contains detergents while breaking buffer does not (see Materials and Methods) . If the extracts are prepared using only breaking buffer, about one-third the amount of p40 is present in the SISI immunoprecipitates (data not shown) as compared to when the extracts are pre-pared using 50% RIPA/50% breaking buffer (Fig. 6) . The use of 100% RIPA buffer does not give any increase in the amounts ofp40 relativeto the amounts seen using 50 % RIPA/ 50% breaking buffer. In all cases, very similar amounts of SISI are present in the immunoprecipitates (only a very small fraction of the SISI protein is associated with p40; see next section) . Therefore, while the solubility of SISI is similar in native buffers compared to detergent containing buffers, the p40/SIS1 complex is much more soluble in buffers containing detergents .
SISI also associates with another protein .
Longer exposure of the gel shows that SISI specifically coimmunoprecipitates with a protein of apparent molecular mass of 88 kD (Fig . 6  C) . In addition, immunoprecipitation of SISI from a strain containing the amino-terminal epitope-tagged SISI gene on a high copy number vector causes two proteins, of apparent molecular masses of 78 and 85 kD, to increase in amounts in the immunoprecipitates (indicated by bracket labeled a in Fig. 6 C) . However, the specificity of the interaction of these proteins with SISI remains to be determined because the same proteins or proteins of the same mobility also precipi-Luke et al . Characterization of SISI Figure 8. Regions of SISI required for association with p40 and p88. (A) Extracts were prepared from 'SS-labeled cells that contained the indicated epitope-tagged SISI deletion or the wild-type SISI gene (No Epitope lane) on a LEU2/cen planmid . Each strain also contained wild-type SISI gene on YCp50. The strains used are listed in Table I . The epitope-tagged SISI derivatives were immunoprecipitaed from 100,u] of extract (all diluted to 1.6 mg protein/ml) using 0.8 'al of ascites of 12CÁ5 monoclonal antibody. The WT SISI arrow indicates the mobility of wild-type SISI that coimmunoprecipitates with the epitope-tagged SISI deletion derivatives. The levels of the p40 protein in the immunoprecipitates of epitope-tagged SISI-84, SISI-85, SISI-86, and SISI-87 are very similar to the levels in the immunoprecipitates of NHZ or COON epitopetagged full-length SISI (data not shown) . Therefore, the majority ofthe p40 protein in the epitope-tagged SISI-84, SISI-85, SISI-86, and SISI-87 immunoprecipitates is not present indirectly by association of p40 with the smaller amount ofwild-type SISI present in the immunoprecipitates . (B) Viability of strains containing the indicated epitope-tagged SIS1 deletion derivative as the only source of SISI. +, cells that have almost a wild-type growth rate and a T §+ phenotype, +/-, cells that have a slow growth phenotype and a Ts phenotype, and -, cells that are inviable. tate nonspecifically in the absence of SISI (first two lanes of Fig . 6 C). Neither p40, p88, nor the two proteins that increase in amounts in the immunoprecipitates when SISI is overexpressed are detected by antiserum (gift from E. Craig) directed against a peptide representing a highly conserved amino terminal region of yeast HSP70 proteins (data not shown) . However, the band labeled HSP70 in Fig . 6 C does react with the anti-HSP70 antiserum .

The p40/SISI Complex Has a Very Large Size
To determine the approximate size of native SISI and the p40/SISI complexes, the cellular extract was fractionated on a Sepharose CL-6B (Pharmacia Inc .) column before immunoprecipitation . The great majority of SISI elutes at -80 kD (Fig . 7) . Therefore, the majority of SISI (predicted monomer molecular mass of 37.6 kD) probably exists as dimers. The E. coli dnaJ protein is also believed to exist as dimers (Zylicz et al., 1985) . In contrast to the majority of SISI, the p40/SIS1 complex elutes at the exclusion limit of this column, which is N2-4 x 106 kD (Fig . 7) . We can not ex- Figure 9. Localization of SISI by indirect immunofluorescence . CY455 cells (wild-type SISI) or CY457 cells (NHZ epitope-tagged SISI) were treated with a 1:350 dilution of monoclonal antibody 12CA5 ascites (1.9 mg protein/ml) and then with goat anti-mouse IgG-FITC to visualize the epitope-tagged SISI and with DAPI to visualize the nucleus (Materials and Methods) . The arrow indicates a budded cell where no staining for SISI is apparent in the bud . clude the possibility that the apparent size of the p40/SISI complex results from its presence in micelles . The very high molecular mass p40/SISI complex seems to contain much more p40 than SISI . Therefore, this complex may contain many p40 molecules for each molecule of SISI .

