..
Corrections
Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistr. In the article "A heat shock gene from Saccharomyces cerevisiae encoding a secretory glycoprotein" by Patrick Russo, Nisse Kalkkinen, Hannele Sareneva, Juha Paakkola, and Marja Makarow, which appeared in number 9, May 1, 1992, of Proc. Natl. Acad. Sci. USA (89, 3671-3675), the authors wish that the following correction be noted. The
8857
nucleotide sequence of the coding region of HSPS50 shown in Fig. 2 lacks A133, C134, and C189, which are included in the corrected Fig. 2 shown here. This changes amino acids 45-62 in the original figure to amino acids 45-63 in the corrected figure. Thus, subunit I of the HSP150 protein has 54 amino acids instead of 53.
-396 AGTGATCTTACTATTTCCTATTTCGGAAATTATTAAAGACAAAAAAGCTCATTAATGGCTTTCCGTCTGTAGTGATAAGTCGCCAACTCAGCCTAATTT -297
TTCATTTCTTTACCAGACAGtAAACTAATAGTACAAATGAGTG
-198
AMTAGAACACTAAAAGGAATGGAAGGTTAAATTATTAGCTTGAATGGTAGGAATCCTCGAGAAGGAACACTTMGTCTAACGACAAT
.................
TTTCTCAAGCGGAACACCACATTTTGAGCTAAATTTAGATTTTGGTCAAAATA
................
.....................
-99 CAATTTCGATTATGTCCTTCCTTTTACCTCAAAGCTCAAAATATCAATAAGAAACTCATATTCCTTTTCTAACCCTAGTACAATAATAATAATATA +1 ATG CAA TAC AAA AAG ACT TTG GTT GCC TCT GCT TTG GCC GCT ACT ACA TTG GCCVGCC TAT GCT CCA TCT GAG CCT 1 Met Gln Tyr Lys Lys Thr Leu Val Ala Ser Ala Leu Ala Ala Thr Thr Leu Ala Ala Tyr Ala Pro Ser Glu Pro +76 TGG TCC ACT TTG ACT CCA ACA GCC ACT TAC AGC GGT GGT GTT ACC GAC TAC GCT TCC ACC TTC GGT ATT GCC GTT 26 Trp Ser Thr Leu Thr Pro Thr Ala Thr Tyr Ser Gly Gly Val Thr Asp Tyr Ala Ser Thr Phe Gly Ile Ala Val +151 CAA CCA ATC TCC ACT ACA TCC AGC GCA TCA TCT GCA GCC ACC ACA GCC TCA TCT AAG GCC AAG AGA'GCT GCT TCC 51 Gln Pro Ile Ser Thr Thr Ser Ser Ala Ser Ser Ala Ala Thr Thr Ala Ser Ser Lye Ala Lys Arg2Ala Ala Ser +226 CAA ATT GGT GAT GGT CAA GTC CAA GCT GCT ACC ACT ACT GCT TCT GTC TCT ACC AAG AGT ACC GCT GCC GCC GTT 76 Gln Ile Gly Asp Gly Gln Val Gln Ala Ala Thr Thr Thr Ala Ser Val Ser Thr Lys Ser Thr Ala Ala Ala Val +301 TCT CAG ATC GGT GAT GGT CAA ATC CAA GCT ACT ACT AAG ACT ACC GCT GCT GCT GTC TCT CAA ATT GGT GAT GGT 101 Ser Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr Thr Lys3Thr Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly +376 CAA ATT CAA GCT ACC ACC AAG ACT ACC TCT GCT AAG ACT ACC GCC GCT GCC GTT TCT CAA ATC AGT GAT GGT CAA 126 Gln Ile Gln Ala Thr Thr Lys Thr Thr Ser Ala Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Ser Asp Gly Gln +451 ATC CAA GCT ACC ACC ACT ACT TTA GCC CCA AAG AGC ACC GCT GCT GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT 151 Ile Gln Ala Thr Thr Thr Thr Leu Ala Pro Lys Ser Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Val
+526 CAA GCT ACC ACC ACT ACT TTA GCC CCA AAG AGC ACC GCT GCT GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT CAA 176 Gln Ala Thr Thr Thr Thr Leu Ala Pro Lys Ser Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Val Gln +601 GCT ACT ACT AAG ACT ACC GCT GCT GCT GTC TTT CAA ATT GGT GAT GGT CAA GTT CTT GCT ACC ACC AAG ACT ACT 201 Ala Thr Thr Lys Thr Thr Ala Ala Ala Val Phe Gln Ile Gly Asp Gly Gln Val Leu Ala Thr Thr Lys Thr Thr
+676 CGT GCC GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT CAA GCT ACT ACC AAG ACT ACC GCT GCT GCT GTC TCT CAA 226 Arg Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Val Gln Ala Thr Thr Ly4Thr Thr Ala Ala Ala Val Ser Gln +751 ATC GGT GAT GGT CAA GTT CAA GCA ACT ACC AAA ACC ACT GCC GCA GCT GTT TCC CAA ATT ACT GAC GGT CAA GTT 251 Ile Gly Asp Gly Gln Val Gln Ala Thr Thr Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Thr Asp Gly Gln Val +826 CAA GCC ACT ACA AAA ACC ACT CAA GCA GCC AGC CAA GTA AGC GAT GGC CAA GTC CAA GCT ACT ACT GCT ACT TCC 276 Gln Ala Thr Thr Lys Thr Thr Gln Ala Ala Ser Gln Val Ser Asp Gly Gln Val Gln Ala Thr Thr Ala Thr Ser
+901 GCT TCT GCA GCC GCT ACC TCC ACT GAC CCA GTC GAT GCT GTC TCC TGT AAG ACT TCT GGT ACC TTA GAA ATG AAC 301 Ala Ser Ala Ala Ala Thr Ser Thr Asp Pro Val Asp Ala Val Ser Cys Lys Thr Ser Gly Thr Leu Glu Met Asn
+976 TTA AAG GGC GGT ATC TTA ACT GAC GGT AAG GGT AGA ATT GGT TCT ATT GTT GCT AAC AGA CAA TTC CAA TTT GAC 326 Leu Lys Gly Gly Ile Leu Thr Asp Gly Lys Gly Arg Ile Gly Ser Ile Val Ala Asn Arg Gln Phe Gln Phe Asp +1051 GGT CCA CCA CCA CAA GCT GGT GCC ATC TAC GCT GCT GGT TGG TCT ATA ACT CCA GAC GGT AAC TTG GCT ATT GGT 351 Gly Pro Pro Pro Gln Ala Gly Ala Ile Tyr Ala Ala Gly Trp Ser Ile Thr Pro Asp Gly Asn Leu Ala Ile Gly
6
+1126 GAC AAT GAT GTC TTC TAC CAA TGT TTG TCC GGT ACT TTC TAC AAC TTG TAC GAC GAA CAC ATT GGT AGT CAA TGT 375 Asp Asn Asp Val Phe Tyr Gln Cys Leu Ser Gly Thr Phe Tyr7Asn Leu Tyr Asp Glu His Ile Gly Ser Gln Cys +1201 ACT CCA GTC CAC TTG GAA GCT ATC GAT TTG ATA GAC TGT TAA GCAGAAAACTATTAGTTCTTrTATCCTGATGACTTTTTCTCAT 401 Thr Pro Val His Leu Glu Ala Ile Asp Leu Ile Asp Cys Stop
+1286 +1385 +1484 +1583
TTGCATTGATTAGAAAGGAAAAAAAGAAGTGTCCTTTTCTACTACTACTCTAGTCGCATCCATTCCTTTGCATTTATCTTTTCTGCGGTTGGCCAATCC ATTCTTCCGAGAATTTGGCTAGCCATACTTGATGTTTTCCCATTATTGGTTCGTTTGGCAATGCTAATTTTCTTAATTGCCCCTTATATACTCTTCCAT AAAATGTTTTTTTTATAACTAATTTTCTGTATATCATTATCTAATAATCTTATAAAATGTTAAAAAGACTTGGAAAGCAACGAGTGATCGTGACCACAT AATITGCCTCGCTACACGGCAAAAATAAGCCAGTCCTAATGTGTATATTAAAGGCTGCATGTGGCTACGTC
FIG. 2. Nucleotide sequence of HSP150 and predicted amino acid sequence of the HSP150 protein. Three putative regulatory elements are indicated in the flanking sequences of the coding region: the TATA element (box), heat shock element (HSE)-like sequences (dotted underline), and two transcription termination signals (double underline). Nucleotides matching the HSE consensus are indicated by asterisks. The open and full arrowheads show the signal sequence and the KEX2 protease-specific cleavage sites, respectively. The underlined and numbered amino acid sequences were confirmed by direct amino acid sequencing of tryptic peptides of mature secreted HSP150. The sequences of peptides 1 and 2 (dashed underline) were obtained by direct N-terminal sequence analysis and thus represent the N termini of subunits I and II.
Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 3671-3675, May 1992
Biochemistry
A heat shock gene from Saccharomyces cerevisiae encoding a secretory glycoprotein PATRICK Russo, NISSE KALKKINEN, HANNELE SARENEVA, JUHA PAAKKOLA, AND MARJA MAKAROW* Institute of Biotechnology, University of Helsinki, Valimotie 7, 00380 Helsinki, Finland
Communicated by Fred Sherman, December 31, 1991
thetic complete (SC), presporulation, and sporulation media were made as in ref. 3. Metabolic Labeling and Immunoprecipitation of HSP150. Cells were labeled for 1 hr in YPD medium with [35S]methionine (>1000 Ci/mmol; 1 Ci = 37 GBq) at 50-100 pCi/ml or [2-3H]mannose (11.5 Ci/mmol; Amersham), at 300 ,uCi/ml, washed, and lysed mechanically with glass beads. Lysates and media were immunoprecipitated, as described before for a-factor (4), with anti-HSP150 antiserum (1:100) raised in rabbits against purified HSP150. The antiserum specifically recognized mature and nonglycosylated HSP150, and the immunoprecipitations were quantitative. Direct Amino Acid Sequencing of HSP150. SF821-8A cells were incubated for 1 hr at 37°C; under these conditions there is a preferential secretion of HSP150. The HSP150 protein (subunits I and II) was purified from the growth medium by concentration on an ion exchanger, followed by dialysis, gel filtration, and anion exchange chromatography. The purified protein was cleaved with trypsin, -and the peptides were purified by reversed-phase chromatography. N-terminal amino acid sequencing was carried out on the native mature protein, and on subunits I and II after separation by reversedphase chromatography. Amino acid sequence analysis was performed with a gas-phase sequencer equipped with an on-line phenylthiohydantoin amino acid analyzer (5). Cloning and Sequencing. Peptide 5 (see Fig. 1) was used to synthesize a 32-mer oligonucleotide probe with inosines at certain ambiguous codon positions (6). The probe was radiolabeled with 32P by T4 polynucleotide kinase (7) and used to screen >300,000 plaques of a yeast cDNA library made in Agtll (7, 8). Putative clones were subcloned into Bluescript II vectors (Stratagene) and subjected to double-stranded DNA sequencing with the chain-termination method (9). A yeast genomic library (10) was screened with the HSP150 cDNA clone, which was radiolabeled with [a-32P]dCTP by the random primer technique (11). Two putative genomic clones were isolated, subcloned, and subjected to sequencing. Both strands of the entire HSP150 coding region and the flanking regions were sequenced. Northern Blot Analysis. Total RNA was isolated from SEY2101a cells, electrophoresed, and subjected to Northern blot analysis as described (12), except that hybridizations were for 12-18 hr at 42°C. The probe used for Northern (and Southern) blots was the same as used for screening the yeast
We report the finding of a secretory heat ABSTRACT shock protein, HSP150, of Saccharomyces cerevisiae, and the characterization of the gene coding for it. HSP150 is constitutively expressed, extensively 0-glycosylated, and secreted efficiently to the growth medium. When cells grown at 25°C were shifted to 37°C, a 7-fold increase in the level of HSP150 was observed within 1 hr. The HSP150 gene encodes a primary translation product of 412 amino acids. Direct amino acid sequencing of the mature secreted protein showed that an N-terminal sequence of 18 amino acids is removed, and a KEX2 protease-specific site is cleaved to yield two subunits of 53 and 341 amino acids, which remain noncovalently associated during secretion. The larger subunit is highly repetitive, containing 11 tandem repeats of a 19-amino acid sequence. Northern blot hybridization analysis showed a substantial increase in HSP150 mRNA level after heat shock. The upstream flanking region of the gene contains several heat shock element-like sequences. Disruption of HSP150 did not lead to inviability or significant effects on growth rate, mating, or thermotolerance. However, heat-regulated antigenic homologs of HSP150 were found in divergent yeasts such as Schizosaccharomyces pombe.
About 15 secretory glycoproteins have been identified from Saccharomyces cerevisiae. The expression of most of them is regulated by nutritional conditions or the mating type of the cells. Most of them remain intercalated in the cell wall, and only a few are secreted across the cell wall to the growth medium (1). No secretory protein has been reported to be regulated by heat stress. Heat-regulated proteins are found in all organisms and fall into two groups: the conserved stress proteins, which include the 70-kDa, 90-kDa, and low molecular weight heat shock proteins, and unclassified ones such as "household" enzymes and ubiquitin. All stress proteins characterized so far are cell-associated (2). In this report we characterize HSP150, a gene coding for a protein that is regulated by heat stress, 0-glycosylated, and secreted to the growth medium.t
MATERIALS AND METHODS Strains and Media. The S. cerevisiae strains SEY2101a (MATal ade2-101 ura3-52 leu2-3,112 suc2-A9 gal2), S288C (MA Ta), SF821-8A (MATa, sec7-1 his4-580 ura3-52 leu23,112 trpl-289), W303D (MATa/MATa ura3-1/ura3-1 his31J/his3-11 his3-15/his3-15 leu2-3/leu2-3 leu2-3, 112/leu23,112 trpl-1/trpl-J ade2-1/ade2-J canl-100/canl-100) and the Schizosaccharomyces pombe (leul-32, h-), Kluyveromyces marxianus (NCYC587), and Torulaspora delbrueckii strains were grown at 25°C in YPD medium containing 1% yeast extract (Oxoid, Basingstoke, U.K.), 2% bacto peptone (Difco), and 2% glucose (BDH), to midlogarithmic phase. Escherichia coli strain XL1-Blue was from Stratagene. Syn-
genomic library. Gene Disruption. The HSP150 chromosomal locus was disrupted by the one-step gene disruption method (13). The genomic HSPI50 clone, with -2-kilobase (kb) and 1-kb flanking sequences at the 5' and 3' ends, respectively, was subcloned into the Sal I site of the Bluescript II SK(±) vector (Stratagene), which had its Pst I site mutated. A Pst I fragment coding for amino acids 61-301 in the HSPJ50 gene was replaced by a HindIII fragment containing a yeast URA3
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
*To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M88698).
3671
3672
Biochemistry: Russo et al.
gene (supplied by Fred Sherman, University of Rochester). The Pst I and HindIII 5' cohesive ends were filled in by the combined 3'-to-5' exonuclease and 5'-to-3' polymerase activities of T4 DNA polymerase in the presence of deoxynucleoside triphosphates, to create blunt ends for ligation (14). The diploid yeast strain W303D was then transformed with this integrating plasmid, URA3' transformants were sporulated, asci were dissected (15), and URA3' spores were analyzed for the disruption of the HSP150 gene. Immunoblotting. Cells grown overnight were washed and resuspended in fresh YPD medium (2 x 108 cells in 400 ,l) and then incubated for 1 hr at either 250C or 370C. The cells were pelleted and lysed, and the medium was precipitated with 14% (wt/vol) trichloroacetic acid. Five percent of each cell lysate and growth-medium sample was subjected to SDS/PAGE, except in the case of Sch. pombe, where 10% of the sample volume was electrophoresed. The proteins were transferred to nitrocellulose filters, which then were probed with anti-HSP150 antiserum (1:1500) and developed by using alkaline phosphatase-conjugated anti-rabbit antibody according to the supplier's instructions (Amersham). Immunostaining was blocked when a 1:10 dilution of growth medium in which S. cerevisiae cells (5 x 108 per ml) had been incubated for 1 hr at 370C was added to the above antiserum preparation. Other Methods. To determine thermotolerance, cells that had been grown overnight at 250C were incubated for up to 20 min at 50°C, with or without a preincubation of 30 min at 37°C. Aliquots were plated on solid YPD media and scored for colony formation at 25°C. SDS/PAGE was done in 7.5-15% polyacrylamide Laemmli gels. Gels were processed for autoradiography and exposed to Kodak X-Omat AR film (Eastman Kodak). Radioactivity in gel slices was quantitated by scintillation counting after solubilization with NCS tissue solubilizer (Amersham). HF treatment was performed to release 0-glycans (16). Trichloroacetic acid (14% TCA) precipitation was for 30 min on ice. Tunicamycin (Sigma; preincubation for 30 min) was used at a concentration of 10
,ug/ml.
RESULTS AND DISCUSSION Heat Shock Increases the Level of a 150-kDa Secretory Glycoprotein. Cells (SEY2101a unless otherwise stated) were labeled with [35S]methionine at various temperatures, and the proteins of the growth media were analyzed in SDS/PAGE. After labeling at 25°C, a minor band of 150 kDa could be detected among other bands. When the labeling was performed at 37°C, the intensity of the 150-kDa band was much higher than at 25°C. Quantitations showed that there was 7 times more radioactivity in the 150-kDa band at 37°C (n = 6; range 5.5-9.3), 6.3 times more at 39°C, and very little at 41°C as compared with 25°C (Fig. 1A). Similar results were obtained with an a mating-type strain, S288C. The 150-kDa protein was designated HSP150. The protein was determined to be a glycosylated secretory protein, since it could be labeled with [3H]mannose. However, tunicamycin had no effect on its [3H]mannosylation or electrophoretic migration (data not shown), suggesting that the protein was not N-glycosylated. Instead, HF treatment, which removes 0-glycans, reduced its apparent molecular mass to 47 kDa (Fig. 1B). Immunoprecipitations of [35S]methionine-labeled growth medium and cell lysate with anti-HSP150 antiserum confirmed that heat stress increased the level of HSP150, and further showed that >90% of the newly synthesized HSP150 was secreted to the medium (Fig. 1C). Thus, HSP150 appears to be an extensively 0-glycosylated protein that is secreted efficiently to the growth medium and regulated by heat stress. To confirm this, we cloned and characterized the HSPI50 gene.
Proc. Natl. Acad. Sci. USA 89 (1992) B
A a
L
0
200-
200
to
b
a
Is
4
92.5-
92.5-
69-
69-
_
3
_ 46-
4630-
c a
b
c
d
e
t
i
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92.5-
69-
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m m
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FIG. 1. Heat stress increases the level of a 150-kDa secretory glycoprotein. (A) Cells were labeled for 1 hr with [35S]methionine at 250C (lane a), 370C (lane b), 390C (lane c), or 41'C (lane d). Proteins were precipitated from the growth media with trichloroacetic acid and analyzed by SDS/PAGE followed by autoradiography. Molecular size markers (kDa) are at left. The arrowhead indicates HSP150. (B) [35S]Methionine-containing HSP150 before (lane a) or after (lane b) HF treatment to remove O-glycans. (C) Cells were labeled as in A at 370C (lanes a-d) or 250C (lanes e-h), and the cell lysates (1) and growth media (m) were subjected to immunoprecipitation with anti-HSP150 antiserum (lanes a, c, e, and g) or preimmune serum (lanes b, d, f, and h).