Glycine-rich Regions
To determine the region of SISI that is required for association with p40, we prepared eight different SISI mutations (termed sisl-79 through sisl-87), each of which encodes an altered SISI protein that lacks a different 22-amino acid region of the protein (see Materials and Methods) . The 22amino acid region deleted in each altered SISI protein is shown in Fig . 3. To eliminate the possibility that the 9-amino acid epitope tag could alter the analysis, each of the 8 SISI The Journal of Cell Biology, Volume 114, 1991 deletions was prepared using the amino-terminal epitopetagged SISI or the carboxyl-terminal epitope-tagged SISI .
The genes encoding the epitope-tagged SISI derivatives on a low copy number LEU2/cen plasmid weretransformed into strain CY455. CY455 has a deletion of SISI at the chromosomal locus (sisl-2) and contains the wild-type SISI gene on a low copy number URA3/cen plasmid . Therefore, each of the resulting strains has both an epitope-tagged deletion derivative of SISI and the wild-type SISI protein without the epitope tag. Immunoprecipitation analysis shows that SISl-84, SISI-85, SISI-86, and SISI-87 retain their association with p40 (Fig . 8 A) . In contrast, SISI-81 (deleted in the region between the glycine-rich region and the glycine/methionine-rich region) and SISI-82 (deleted for the glycine/methionine-rich region) do not associate with p40. Although the amount of SISI-80 (deleted for the glycine-rich region) in the immunoprecipitates is reduced (probably due to instability of the altered SISI-80 protein), prolonged exposure of the gels show no detectable p40 in these immunoprecipitations. Both SISI-79 derivatives, which are deleted in the aminoterminal region that is highly conserved between dnaJ proteins and dnaJ homologues, are not detected in the immunoprecipitates and are probably unstable . Therefore, we are not able to determine if this highly conserved aminoterminal region of SISI is required for association with p40. Increased exposure of the same gels shows that SISI-84, SISI-85, SISI-86, and SISI-87 retain their association with p88 while SISI-81 and SISI-82 do not associate with p88 (data not shown) . We cannot determine if SIS179 and SISl-80 associate with p88 due to their much reduced levels in the immunoprecipitates . It is possible that the regions of SISI that are required for its association with p88 are the same regions of SISI that are required for its association with p40.
The immunoprecipitation analysis also directly shows that SISI is multimeric. The mobility ofeach of the SISI deletion derivatives is somewhat variable. In some of the lanes, SISI is present as two separate bands : one band results from the epitope-tagged SISI deletion derivative and the other band results from untagged wild-type SISI (position indicated by arrow, Fig 8 A) that coinnnunoprecipitates with the epitopetagged SIS1. These results show that the regions of SISI deleted in the SISI-84, SISI-85, and SISI-86 derivatives are not required for multimerization of SISI.

Analysis ofStrains Containing Deletion Derivatives ofSISI
We also tested each of the SISI deletion derivatives to see ifthey could substitute for wild-type SISI. Strains containing only SISI-79, SISI-84, or SISI-87 are not viable; strains containing only SISI-85 of SISI-86 grow very slowly and are temperature sensitive ; and strains containing only SISI-81 or SISI-82 have only a very slight growth defect (Fig. 8 B) . For SISI-80, the ability to substitute for SISI depends on the position of the epitope tag: a strain containing only NH2 epitope-tagged SISI-80 is not viable while a strain containing only COOH epitope-tagged SISI-80 has only a slight growth defect . Perhaps the 22-amino acid deletion in SISI-80 is close enough to the amino-terminal end so that, when combined with the insertion ofthe epitope at the amino-terminal end, it results in a nonfunctional protein .