Cloning and Primary Sequence Analysis. A S. cerevisiae cDNA library made in Agtll (7) was screened with an oligonucleotide probe, which was synthesized according to the sequence of a peptide obtained from trypsin-digested purified HSP150 protein. Six putative clones were isolated and found to have the same restriction endonuclease digestion fragments. Two of them were sequenced, and both clones had an open reading frame of 1236 nucleotides that included the amino acid sequence of the peptide used for design of the oligonucleotide probe, as well as the sequences of six other peptides determined from the purified protein (Fig. 2). The HSPI50 gene encodes a primary translation product of 412 amino acids with a calculated molecular mass of 41,633 Da (Fig. 2). The polypeptide starts with a putative signal sequence of 18 amino acids. It has the following features common to yeast signal sequences: two lysine residues in the N-terminal region, a hydrophobic core, and small uncharged amino acids at positions -1 and -3 relative to the cleavage site (17). Direct N-terminal sequence analysis of the native mature protein harvested from the growth medium gave a double sequence corresponding to peptides 1 and 2, in approximately equimolar ratio. Reversed-phase chromatography resolved the native protein into two components, subunits I and II, which in N-terminal sequence analysis gave single sequences
Biochemistry: Russo et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
3673
-396 AGTGATCTTACTATTTCCTATTTCGGAAATTATTAAAGACAAAAAAGCTCATTAATGGCTTTCCGTCTGTAGTGATAAGTCGCCAACTCAGCCTAATTT
**
***
*
-297 TTCATTTCTTTACCAGATCAGGAAAACTAATAGTACAAATGAGTGTTTTCTCAAGCGGAACACCACATTTTGAGCTAAATTTAGATTTTGGTCAAAATA -198
***
**
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***
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AGAAAGATCCTAAAAAAGGAATGGTTGGTGAAAAATTTATTAGCTTGAATGGTAGGAATCCTCGAG. AT... ...............
.
................
***
*
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GGAACACTTGAAGTCTAACGACAAT .................... .......
-99 CAATTTCGATTATGTCCTTCCTTTTACCTCAAAGCTCAAAAAAATATCAATAAGAAACTCATATTCCT rTCTAACCCTAGTACAATAATAATAATATA +1 ATG CAA TAC AAA AAG ACT TTG GTT GCC TCT GCT TTG GCC GCT ACT ACA TTG GCCVGCC TAT GCT CCA TCT GAG CCT 1 Met Gln Tyr Lys Lys Thr Leu Val Ala Ser Ala Leu Ala Ala Thr Thr Leu Ala Ala Tyr Ala Pro Ser Glu Pro +76 TGG TCC ACT TTG ACT CCA ACA GCC ACT TAC AGC GGT GGT GTT ACC GAC TAC GCT TCC CTT CGG TAT TGC CGT TCA 26 Trp Ser Thr Leu Thr Pro Thr Ala Thr Tyr Ser Gly Gly Val Thr Asp Tyr Ala Ser Leu Arg Tyr Cys Arg Ser +151 ACC AAT CTC CAC TAC ATC CAG CGC ATC ATC TGC AGC ACC ACA GCC TCA TCT AAG GCC AAG AGATGCT GCT TCC CAA 51 Thr Asn Leu His Tyr Ile Gln Arg Ile Ile Cys Ser Thr Thr Ala Ser Ser Lys Ala Lys Arg Ala Ala Ser Gln
+226 ATT GGT GAT GGT CAA GTC CAA GCT GCT ACC ACT ACT GCT TCT GTC TCT ACC AAG AGT ACC GCT GCC GCC GTT TCT 76 Ile Gly Asp Gly Gln Val Gln Ala Ala Thr Thr Thr Ala Ser Val Ser Thr Lys Ser Thr Ala Ala Ala Val Ser +301 CAG ATC GGT GAT GGT CAA ATC CAA GCT ACT ACT AAG ACT ACC GCT GCT GCT GTC TCT CAA ATT GGT GAT GGT CAA 101 Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr Thr Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln +376 ATT CAA GCT ACC ACC AAG ACT ACC TCT GCT AAG ACT ACC GCC GCT GCC GTT TCT CAA ATC AGT GAT GGT CAA ATC 126 Ile Gln hla Thr Thr Lys Thr Thr Ser Ala Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Ser Asp Gly Gin Ile +451 CAA GCT ACC ACC ACT ACT TTA GCC CCA AAG AGC ACC GCT GCT GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT CAA 151 Gln Ala Thr Thr Thr Thr Leu Ala Pro Lys Ser Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Val Gin +526 GCT ACC ACC ACT ACT TTA GCC CCA AAG AGC ACC GCT GCT GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT CAA GCT 176 Ala Thr Thr Thr Thr Leu Ala Pro Lys Ser Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Val Gln Ala
+601 ACT ACT AAG ACT ACC GCT GCT GCT GTC TTT CAA ATT GGT GAT GGT CAA GTT CTT GCT ACC ACC AAG ACT ACT CGT 201 Thr Thr Lys Thr Thr Ala Ala Ala Val Phe Gln Ile Gly Asp Gly Gln Val Leu Ala Thr Thr Lys Thr Thr Arg +676 GCC GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT CAA GCT ACT ACC AAG ACT ACC GCT GCT GCT GTC TCT CAA ATC 226 Ala Ala Val Ser Gln Ile Gly Asp Gly Gin Val Gln Ala Thr Thr Lys Thr Thr Ala Ala Ala Val Ser Gin Ile
+751 GGT GAT GGT CAA GTT CAA GCA ACT ACC AAA ACC ACT GCC GCA GCT GTT TCC CAA ATT ACT GAC GGT CAA GTT CAA 251 Gly Asp Gly Gln Val Gln Ala Thr Thr Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Thr Asp Gly Gln Val Gln +826 GCC ACT ACA AAA ACC ACT CAA GCA GCC AGC CAA GTA AGC GAT GGC CAA GTC CAA GCT ACT ACT GCT ACT TCC GCT 276 Ala Thr Thr Lys Thr Thr Gln Ala Ala Ser Gln Val Ser Asp Gly Gln Val Gln Ala Thr Thr Ala Thr Ser Ala +901 TCT GCA GCC GCT ACC TCC ACT GAC CCA GTC GAT GCT GTC TCC TGT AAG ACT TCT GGT ACC TTA GAA ATG AAC TTA 301 Ser Ala Ala Ala Thr Ser Thr Asp Pro Val Asp Ala Val Ser Cys Lys Thr Ser Gly Thr Leu Glu Met Asn Leu +976 AAG GGC GGT ATC TTA ACT GAC GGT AAG GGT AGA ATT GGT TCT ATT GTT GCT AAC AGA CAA TTC CAA TTT GAC GGT 326 Lys Gly Gly Ile Leu Thr Asp Gly Lys Gly Arg Ile Gly Ser Ile Val Ala Asn Arg Gln Phe Gln Phe Asp Gly
+1051 CCA CCA CCA CAA GCT GGT GCC ATC TAC GCT GCT GGT TGG TCT ATA ACT CCA GAC GGT AAC TTG GCT ATT GGT GAC 351 Pro Pro Pro Gln Ala Gly Ala Ile Tyr Ala Ala Gly Trp Ser Ile Thr Pro Asp Gly Asn Leu Ala Ile Gly Asp
+1126 AAT GAT GTC TTC TAC CAA TGT TTG TCC GGT ACT TTC TAC AAC TTG TAC GAC GAA CAC ATT GGT AGT CAA TGT ACT 375 Asn Asp Val Phe Tyr Gln Cys Leu Ser Gly Thr Phe Tyr Asn Leu Tyr Asp Glu His Ile Gly Ser Gln Cys Thr
+1201 CCA GTC CAC TTG GAA GCT ATC GAT TTG ATA GAC TGT TAA GCAGAAAACTATTAGTTCTTTTATCCTGATGACTTTTTCTCATTTGC 401 Pro Val His Leu Glu Ala Ile Asp Leu Ile Asp Cys Stop +1287 ATTGATTAGAAAGGAAAAAAAGAAGTGTCCTTTTCTACTACTACTCTAGTCGCATCCATTCCTTTGCATTTATCTTTTCTGCGGTTGGCCAATCCATTC +1386 TTCCGAGAATTTGGCTAGCCATACTTGATGTTTTCCCATTATTGGTTCGTTTGGCAATGCTAATTTTCTTAATTGCCCCTTATATACTCTTCCATAAAA
+1485 TGTTTTTTTTATAACTAATTTTCTGTATATCATTATCTAATAATCTTATAAAATGTTAAAAAGACTTGGAAAGCAACGAGTGATCGTGACCACATAATT +1584
GCCTCGCTACACGGCAAAAATAAGCCAGTCCTAATGTGTATATTAAAGGCTGCATGTGGCTACGTC
FIG. 2. Nucleotide sequence of HSP150 and predicted amino acid sequence of the HSP150 protein. Three putative regulatory elements are indicated in the flanking sequences of the coding region: the TATA element (box), heat shock element (HSE)-like sequences (dotted underline), and two transcription termination signals (double underline). Nucleotides matching the HSE consensus are indicated by asterisks. The open and full arrowheads show the signal sequence and the KEX2 protease-specific cleavage sites, respectively. The underlined and numbered amino acid sequences were confirmed by direct amino acid sequencing of tryptic peptides of mature secreted HSP150. The sequences of peptides 1 and 2 (dashed underline) were obtained by direct N-terminal sequence analysis and thus represent the N termini of subunits I and II.
starting at amino acids 19 (peptide 1) and 72 (peptide 2), respectively (Fig. 2). This suggests that the signal sequence was cleaved and that a second cleavage took place directly after Lys70-Arg71, a putative KEX2 protease-recognition site, to yield subunits I and II, which remained noncovalently associated in the mature secreted protein. Whether the C terminus of subunit II remained intact was not studied. Thus, the 150-kDa band in SDS/PAGE (Fig. 1) represents glycosylated subunit II, whose apparent molecular mass is highly exaggerated, since the calculated molecular mass of the apoprotein is -34 kD. Subunits I and II lack potential N-glycosylation sites (Asn-Xaa-Ser/Thr), whereas 25% of their amino acids are serines or threonines, which are potential O-glycosylation sites. The cleavage of subunits I and II may have been carried out by the serine protease KEX2, which cleaves the precursors of the mating pheromone a-factor and the M1 killer toxin at pairs of basic amino acids (18, 19). The KEX2 protease is thought to be located in the late Golgi compartment (20). In the case of a-factor and killer toxin, the released effector
molecule is secreted to the growth medium, whereas the rest of the precursor remains cell-associated. The N-terminal portion of subunit II, 227 out of 341 amino acids, is highly repetitive. It consists of 11 tandem repeats of a 19-amino acid sequence (Fig. 3). Some of the repeats are separated by 5 or 7 amino acids. Although the nucleotide sequences coding for the repeats vary, the amino acid sequences are conserved (Fig. 3B). Regulation of the HSP150 Gene. Sequence analysis of the genomic clones revealed the same open reading frame found in the cDNA clones with no introns, plus putative regulatory signals in the sequences flanking the coding region (Fig. 2). The flanking region downstream of HSP150 contains two putative yeast mRNA 3' end-forming signals, TATATA (21) and T-ll--lTT-TTATA (22), starting at nucleotides + 1466 and +1487, respectively. The region upstream from the translation initiation codon revealed a putative TATA box (TATAA) starting at position -131. The 396 nucleotides 5' to the translation initiation codon contain four nucleotide sequences that resemble the consensus heat shock element,
Biochemistry: Russo et A
3674
Proc. Natl. Acad. Sci. USA 89 (1992)
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262TAAAVSQITDGQVQATTKT'80
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281T6AA.SQVSDGQVQATTAT298 FIG. 3. (A) Primary translation product of the HSP150 gene. The putative signal sequence (Ss) is removed by a cleavage between Ala18 and Ala'9. Subunit I is cleaved directly after a KEX2 proteasespecific site (Lys70-Arg71), between Arg7l and Ala72. Subunit II contains 11 repeats of a 19-amino acid peptide (hatched boxes). The numbers 18, 53, and 341 denote the number of amino acids in the signal sequence and the subunits. (B) Amino acid sequence of the repetitive area in subunit II. Dots above the amino acids indicate nonconservative changes, and dashes denote gaps. The sequences between the repeats are underlined.
three or more contiguous repeats of the element NGAAN in alternating orientations (23, 24). Northern analysis of total RNA revealed a constitutively expressed HSP150 transcript of -1.6 kb that which showed a substantial increase in steady-state level after heat shock (Fig. 4). This, along with the presence of heat shock elements, suggests that heat regulation of HSP150 occurs at the transcriptional level. Disruption of the HSPISO Gene. A single-step gene disruption experiment (13) was carried out to assess whether HSP150 is essential. The genomic copy of HSP150 was disrupted with a URA3 gene (Fig. 5A), and the hspJS0:: URA3 construction was transformed into the diploid strain W303D. Integrants were isolated on SC plates lacking uracil. Four URA3' integrants were sporulated and tetrad analysis was performed on 20 asci from each integrant. Greater than 90% spore viability for each integrant suggests that HSP150 is not essential. The disruption of HSP150 was confirmed by Southern and Northern analyses (data not shown), and immuno-
37° 25(1
25 S 18 S
_
FIG. 4. Northern blot analysis of HSPl50 mRNA levels under normal and heat shock conditions. Cells were grown to midlogarithmic phase, and then the culture was divided in two and incubated for 1 hr at 37°C or 25°C. Total RNA was isolated and subjected to Northern blot analysis. The 32P-labeled HSPl50 cDNA clone was used as the probe. The positions of the 25S and 18S ribosomal RNAs
are
indicated at left.
m
FIG. 5. Disruption of the HSPJ50 gene. (A) The nucleotides coding for amino acids 61-302 in the genomic copy of HSPI50 were replaced by a URA3 gene. (B) A HSP150 strain (lanes a and b) and a hsplSO::URA3 strain (lanes c and d) were labeled with [35S]methionine for 1 hr at 37TC, and the cell lysates (1; lanes a and c) and growth media (m; lanes b and d) were subjected to immunoprecipitation with anti-HSP150 antiserum. The precipitates were subjected to SDS/PAGE analysis. Molecular size markers (kDa) are at right.
precipitation experiments demonstrated the lack of HSP150 protein in both cell lysates and media (Fig. 5B). The disruption of HSP150 gene had no effect on growth at 25°C or 37°C, mating, and thermotolerance (data not shown). In comparison, two members of the 70-kDa heat shock gene family, SSCI and KAR2, have been found to be essential (25-27). Conservation of the HSP150 protein. Computer-aided searches of nucleic acid and protein files in the EMBL data base showed no significant homologies between HSP150 and any known genes or proteins, thus providing no hypothesis for the function of the HSP150 protein. Thus, we studied whether HSP150 is conserved, by searching for antigenic homologs in other yeasts. T. delbrueckii, K. marxianus and Sch. pombe cells were incubated at 25°C and 37°C, and the growth media and cell lysates were subjected to immunoblot analysis using anti-HSP150 antiserum. Antigenic homologs were discovered in all three yeasts. T. delbueckii (Fig. 6A) and K. marxianus (Fig. 6B) synthesized secretory homologs with apparent molecular masses of 54 kDa and 54-150 kDa, respectively. In both cases, similar homologs were found in the cell lysates, together with putative precursors of smaller molecular size. The heterogeneous electrophoretic migration of the homolog of K. marxianus may be due to heterogeneous N-glycosylation. A homolog of 120 kDa was found in Sch. pombe; however, it was not secreted but remained cellassociated (Fig. 6C). Excess HSP150 protein of S. cerevisiae inhibited the immunostaining of the Sch. pombe protein. The levels of the antigenic homologs of each organism were increased by heat shock. Several heat shock proteins have previously been found to serve important physiological functions as chaperonins, which are involved in the maintenance or change of the conformation of other proteins (28, 29). Most stress proteins reside in the cytoplasm, nucleus, or mitochondria (2), and some are known to be segregated to the secretory pathway; however, they are not secreted. The BiP protein and grp94 are resident proteins of the endoplasmic reticulum (30, 31) and a 118-kDa glycoprotein of yeast has an unknown intra-
Biochemistry: Russo et al.
A 1
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Proc. Natl. Acad. Sci. USA 89 (1992)
2
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FIG. 6. Antigenic homologs of HSP150 in other yeasts. T. delbrueckii (A), K. marxianus (B), and Sch. pombe (C) cells were incubated for 1 hr at 250C (lanes 2 and 4) or 370C (lanes 1 and 3). The growth media (m) and lysed cells (I) were subjected to SDS/PAGE followed by immunoblot analysis with anti-HSP150 antiserum. HSP150 (subunit II) secreted to the medium by S. cerevisiae cells served as a control (D). Preimmune serum gave negative results for all samples (data not shown). Molecular size markers (kDa) are at right.
cellular location (32). Thus, HSP150 appears to be the first secretory stress protein reported. Though we have no data to suggest a possible function for HSP150, the apparent conservation of HSP150 in divergent yeasts suggests that HSP150-like proteins may constitute a class of secretory stress proteins. We thank Drs. Gennadi and Elena Naumov (All-Union Scientific Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow) for kindly helping with the tetrad dissections. We thank Dr. Manfred Watzele (University of Regensburg) for carrying out the HF treatment. The Academy of Finland, the Nordic Yeast Research Program, the University of Helsinki, and the Foundation for Biotechnical and Fermentation Research are acknowledged for financial support. 1. Tanner, W. & Lehle, L. (1987) Biochim. Biophys. Acta 906, 81-99. 2. Lindquist, S. & Craig, E. A. (1988) Annu. Rev. Genet. 22, 631-677. 3. Sherman, F. (1991) Methods Enzymol. 194, 12-17.
3675
4. Nevalainen, L. T., Louhelainen, J. & Makarow, M. (1989) Eur. J. Biochem. 184, 165-172. 5. Kalkkinen, N. & Tilgman, C. (1988) J. Prot. Chem. 7, 242-243. 6. Takahashi, Y., Kato, K., Hayashizaki, Y., Wakabayashi, T., Ohtsuka, E., Matsuki, S., Ikehara, M. & Matsubara, K. (1985) Proc. Natl. Acad. Sci. USA 82, 1931-1935. 7. Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA 80, 1194-1198. 8. Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182. 9. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 10. Carlson, M. & Botstein, D. (1982) Cell 28, 145-154. 11. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 12. Russo, P. & Sherman, F. (1989) Proc. Natl. Acad. Sci. USA 86, 8348-8352. 13. Rothstein, R. J. (1983) Methods Enzymol. 101, 202-211. 14. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 15. Sherman, F. & Hicks, J. (1991) Methods Enzymol. 194, 21-37. 16. Watzele, M., Klis, F. & Tanner, W. (1988) EMBO J. 5, 1483-1488. 17. von Hejne, G. & Abrahamsdn, L. (1989) FEBS Lett. 244, 439-446. 18. Julius, D. A., Brake, A., Blair, L., Kunisawa, R. & Thorner, J. (1984) Cell 37, 1075-1089. 19. Fuller, R. S., Brake, A. & Thorner, J. (1989) Proc. Natl. Acad. Sci. USA 86, 1434-1438. 20. Redding, K., Holcomb, C. & Fuller, S. (1991) J. Cell Biol. 113, 527-538. 21. Russo, P., Li, W.-Z., Hampsey, D. M., Zaret, K. S. & Sherman, F. (1991) EMBO J. 10, 563-571. 22. Henikoff, S. & Cohen, E. H. (1984) Mol. Cell. Biol. 4, 15151520. 23. Amin, J., Ananthan, J. & Voellmy, R. (1988) Mol. Cell. Biol. 8, 3761-3769. 24. Susek, R. D. & Craig, S. (1990) Mol. Cell. Biol. 10, 6362-6373. 25. Craig, E. A., Kramer, J. & Kosic-Smithers, J. (1987) Proc. Natl. Acad. Sci. USA 84, 4156-4160. 26. Normington, K., Kohno, K., Kozutsumi, Y., Gething, M.-J. & Sambrook, J. (1989) Cell 57, 1223-1247. 27. Rose, M. D., Misra, L. M. & Vogel, J. P. (1989) Cell 57, 1211-1221. 28. Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A. & Schekman, R. (1988) Nature (London) 332, 800-805. 29. Cheng, M. Y., Hartd, F.-U., Martin, J., Pollock, R. A., Kalousek, F., Neupert, W., Hallberg, E. M., Hallberg, R. L. & Horwich, A. L. (1989) Nature (London) 337, 620-625. 30. Munro, S. & Pelham, H. R. B. (1986) Cell 46, 291-300. 31. Mazzarella, R. A. & Green, M. (1987) J. Biol. Chem. 262, 8875-8883. 32. Verma, R., fida, H. & Pardee, A. (1988) J. Biol. Chem. 263, 8569-8575.