Some of these SISI deletion derivatives cause alterations in plasmid stability. An isogenic set of strains was prepared that has either wild-type SISI or the SISI-81, SISI-82, SISI-85, or SISI-86 gene (all NH2 epitope tagged) integrated at the LEU2 locus. Each of these strains was transformed with the URA3/centromere plasmid YCp50. After growing the strains for 10 generations on medium containing uracil, the percentage of cells containing YCp50 was determined. At least six measurements were performed for each strain . For the strain containing wild-type SISI, 34% (standard deviation = SD of 7 %) of the cells had lost the plasmid . In contrast, the strain containing SISI-81 had a reproducibly higher rate of plasmid loss: 55 % (SD of 11%) of the cells had lost the plasmid . In contrast, the strain containing SISI-85 had a reproducibly lower rate of plasmid loss: 18% (SD of 6%) of the cells had lost the plasmid . The SISI-82 strain and the SISI-86 strain lost the plasmid at a rate similar to the strain containing wild-type SISI (31% with SD of 7 % and 29 with a SD of 7%, respectively) .

Localization of SISI by Immunofuorescence
To determine the subcellular localization of SISI by indirect immunofluorescence microscopy, we used three isogenic strains : strain CY455 contained only wild-type SISI, strain CY457 contained only NH2 epitope-tagged SISI, and strain CY461 contained only COOH epitope-tagged SISI. Immunofluorescence microscopy was performed using the monoclonal antibody 12CA5 which is directed against the epitope tag. Therefore, any difference in the staining between these strains will be due only to the epitope-tagged SISI proteins. In the absence of the epitope tag, wild-type yeast cells (strain CY455) show only very weak straining (Fig. 9). The staining for strain CY457 shows that the NH2-tagged SISI is localized throughout the cell but is more concentrated in the region of the nucleus, whose position is visualized by DAPI straining (Fig. 9). In almost every case, the staining for SISI is very weak or absent in the bud until it gets a nucleus transferred from the mother cell (see arrow in Fig. 9). Staining indistinguishable from that shown for NH2 epitope-tagged SISI containing cells is obtained with cells containing COOH epitope-tagged SISI protein (data not shown) .

Distribution of SISI in Subcellular Fractions
Since SISI is more concentrated in the region ofthe nucleus, we determined how SISI fractionated with nuclei using the method of Kalinich and Douglas (1989) . Yeast spheroplasts containing wild-type SISI were lysed in a hypotonic buffer (0.02 M Hepes, pH 7.4, 5 MM MgC12). The lysate (Cells lane in Fig . 10 A) was layered onto 20% /30 % /40% glycerol step gradients. These gradients separated the lysate into a pellet fraction containing membranes, nuclei, and unlysed cells (P in Fig . 10 A) and a soluble cytoplasmic fraction (S in Fig. 10 A). In this procedure, -90% of the SISI fractionates with the pellet . If higher ionic strength buffers are used to lyse the cells, more of the SISI fractionates with the supernatant (data not shown) . The extracts for the immunoprecipitation analysis shown in Figs. 6-8 were prepared using glass bead lysis and higher ionic strength buffers (0.2 M NaCl, 0.075 M Tris-HCI), which solubilizes >90% of the SISI .
The pellet from the glycerol gradient was subjected to two rounds ofdifferential centrifugation in 18% Ficoll to remove unlysed cells and to purify nuclei (Kalinich and Douglas, 1989) . The final nuclear pellet fraction contained almost exclusively nuclei (and associated membranes) as determined by DAN staining. Almost all of the SISI in the glycerol gradient pellet is recovered in this nuclear fraction (data not shown) . The yeast nuclear fraction was divided into aliquots which were treated separately with DNase plus RNase to digest chromatin and RNA, 1 M NaCl to remove salt soluble components, or 2% Triton X-100 to remove membranes (procedure of Allen and Douglas, 1989) . After each treatment, the residual nuclei (P or pellet) were separated from the soluble components (S or supernatant) by centrifugation. Interestingly, when the nuclear fraction was treated with DNase I and RNase A, about one-fourth of the SISI was released (Fig. 10 B). Separate treatment of the nuclear fraction with RNase A or DNase I shows that treatment with RNase A releases SISI while treatment with DNase I does not (Fig. 10 B). Subsequent probing of these blots with antiserum against YDJ1 (gift of A. Caplan and M. Douglas) shows that YDJI is not released by treatment with either Figure 10. Fractionation of yeast cells and localization of SISI . (A) Cells of strain BJ926 were fractionated as described in Kalinich and Douglas (1989) . 20 ml of lysed spheroplasts were layered on a glycerol step gradient and centrifuged . The resulting pellet was resuspended in 20 ml lysis buffer. Then 1 .2 ml of the original cell lysate (cells lane), 1 .2 nil of the gradient top layer (S lane), or 1 .2 nil of the resuspended pellet were TCA precipitated . (B) 0.45-ml aliquots of the nuclear fraction (in digestion buffer) were separately treated in a total volume of 4 nil with buffer (Control lanes), RNase + DNase, RNase, DNase, i M NaCl, or 2% Triton as described in Allen and Douglas (1989) . The samples were centrifuged and the pellets were resuspended in 4 ml of buffer. Then, 1 .2 ml of the supernatant fraction (of 4 nil) and 1 .2 nil of the pellet fraction (of 4 ml) was TCA precipitated . The TCA precipitates were separated on a SDS/10% polyacrylamide gel, transferred to nitrocellulose membrane, and probed with anti-SISI No. 252 antiserum . The sample for the control lanes shown in the figure was maintained at 37°C for 25 min as were the RNase + DNase, RNase, and DNase samples. The RNase buffer itself did not release SISI into the supernatant because a parallel sample treated with RNase buffer alone gave results identical to the Control lanes .
RNase A or DNase I (data not shown ; also see Caplan and Douglas, 1991) . Treatment of the nuclear fraction with 1M NaCl releases at least two-thirds of the SISI protein, confirming the ability of high salt to solubilize SISI . Treatment of the nuclear fraction with 2 % Triton X-100 releases about one-third of the SISI . When yeast nuclei are treated sequentially with RNase + DNase, NaCl, and then Triton X-100, the residual material is termed the "matrix lamina pore complex ." Less than 10% of SISI remains associated with this complex (data not shown) .

YDJI Can Not Substitutefor SISI
The SIR gene is essential for viability. To determine if overexpression of the yeast dnaJ homologue YDR could substitute for SISI, a sisl-2/SISI diploid containing the YDJI gene on a high copy number plasmid (pAV5 ; gift of A . Caplan and M . Douglas) was sporulated and 30 tetrads dissected . All of the sisl-2 haploid progeny gave colonies of very similar size, each containing -500 inviable cells (data not shown) . We expect -75 % of the sisl-2 haploid progeny to contain pAV5 because about 75 % of the SISI haploid progeny contained pAV5 (are Ura+) . Strains containing pAV5 overexpress YDR about fivefold compared to wild-type strains (Caplan, A ., personal communication) . Therefore, overexpression of YDJI cannot cure the lethality due to deletion of SISI .
We also tested if overexpression of YDR could partially cure the slow growth phenotype of a sisl-85 strain or a sisl-86 strain . When a sisl-85 or sisl-86 strain was transformed with the high copy number plasmid with no insert, the transformants grew at the same rate as the untransformed strains . In contrast, the sisl-85 strain or the sisl-86 strain transformed with the YDJ1 gene on a high copy number plasmid (pAV5) grew much slower than the untransformed strains (data not shown) . An isogenic wild-type SISI strain transformed with pAV5 did not grow slower than when transformed with the control plasmid . Therefore, not only can YDR not substitute for SISI, but overexpression of YDR interferes with the functions of the SISI-85 and SISI-86 proteins . This result contrasts with the suppression of YDJI mutants by high copy number SISI (see Caplan and Douglas, 1991

Discussion
In addition to SISI, three proteins with similarity to dnaJ have been identified in the yeast S. cerevisiae. The yeast SEC63 protein contains a 70-amino acid region similar to the amino-terminal region of dnaJ and is required for import of proteins into the endoplasmic reticulum Sadler et al ., 1989) . One possible model is that SEC63 functions to unfold proteins for transport across the ER membrane . The yeast YDJI and SCR proteins are similar to dnaJ over their entire lengths and, like dnaJ, contain four cysteine rich repeats . Therefore, YDJ1 and SCR may be true dnaJ homologues. The YDJI protein was isolated as a component of the matrix lamina pore complex (Caplan and Douglas, 1991) . The SCR gene encodes a protein that is localized in mitochondria and was identified as a high copy number suppressor in a screen to isolate genes whose products alter protein sorting (Blumberg and Silver, 1991) . Although it is possible that YDJI and SCH function for protein unfolding and/or protein import into various organelles, the functions of YDJI and SCJI are not known .