Corrections
Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistr. In the article "A heat shock gene from Saccharomyces cerevisiae encoding a secretory glycoprotein" by Patrick Russo, Nisse Kalkkinen, Hannele Sareneva, Juha Paakkola, and Marja Makarow, which appeared in number 9, May 1, 1992, of Proc. Natl. Acad. Sci. USA (89, 3671-3675), the authors wish that the following correction be noted. The
8857
nucleotide sequence of the coding region of HSPS50 shown in Fig. 2 lacks A133, C134, and C189, which are included in the corrected Fig. 2 shown here. This changes amino acids 45-62 in the original figure to amino acids 45-63 in the corrected figure. Thus, subunit I of the HSP150 protein has 54 amino acids instead of 53.
-396 AGTGATCTTACTATTTCCTATTTCGGAAATTATTAAAGACAAAAAAGCTCATTAATGGCTTTCCGTCTGTAGTGATAAGTCGCCAACTCAGCCTAATTT -297
TTCATTTCTTTACCAGACAGtAAACTAATAGTACAAATGAGTG
-198
AMTAGAACACTAAAAGGAATGGAAGGTTAAATTATTAGCTTGAATGGTAGGAATCCTCGAGAAGGAACACTTMGTCTAACGACAAT
.................
TTTCTCAAGCGGAACACCACATTTTGAGCTAAATTTAGATTTTGGTCAAAATA
................
.....................
-99 CAATTTCGATTATGTCCTTCCTTTTACCTCAAAGCTCAAAATATCAATAAGAAACTCATATTCCTTTTCTAACCCTAGTACAATAATAATAATATA +1 ATG CAA TAC AAA AAG ACT TTG GTT GCC TCT GCT TTG GCC GCT ACT ACA TTG GCCVGCC TAT GCT CCA TCT GAG CCT 1 Met Gln Tyr Lys Lys Thr Leu Val Ala Ser Ala Leu Ala Ala Thr Thr Leu Ala Ala Tyr Ala Pro Ser Glu Pro +76 TGG TCC ACT TTG ACT CCA ACA GCC ACT TAC AGC GGT GGT GTT ACC GAC TAC GCT TCC ACC TTC GGT ATT GCC GTT 26 Trp Ser Thr Leu Thr Pro Thr Ala Thr Tyr Ser Gly Gly Val Thr Asp Tyr Ala Ser Thr Phe Gly Ile Ala Val +151 CAA CCA ATC TCC ACT ACA TCC AGC GCA TCA TCT GCA GCC ACC ACA GCC TCA TCT AAG GCC AAG AGA'GCT GCT TCC 51 Gln Pro Ile Ser Thr Thr Ser Ser Ala Ser Ser Ala Ala Thr Thr Ala Ser Ser Lye Ala Lys Arg2Ala Ala Ser +226 CAA ATT GGT GAT GGT CAA GTC CAA GCT GCT ACC ACT ACT GCT TCT GTC TCT ACC AAG AGT ACC GCT GCC GCC GTT 76 Gln Ile Gly Asp Gly Gln Val Gln Ala Ala Thr Thr Thr Ala Ser Val Ser Thr Lys Ser Thr Ala Ala Ala Val +301 TCT CAG ATC GGT GAT GGT CAA ATC CAA GCT ACT ACT AAG ACT ACC GCT GCT GCT GTC TCT CAA ATT GGT GAT GGT 101 Ser Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr Thr Lys3Thr Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly +376 CAA ATT CAA GCT ACC ACC AAG ACT ACC TCT GCT AAG ACT ACC GCC GCT GCC GTT TCT CAA ATC AGT GAT GGT CAA 126 Gln Ile Gln Ala Thr Thr Lys Thr Thr Ser Ala Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Ser Asp Gly Gln +451 ATC CAA GCT ACC ACC ACT ACT TTA GCC CCA AAG AGC ACC GCT GCT GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT 151 Ile Gln Ala Thr Thr Thr Thr Leu Ala Pro Lys Ser Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Val
+526 CAA GCT ACC ACC ACT ACT TTA GCC CCA AAG AGC ACC GCT GCT GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT CAA 176 Gln Ala Thr Thr Thr Thr Leu Ala Pro Lys Ser Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Val Gln +601 GCT ACT ACT AAG ACT ACC GCT GCT GCT GTC TTT CAA ATT GGT GAT GGT CAA GTT CTT GCT ACC ACC AAG ACT ACT 201 Ala Thr Thr Lys Thr Thr Ala Ala Ala Val Phe Gln Ile Gly Asp Gly Gln Val Leu Ala Thr Thr Lys Thr Thr
+676 CGT GCC GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT CAA GCT ACT ACC AAG ACT ACC GCT GCT GCT GTC TCT CAA 226 Arg Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Val Gln Ala Thr Thr Ly4Thr Thr Ala Ala Ala Val Ser Gln +751 ATC GGT GAT GGT CAA GTT CAA GCA ACT ACC AAA ACC ACT GCC GCA GCT GTT TCC CAA ATT ACT GAC GGT CAA GTT 251 Ile Gly Asp Gly Gln Val Gln Ala Thr Thr Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Thr Asp Gly Gln Val +826 CAA GCC ACT ACA AAA ACC ACT CAA GCA GCC AGC CAA GTA AGC GAT GGC CAA GTC CAA GCT ACT ACT GCT ACT TCC 276 Gln Ala Thr Thr Lys Thr Thr Gln Ala Ala Ser Gln Val Ser Asp Gly Gln Val Gln Ala Thr Thr Ala Thr Ser
+901 GCT TCT GCA GCC GCT ACC TCC ACT GAC CCA GTC GAT GCT GTC TCC TGT AAG ACT TCT GGT ACC TTA GAA ATG AAC 301 Ala Ser Ala Ala Ala Thr Ser Thr Asp Pro Val Asp Ala Val Ser Cys Lys Thr Ser Gly Thr Leu Glu Met Asn
+976 TTA AAG GGC GGT ATC TTA ACT GAC GGT AAG GGT AGA ATT GGT TCT ATT GTT GCT AAC AGA CAA TTC CAA TTT GAC 326 Leu Lys Gly Gly Ile Leu Thr Asp Gly Lys Gly Arg Ile Gly Ser Ile Val Ala Asn Arg Gln Phe Gln Phe Asp +1051 GGT CCA CCA CCA CAA GCT GGT GCC ATC TAC GCT GCT GGT TGG TCT ATA ACT CCA GAC GGT AAC TTG GCT ATT GGT 351 Gly Pro Pro Pro Gln Ala Gly Ala Ile Tyr Ala Ala Gly Trp Ser Ile Thr Pro Asp Gly Asn Leu Ala Ile Gly
6
+1126 GAC AAT GAT GTC TTC TAC CAA TGT TTG TCC GGT ACT TTC TAC AAC TTG TAC GAC GAA CAC ATT GGT AGT CAA TGT 375 Asp Asn Asp Val Phe Tyr Gln Cys Leu Ser Gly Thr Phe Tyr7Asn Leu Tyr Asp Glu His Ile Gly Ser Gln Cys +1201 ACT CCA GTC CAC TTG GAA GCT ATC GAT TTG ATA GAC TGT TAA GCAGAAAACTATTAGTTCTTrTATCCTGATGACTTTTTCTCAT 401 Thr Pro Val His Leu Glu Ala Ile Asp Leu Ile Asp Cys Stop
+1286 +1385 +1484 +1583
TTGCATTGATTAGAAAGGAAAAAAAGAAGTGTCCTTTTCTACTACTACTCTAGTCGCATCCATTCCTTTGCATTTATCTTTTCTGCGGTTGGCCAATCC ATTCTTCCGAGAATTTGGCTAGCCATACTTGATGTTTTCCCATTATTGGTTCGTTTGGCAATGCTAATTTTCTTAATTGCCCCTTATATACTCTTCCAT AAAATGTTTTTTTTATAACTAATTTTCTGTATATCATTATCTAATAATCTTATAAAATGTTAAAAAGACTTGGAAAGCAACGAGTGATCGTGACCACAT AATITGCCTCGCTACACGGCAAAAATAAGCCAGTCCTAATGTGTATATTAAAGGCTGCATGTGGCTACGTC
FIG. 2. Nucleotide sequence of HSP150 and predicted amino acid sequence of the HSP150 protein. Three putative regulatory elements are indicated in the flanking sequences of the coding region: the TATA element (box), heat shock element (HSE)-like sequences (dotted underline), and two transcription termination signals (double underline). Nucleotides matching the HSE consensus are indicated by asterisks. The open and full arrowheads show the signal sequence and the KEX2 protease-specific cleavage sites, respectively. The underlined and numbered amino acid sequences were confirmed by direct amino acid sequencing of tryptic peptides of mature secreted HSP150. The sequences of peptides 1 and 2 (dashed underline) were obtained by direct N-terminal sequence analysis and thus represent the N termini of subunits I and II.
Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 3671-3675, May 1992
Biochemistry
A heat shock gene from Saccharomyces cerevisiae encoding a secretory glycoprotein PATRICK Russo, NISSE KALKKINEN, HANNELE SARENEVA, JUHA PAAKKOLA, AND MARJA MAKAROW* Institute of Biotechnology, University of Helsinki, Valimotie 7, 00380 Helsinki, Finland
Communicated by Fred Sherman, December 31, 1991
thetic complete (SC), presporulation, and sporulation media were made as in ref. 3. Metabolic Labeling and Immunoprecipitation of HSP150. Cells were labeled for 1 hr in YPD medium with [35S]methionine (>1000 Ci/mmol; 1 Ci = 37 GBq) at 50-100 pCi/ml or [2-3H]mannose (11.5 Ci/mmol; Amersham), at 300 ,uCi/ml, washed, and lysed mechanically with glass beads. Lysates and media were immunoprecipitated, as described before for a-factor (4), with anti-HSP150 antiserum (1:100) raised in rabbits against purified HSP150. The antiserum specifically recognized mature and nonglycosylated HSP150, and the immunoprecipitations were quantitative. Direct Amino Acid Sequencing of HSP150. SF821-8A cells were incubated for 1 hr at 37°C; under these conditions there is a preferential secretion of HSP150. The HSP150 protein (subunits I and II) was purified from the growth medium by concentration on an ion exchanger, followed by dialysis, gel filtration, and anion exchange chromatography. The purified protein was cleaved with trypsin, -and the peptides were purified by reversed-phase chromatography. N-terminal amino acid sequencing was carried out on the native mature protein, and on subunits I and II after separation by reversedphase chromatography. Amino acid sequence analysis was performed with a gas-phase sequencer equipped with an on-line phenylthiohydantoin amino acid analyzer (5). Cloning and Sequencing. Peptide 5 (see Fig. 1) was used to synthesize a 32-mer oligonucleotide probe with inosines at certain ambiguous codon positions (6). The probe was radiolabeled with 32P by T4 polynucleotide kinase (7) and used to screen >300,000 plaques of a yeast cDNA library made in Agtll (7, 8). Putative clones were subcloned into Bluescript II vectors (Stratagene) and subjected to double-stranded DNA sequencing with the chain-termination method (9). A yeast genomic library (10) was screened with the HSP150 cDNA clone, which was radiolabeled with [a-32P]dCTP by the random primer technique (11). Two putative genomic clones were isolated, subcloned, and subjected to sequencing. Both strands of the entire HSP150 coding region and the flanking regions were sequenced. Northern Blot Analysis. Total RNA was isolated from SEY2101a cells, electrophoresed, and subjected to Northern blot analysis as described (12), except that hybridizations were for 12-18 hr at 42°C. The probe used for Northern (and Southern) blots was the same as used for screening the yeast
We report the finding of a secretory heat ABSTRACT shock protein, HSP150, of Saccharomyces cerevisiae, and the characterization of the gene coding for it. HSP150 is constitutively expressed, extensively 0-glycosylated, and secreted efficiently to the growth medium. When cells grown at 25°C were shifted to 37°C, a 7-fold increase in the level of HSP150 was observed within 1 hr. The HSP150 gene encodes a primary translation product of 412 amino acids. Direct amino acid sequencing of the mature secreted protein showed that an N-terminal sequence of 18 amino acids is removed, and a KEX2 protease-specific site is cleaved to yield two subunits of 53 and 341 amino acids, which remain noncovalently associated during secretion. The larger subunit is highly repetitive, containing 11 tandem repeats of a 19-amino acid sequence. Northern blot hybridization analysis showed a substantial increase in HSP150 mRNA level after heat shock. The upstream flanking region of the gene contains several heat shock element-like sequences. Disruption of HSP150 did not lead to inviability or significant effects on growth rate, mating, or thermotolerance. However, heat-regulated antigenic homologs of HSP150 were found in divergent yeasts such as Schizosaccharomyces pombe.
About 15 secretory glycoproteins have been identified from Saccharomyces cerevisiae. The expression of most of them is regulated by nutritional conditions or the mating type of the cells. Most of them remain intercalated in the cell wall, and only a few are secreted across the cell wall to the growth medium (1). No secretory protein has been reported to be regulated by heat stress. Heat-regulated proteins are found in all organisms and fall into two groups: the conserved stress proteins, which include the 70-kDa, 90-kDa, and low molecular weight heat shock proteins, and unclassified ones such as "household" enzymes and ubiquitin. All stress proteins characterized so far are cell-associated (2). In this report we characterize HSP150, a gene coding for a protein that is regulated by heat stress, 0-glycosylated, and secreted to the growth medium.t
MATERIALS AND METHODS Strains and Media. The S. cerevisiae strains SEY2101a (MATal ade2-101 ura3-52 leu2-3,112 suc2-A9 gal2), S288C (MA Ta), SF821-8A (MATa, sec7-1 his4-580 ura3-52 leu23,112 trpl-289), W303D (MATa/MATa ura3-1/ura3-1 his31J/his3-11 his3-15/his3-15 leu2-3/leu2-3 leu2-3, 112/leu23,112 trpl-1/trpl-J ade2-1/ade2-J canl-100/canl-100) and the Schizosaccharomyces pombe (leul-32, h-), Kluyveromyces marxianus (NCYC587), and Torulaspora delbrueckii strains were grown at 25°C in YPD medium containing 1% yeast extract (Oxoid, Basingstoke, U.K.), 2% bacto peptone (Difco), and 2% glucose (BDH), to midlogarithmic phase. Escherichia coli strain XL1-Blue was from Stratagene. Syn-
genomic library. Gene Disruption. The HSP150 chromosomal locus was disrupted by the one-step gene disruption method (13). The genomic HSPI50 clone, with -2-kilobase (kb) and 1-kb flanking sequences at the 5' and 3' ends, respectively, was subcloned into the Sal I site of the Bluescript II SK(±) vector (Stratagene), which had its Pst I site mutated. A Pst I fragment coding for amino acids 61-301 in the HSPJ50 gene was replaced by a HindIII fragment containing a yeast URA3
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
*To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M88698).
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gene (supplied by Fred Sherman, University of Rochester). The Pst I and HindIII 5' cohesive ends were filled in by the combined 3'-to-5' exonuclease and 5'-to-3' polymerase activities of T4 DNA polymerase in the presence of deoxynucleoside triphosphates, to create blunt ends for ligation (14). The diploid yeast strain W303D was then transformed with this integrating plasmid, URA3' transformants were sporulated, asci were dissected (15), and URA3' spores were analyzed for the disruption of the HSP150 gene. Immunoblotting. Cells grown overnight were washed and resuspended in fresh YPD medium (2 x 108 cells in 400 ,l) and then incubated for 1 hr at either 250C or 370C. The cells were pelleted and lysed, and the medium was precipitated with 14% (wt/vol) trichloroacetic acid. Five percent of each cell lysate and growth-medium sample was subjected to SDS/PAGE, except in the case of Sch. pombe, where 10% of the sample volume was electrophoresed. The proteins were transferred to nitrocellulose filters, which then were probed with anti-HSP150 antiserum (1:1500) and developed by using alkaline phosphatase-conjugated anti-rabbit antibody according to the supplier's instructions (Amersham). Immunostaining was blocked when a 1:10 dilution of growth medium in which S. cerevisiae cells (5 x 108 per ml) had been incubated for 1 hr at 370C was added to the above antiserum preparation. Other Methods. To determine thermotolerance, cells that had been grown overnight at 250C were incubated for up to 20 min at 50°C, with or without a preincubation of 30 min at 37°C. Aliquots were plated on solid YPD media and scored for colony formation at 25°C. SDS/PAGE was done in 7.5-15% polyacrylamide Laemmli gels. Gels were processed for autoradiography and exposed to Kodak X-Omat AR film (Eastman Kodak). Radioactivity in gel slices was quantitated by scintillation counting after solubilization with NCS tissue solubilizer (Amersham). HF treatment was performed to release 0-glycans (16). Trichloroacetic acid (14% TCA) precipitation was for 30 min on ice. Tunicamycin (Sigma; preincubation for 30 min) was used at a concentration of 10
,ug/ml.
RESULTS AND DISCUSSION Heat Shock Increases the Level of a 150-kDa Secretory Glycoprotein. Cells (SEY2101a unless otherwise stated) were labeled with [35S]methionine at various temperatures, and the proteins of the growth media were analyzed in SDS/PAGE. After labeling at 25°C, a minor band of 150 kDa could be detected among other bands. When the labeling was performed at 37°C, the intensity of the 150-kDa band was much higher than at 25°C. Quantitations showed that there was 7 times more radioactivity in the 150-kDa band at 37°C (n = 6; range 5.5-9.3), 6.3 times more at 39°C, and very little at 41°C as compared with 25°C (Fig. 1A). Similar results were obtained with an a mating-type strain, S288C. The 150-kDa protein was designated HSP150. The protein was determined to be a glycosylated secretory protein, since it could be labeled with [3H]mannose. However, tunicamycin had no effect on its [3H]mannosylation or electrophoretic migration (data not shown), suggesting that the protein was not N-glycosylated. Instead, HF treatment, which removes 0-glycans, reduced its apparent molecular mass to 47 kDa (Fig. 1B). Immunoprecipitations of [35S]methionine-labeled growth medium and cell lysate with anti-HSP150 antiserum confirmed that heat stress increased the level of HSP150, and further showed that >90% of the newly synthesized HSP150 was secreted to the medium (Fig. 1C). Thus, HSP150 appears to be an extensively 0-glycosylated protein that is secreted efficiently to the growth medium and regulated by heat stress. To confirm this, we cloned and characterized the HSPI50 gene.
Proc. Natl. Acad. Sci. USA 89 (1992) B
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FIG. 1. Heat stress increases the level of a 150-kDa secretory glycoprotein. (A) Cells were labeled for 1 hr with [35S]methionine at 250C (lane a), 370C (lane b), 390C (lane c), or 41'C (lane d). Proteins were precipitated from the growth media with trichloroacetic acid and analyzed by SDS/PAGE followed by autoradiography. Molecular size markers (kDa) are at left. The arrowhead indicates HSP150. (B) [35S]Methionine-containing HSP150 before (lane a) or after (lane b) HF treatment to remove O-glycans. (C) Cells were labeled as in A at 370C (lanes a-d) or 250C (lanes e-h), and the cell lysates (1) and growth media (m) were subjected to immunoprecipitation with anti-HSP150 antiserum (lanes a, c, e, and g) or preimmune serum (lanes b, d, f, and h).