The yeast SISI protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins . However, the central third of SISI is not similar to any of the other known dnaJ proteins or homologues . This central third of SISI contains a striking glycine/methionine-rich sequence that, along with the more aminoterminal glycine-rich region and the sequences in between, is required for association of SISI with p40 (and probably p88) . Perhaps the corresponding region of bacterial dnaJ proteins and the yeast YDR and SCH proteins, all of which contain the cysteine-rich repeats that are absent in SISI, mediates the association of these proteins with other specific cellular components .
The functions of the SISI associated proteins p40 and p88 are not known . The p40/SISI complex elutes from a sizing column as a very large complex and may contain many p40 molecules for each SISI molecule. Since the p40/SISI complex is more soluble in buffers containing detergents, it may be membrane associated . The soluble SISI protein present in the cell extracts used for immunoprecipitation analysis is not detectably associated with yeast HSP70 proteins. Whether or not SISI associates with or functions in conjunction with yeast HSP70 proteins in vivo is not known. The E. coli dnaJ protein interacts with dnaK, a bacterial homologue of eukaryotic HSP70 proteins (Sell et al., 1990).
We currently do not know the specific function(s) of SISI. Many of the cells in strains limited for SISI appear blocked for nuclear migration and/or become very large in size and contain a large vacuole. However, we can not distinguish if these phenotypic defects result directly or indirectly from limiting SISI. The very large cells containing a large vacuole could result nonspecifically from cell death . The block in nuclear migration resulting from limiting the SISI protein is similar to that seen for cdc mutants (such as cdc2, cdc7, and cdc9 mutants) that are defective in DNA replication (Pringle and Hartwell, 1981) . Specific mutations in SISI cause alterations in plasmid stability, which could result from defects in plasmid replication or segregation . The alterations in plasmid stability resulting from the altered SISI proteins could be related to the blocked nuclear migration phenotype in cells limited for SIS1.
The SISI protein is localized throughout the cell but is more concentrated in the region of the nucleus. In addition, about a fourth of SISI (but none of the YDJI) is released from a nuclear fraction upon treatment with RNase . This finding may be relevant to the screen used to identify the SISI gene. High copy number SISI dramatically increases the growth rate of strains containing transcriptional suppressor sit4 mutations . These sit4 strains have alterations in the lengths and amounts of the mRNA for many diverse genes  . Therefore, the slow growth phenotype of sit4 strains may result from the limitation of one or more essential gene products whose transcription is altered due to the sit4 mutation. Such a limited gene product can not be SISI itself since isogenic wild-type and sit4 strains have very similar amounts of the SISI protein (Luke, M.M ., A. Sutton, and K . T. Arndt, unpublished data). It is possible that high copy number SISI increases the growth rate of sit4 strains by increasing the functional amount of a limiting mRNA . That about a fourth of the SISI protein is released from a nuclear fraction upon treatment with RNase could be due to the involvement of SISI in some aspect of mRNA transcription, processing, or transport. Whether or not SIT4 and SISI are both involved in some common step of mRNA transcription remains to be determined. Could SISI be a substrate of SIT4? A small minority (<10%) of the SISI protein is phosphorylated in vivo such that this form of SISI migrates slightly slower than the majority of SISI (seen by I'S labeling) . Although the overall in vivo phosphorylation state of SISI is very similar in wild-type strains as compared to sit4 strains (Luke, M . M., A. Sutton, and K. T. Arndt, unpublished data), we can not rule out the possibility that SIT4 dephosphorylate only one of many possible phosphorylated residues on the phosphorylated form of SISI.
SISI may have multiple functions required for cellular growth . Some insight into how SISI functions may be inferred from studies with E. coli dnaJ protein using the in vitro X phage DNA replication system. In this system, dnaJ seems to be mediating a specific protein/protein dissociation and interacts with the dnaB helicase (Zylicz et al., 1989;Liberek et al., 1990). Perhaps, like dnaJ, the SISI protein also mediates specific protein/protein dissociations . What-ever the mechanism of SISI action, the function of SISI is unique : SISI is an essential gene. Overexpression ofthe yeast dnaJ homologue YDJI (Caplan and Douglas, 1991) cannot substitute for SISI and actually inhibits the growth of strains containing sisl-85 or sisl-86 mutations . Further genetic and biochemical analysis will be required to determine the precise cellular functions of SISI.