Cloning and Primary Sequence Analysis. A S. cerevisiae cDNA library made in Agtll (7) was screened with an oligonucleotide probe, which was synthesized according to the sequence of a peptide obtained from trypsin-digested purified HSP150 protein. Six putative clones were isolated and found to have the same restriction endonuclease digestion fragments. Two of them were sequenced, and both clones had an open reading frame of 1236 nucleotides that included the amino acid sequence of the peptide used for design of the oligonucleotide probe, as well as the sequences of six other peptides determined from the purified protein (Fig. 2). The HSPI50 gene encodes a primary translation product of 412 amino acids with a calculated molecular mass of 41,633 Da (Fig. 2). The polypeptide starts with a putative signal sequence of 18 amino acids. It has the following features common to yeast signal sequences: two lysine residues in the N-terminal region, a hydrophobic core, and small uncharged amino acids at positions -1 and -3 relative to the cleavage site (17). Direct N-terminal sequence analysis of the native mature protein harvested from the growth medium gave a double sequence corresponding to peptides 1 and 2, in approximately equimolar ratio. Reversed-phase chromatography resolved the native protein into two components, subunits I and II, which in N-terminal sequence analysis gave single sequences
Biochemistry: Russo et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
3673
-396 AGTGATCTTACTATTTCCTATTTCGGAAATTATTAAAGACAAAAAAGCTCATTAATGGCTTTCCGTCTGTAGTGATAAGTCGCCAACTCAGCCTAATTT
**
***
*
-297 TTCATTTCTTTACCAGATCAGGAAAACTAATAGTACAAATGAGTGTTTTCTCAAGCGGAACACCACATTTTGAGCTAAATTTAGATTTTGGTCAAAATA -198
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AGAAAGATCCTAAAAAAGGAATGGTTGGTGAAAAATTTATTAGCTTGAATGGTAGGAATCCTCGAG. AT... ...............
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-99 CAATTTCGATTATGTCCTTCCTTTTACCTCAAAGCTCAAAAAAATATCAATAAGAAACTCATATTCCT rTCTAACCCTAGTACAATAATAATAATATA +1 ATG CAA TAC AAA AAG ACT TTG GTT GCC TCT GCT TTG GCC GCT ACT ACA TTG GCCVGCC TAT GCT CCA TCT GAG CCT 1 Met Gln Tyr Lys Lys Thr Leu Val Ala Ser Ala Leu Ala Ala Thr Thr Leu Ala Ala Tyr Ala Pro Ser Glu Pro +76 TGG TCC ACT TTG ACT CCA ACA GCC ACT TAC AGC GGT GGT GTT ACC GAC TAC GCT TCC CTT CGG TAT TGC CGT TCA 26 Trp Ser Thr Leu Thr Pro Thr Ala Thr Tyr Ser Gly Gly Val Thr Asp Tyr Ala Ser Leu Arg Tyr Cys Arg Ser +151 ACC AAT CTC CAC TAC ATC CAG CGC ATC ATC TGC AGC ACC ACA GCC TCA TCT AAG GCC AAG AGATGCT GCT TCC CAA 51 Thr Asn Leu His Tyr Ile Gln Arg Ile Ile Cys Ser Thr Thr Ala Ser Ser Lys Ala Lys Arg Ala Ala Ser Gln
+226 ATT GGT GAT GGT CAA GTC CAA GCT GCT ACC ACT ACT GCT TCT GTC TCT ACC AAG AGT ACC GCT GCC GCC GTT TCT 76 Ile Gly Asp Gly Gln Val Gln Ala Ala Thr Thr Thr Ala Ser Val Ser Thr Lys Ser Thr Ala Ala Ala Val Ser +301 CAG ATC GGT GAT GGT CAA ATC CAA GCT ACT ACT AAG ACT ACC GCT GCT GCT GTC TCT CAA ATT GGT GAT GGT CAA 101 Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr Thr Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln +376 ATT CAA GCT ACC ACC AAG ACT ACC TCT GCT AAG ACT ACC GCC GCT GCC GTT TCT CAA ATC AGT GAT GGT CAA ATC 126 Ile Gln hla Thr Thr Lys Thr Thr Ser Ala Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Ser Asp Gly Gin Ile +451 CAA GCT ACC ACC ACT ACT TTA GCC CCA AAG AGC ACC GCT GCT GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT CAA 151 Gln Ala Thr Thr Thr Thr Leu Ala Pro Lys Ser Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Val Gin +526 GCT ACC ACC ACT ACT TTA GCC CCA AAG AGC ACC GCT GCT GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT CAA GCT 176 Ala Thr Thr Thr Thr Leu Ala Pro Lys Ser Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Val Gln Ala
+601 ACT ACT AAG ACT ACC GCT GCT GCT GTC TTT CAA ATT GGT GAT GGT CAA GTT CTT GCT ACC ACC AAG ACT ACT CGT 201 Thr Thr Lys Thr Thr Ala Ala Ala Val Phe Gln Ile Gly Asp Gly Gln Val Leu Ala Thr Thr Lys Thr Thr Arg +676 GCC GCC GTT TCT CAA ATC GGT GAT GGT CAA GTT CAA GCT ACT ACC AAG ACT ACC GCT GCT GCT GTC TCT CAA ATC 226 Ala Ala Val Ser Gln Ile Gly Asp Gly Gin Val Gln Ala Thr Thr Lys Thr Thr Ala Ala Ala Val Ser Gin Ile
+751 GGT GAT GGT CAA GTT CAA GCA ACT ACC AAA ACC ACT GCC GCA GCT GTT TCC CAA ATT ACT GAC GGT CAA GTT CAA 251 Gly Asp Gly Gln Val Gln Ala Thr Thr Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Thr Asp Gly Gln Val Gln +826 GCC ACT ACA AAA ACC ACT CAA GCA GCC AGC CAA GTA AGC GAT GGC CAA GTC CAA GCT ACT ACT GCT ACT TCC GCT 276 Ala Thr Thr Lys Thr Thr Gln Ala Ala Ser Gln Val Ser Asp Gly Gln Val Gln Ala Thr Thr Ala Thr Ser Ala +901 TCT GCA GCC GCT ACC TCC ACT GAC CCA GTC GAT GCT GTC TCC TGT AAG ACT TCT GGT ACC TTA GAA ATG AAC TTA 301 Ser Ala Ala Ala Thr Ser Thr Asp Pro Val Asp Ala Val Ser Cys Lys Thr Ser Gly Thr Leu Glu Met Asn Leu +976 AAG GGC GGT ATC TTA ACT GAC GGT AAG GGT AGA ATT GGT TCT ATT GTT GCT AAC AGA CAA TTC CAA TTT GAC GGT 326 Lys Gly Gly Ile Leu Thr Asp Gly Lys Gly Arg Ile Gly Ser Ile Val Ala Asn Arg Gln Phe Gln Phe Asp Gly
+1051 CCA CCA CCA CAA GCT GGT GCC ATC TAC GCT GCT GGT TGG TCT ATA ACT CCA GAC GGT AAC TTG GCT ATT GGT GAC 351 Pro Pro Pro Gln Ala Gly Ala Ile Tyr Ala Ala Gly Trp Ser Ile Thr Pro Asp Gly Asn Leu Ala Ile Gly Asp
+1126 AAT GAT GTC TTC TAC CAA TGT TTG TCC GGT ACT TTC TAC AAC TTG TAC GAC GAA CAC ATT GGT AGT CAA TGT ACT 375 Asn Asp Val Phe Tyr Gln Cys Leu Ser Gly Thr Phe Tyr Asn Leu Tyr Asp Glu His Ile Gly Ser Gln Cys Thr
+1201 CCA GTC CAC TTG GAA GCT ATC GAT TTG ATA GAC TGT TAA GCAGAAAACTATTAGTTCTTTTATCCTGATGACTTTTTCTCATTTGC 401 Pro Val His Leu Glu Ala Ile Asp Leu Ile Asp Cys Stop +1287 ATTGATTAGAAAGGAAAAAAAGAAGTGTCCTTTTCTACTACTACTCTAGTCGCATCCATTCCTTTGCATTTATCTTTTCTGCGGTTGGCCAATCCATTC +1386 TTCCGAGAATTTGGCTAGCCATACTTGATGTTTTCCCATTATTGGTTCGTTTGGCAATGCTAATTTTCTTAATTGCCCCTTATATACTCTTCCATAAAA
+1485 TGTTTTTTTTATAACTAATTTTCTGTATATCATTATCTAATAATCTTATAAAATGTTAAAAAGACTTGGAAAGCAACGAGTGATCGTGACCACATAATT +1584
GCCTCGCTACACGGCAAAAATAAGCCAGTCCTAATGTGTATATTAAAGGCTGCATGTGGCTACGTC
FIG. 2. Nucleotide sequence of HSP150 and predicted amino acid sequence of the HSP150 protein. Three putative regulatory elements are indicated in the flanking sequences of the coding region: the TATA element (box), heat shock element (HSE)-like sequences (dotted underline), and two transcription termination signals (double underline). Nucleotides matching the HSE consensus are indicated by asterisks. The open and full arrowheads show the signal sequence and the KEX2 protease-specific cleavage sites, respectively. The underlined and numbered amino acid sequences were confirmed by direct amino acid sequencing of tryptic peptides of mature secreted HSP150. The sequences of peptides 1 and 2 (dashed underline) were obtained by direct N-terminal sequence analysis and thus represent the N termini of subunits I and II.
starting at amino acids 19 (peptide 1) and 72 (peptide 2), respectively (Fig. 2). This suggests that the signal sequence was cleaved and that a second cleavage took place directly after Lys70-Arg71, a putative KEX2 protease-recognition site, to yield subunits I and II, which remained noncovalently associated in the mature secreted protein. Whether the C terminus of subunit II remained intact was not studied. Thus, the 150-kDa band in SDS/PAGE (Fig. 1) represents glycosylated subunit II, whose apparent molecular mass is highly exaggerated, since the calculated molecular mass of the apoprotein is -34 kD. Subunits I and II lack potential N-glycosylation sites (Asn-Xaa-Ser/Thr), whereas 25% of their amino acids are serines or threonines, which are potential O-glycosylation sites. The cleavage of subunits I and II may have been carried out by the serine protease KEX2, which cleaves the precursors of the mating pheromone a-factor and the M1 killer toxin at pairs of basic amino acids (18, 19). The KEX2 protease is thought to be located in the late Golgi compartment (20). In the case of a-factor and killer toxin, the released effector
molecule is secreted to the growth medium, whereas the rest of the precursor remains cell-associated. The N-terminal portion of subunit II, 227 out of 341 amino acids, is highly repetitive. It consists of 11 tandem repeats of a 19-amino acid sequence (Fig. 3). Some of the repeats are separated by 5 or 7 amino acids. Although the nucleotide sequences coding for the repeats vary, the amino acid sequences are conserved (Fig. 3B). Regulation of the HSP150 Gene. Sequence analysis of the genomic clones revealed the same open reading frame found in the cDNA clones with no introns, plus putative regulatory signals in the sequences flanking the coding region (Fig. 2). The flanking region downstream of HSP150 contains two putative yeast mRNA 3' end-forming signals, TATATA (21) and T-ll--lTT-TTATA (22), starting at nucleotides + 1466 and +1487, respectively. The region upstream from the translation initiation codon revealed a putative TATA box (TATAA) starting at position -131. The 396 nucleotides 5' to the translation initiation codon contain four nucleotide sequences that resemble the consensus heat shock element,
Biochemistry: Russo et A
3674
Proc. Natl. Acad. Sci. USA 89 (1992)
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three or more contiguous repeats of the element NGAAN in alternating orientations (23, 24). Northern analysis of total RNA revealed a constitutively expressed HSP150 transcript of -1.6 kb that which showed a substantial increase in steady-state level after heat shock (Fig. 4). This, along with the presence of heat shock elements, suggests that heat regulation of HSP150 occurs at the transcriptional level. Disruption of the HSPISO Gene. A single-step gene disruption experiment (13) was carried out to assess whether HSP150 is essential. The genomic copy of HSP150 was disrupted with a URA3 gene (Fig. 5A), and the hspJS0:: URA3 construction was transformed into the diploid strain W303D. Integrants were isolated on SC plates lacking uracil. Four URA3' integrants were sporulated and tetrad analysis was performed on 20 asci from each integrant. Greater than 90% spore viability for each integrant suggests that HSP150 is not essential. The disruption of HSP150 was confirmed by Southern and Northern analyses (data not shown), and immuno-
37° 25(1
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FIG. 4. Northern blot analysis of HSPl50 mRNA levels under normal and heat shock conditions. Cells were grown to midlogarithmic phase, and then the culture was divided in two and incubated for 1 hr at 37°C or 25°C. Total RNA was isolated and subjected to Northern blot analysis. The 32P-labeled HSPl50 cDNA clone was used as the probe. The positions of the 25S and 18S ribosomal RNAs
are
indicated at left.
m
FIG. 5. Disruption of the HSPJ50 gene. (A) The nucleotides coding for amino acids 61-302 in the genomic copy of HSPI50 were replaced by a URA3 gene. (B) A HSP150 strain (lanes a and b) and a hsplSO::URA3 strain (lanes c and d) were labeled with [35S]methionine for 1 hr at 37TC, and the cell lysates (1; lanes a and c) and growth media (m; lanes b and d) were subjected to immunoprecipitation with anti-HSP150 antiserum. The precipitates were subjected to SDS/PAGE analysis. Molecular size markers (kDa) are at right.
precipitation experiments demonstrated the lack of HSP150 protein in both cell lysates and media (Fig. 5B). The disruption of HSP150 gene had no effect on growth at 25°C or 37°C, mating, and thermotolerance (data not shown). In comparison, two members of the 70-kDa heat shock gene family, SSCI and KAR2, have been found to be essential (25-27). Conservation of the HSP150 protein. Computer-aided searches of nucleic acid and protein files in the EMBL data base showed no significant homologies between HSP150 and any known genes or proteins, thus providing no hypothesis for the function of the HSP150 protein. Thus, we studied whether HSP150 is conserved, by searching for antigenic homologs in other yeasts. T. delbrueckii, K. marxianus and Sch. pombe cells were incubated at 25°C and 37°C, and the growth media and cell lysates were subjected to immunoblot analysis using anti-HSP150 antiserum. Antigenic homologs were discovered in all three yeasts. T. delbueckii (Fig. 6A) and K. marxianus (Fig. 6B) synthesized secretory homologs with apparent molecular masses of 54 kDa and 54-150 kDa, respectively. In both cases, similar homologs were found in the cell lysates, together with putative precursors of smaller molecular size. The heterogeneous electrophoretic migration of the homolog of K. marxianus may be due to heterogeneous N-glycosylation. A homolog of 120 kDa was found in Sch. pombe; however, it was not secreted but remained cellassociated (Fig. 6C). Excess HSP150 protein of S. cerevisiae inhibited the immunostaining of the Sch. pombe protein. The levels of the antigenic homologs of each organism were increased by heat shock. Several heat shock proteins have previously been found to serve important physiological functions as chaperonins, which are involved in the maintenance or change of the conformation of other proteins (28, 29). Most stress proteins reside in the cytoplasm, nucleus, or mitochondria (2), and some are known to be segregated to the secretory pathway; however, they are not secreted. The BiP protein and grp94 are resident proteins of the endoplasmic reticulum (30, 31) and a 118-kDa glycoprotein of yeast has an unknown intra-
Biochemistry: Russo et al.
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Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 6. Antigenic homologs of HSP150 in other yeasts. T. delbrueckii (A), K. marxianus (B), and Sch. pombe (C) cells were incubated for 1 hr at 250C (lanes 2 and 4) or 370C (lanes 1 and 3). The growth media (m) and lysed cells (I) were subjected to SDS/PAGE followed by immunoblot analysis with anti-HSP150 antiserum. HSP150 (subunit II) secreted to the medium by S. cerevisiae cells served as a control (D). Preimmune serum gave negative results for all samples (data not shown). Molecular size markers (kDa) are at right.
cellular location (32). Thus, HSP150 appears to be the first secretory stress protein reported. Though we have no data to suggest a possible function for HSP150, the apparent conservation of HSP150 in divergent yeasts suggests that HSP150-like proteins may constitute a class of secretory stress proteins. We thank Drs. Gennadi and Elena Naumov (All-Union Scientific Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow) for kindly helping with the tetrad dissections. We thank Dr. Manfred Watzele (University of Regensburg) for carrying out the HF treatment. The Academy of Finland, the Nordic Yeast Research Program, the University of Helsinki, and the Foundation for Biotechnical and Fermentation Research are acknowledged for financial support. 1. Tanner, W. & Lehle, L. (1987) Biochim. Biophys. Acta 906, 81-99. 2. Lindquist, S. & Craig, E. A. (1988) Annu. Rev. Genet. 22, 631-677. 3. Sherman, F. (1991) Methods Enzymol. 194, 12-17.
3675
4. Nevalainen, L. T., Louhelainen, J. & Makarow, M. (1989) Eur. J. Biochem. 184, 165-172. 5. Kalkkinen, N. & Tilgman, C. (1988) J. Prot. Chem. 7, 242-243. 6. Takahashi, Y., Kato, K., Hayashizaki, Y., Wakabayashi, T., Ohtsuka, E., Matsuki, S., Ikehara, M. & Matsubara, K. (1985) Proc. Natl. Acad. Sci. USA 82, 1931-1935. 7. Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA 80, 1194-1198. 8. Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182. 9. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 10. Carlson, M. & Botstein, D. (1982) Cell 28, 145-154. 11. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 12. Russo, P. & Sherman, F. (1989) Proc. Natl. Acad. Sci. USA 86, 8348-8352. 13. Rothstein, R. J. (1983) Methods Enzymol. 101, 202-211. 14. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 15. Sherman, F. & Hicks, J. (1991) Methods Enzymol. 194, 21-37. 16. Watzele, M., Klis, F. & Tanner, W. (1988) EMBO J. 5, 1483-1488. 17. von Hejne, G. & Abrahamsdn, L. (1989) FEBS Lett. 244, 439-446. 18. Julius, D. A., Brake, A., Blair, L., Kunisawa, R. & Thorner, J. (1984) Cell 37, 1075-1089. 19. Fuller, R. S., Brake, A. & Thorner, J. (1989) Proc. Natl. Acad. Sci. USA 86, 1434-1438. 20. Redding, K., Holcomb, C. & Fuller, S. (1991) J. Cell Biol. 113, 527-538. 21. Russo, P., Li, W.-Z., Hampsey, D. M., Zaret, K. S. & Sherman, F. (1991) EMBO J. 10, 563-571. 22. Henikoff, S. & Cohen, E. H. (1984) Mol. Cell. Biol. 4, 15151520. 23. Amin, J., Ananthan, J. & Voellmy, R. (1988) Mol. Cell. Biol. 8, 3761-3769. 24. Susek, R. D. & Craig, S. (1990) Mol. Cell. Biol. 10, 6362-6373. 25. Craig, E. A., Kramer, J. & Kosic-Smithers, J. (1987) Proc. Natl. Acad. Sci. USA 84, 4156-4160. 26. Normington, K., Kohno, K., Kozutsumi, Y., Gething, M.-J. & Sambrook, J. (1989) Cell 57, 1223-1247. 27. Rose, M. D., Misra, L. M. & Vogel, J. P. (1989) Cell 57, 1211-1221. 28. Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A. & Schekman, R. (1988) Nature (London) 332, 800-805. 29. Cheng, M. Y., Hartd, F.-U., Martin, J., Pollock, R. A., Kalousek, F., Neupert, W., Hallberg, E. M., Hallberg, R. L. & Horwich, A. L. (1989) Nature (London) 337, 620-625. 30. Munro, S. & Pelham, H. R. B. (1986) Cell 46, 291-300. 31. Mazzarella, R. A. & Green, M. (1987) J. Biol. Chem. 262, 8875-8883. 32. Verma, R., fida, H. & Pardee, A. (1988) J. Biol. Chem. 263, 8569-8575.
