Disruption of YHC8, a member of the TSR1 gene family, reveals its direct involvement in yeast protein translocation

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Disruption of YHC8, a member of the TSR1 gene

family, reveals its direct involvement in yeast protein

translocation

Choukri Ben Mamoun, Jean Marie Beckerich, Claude Gaillardin, Francois

Kepes

To cite this version:

Choukri Ben Mamoun, Jean Marie Beckerich, Claude Gaillardin, Francois Kepes. Disruption of YHC8,

a member of the TSR1 gene family, reveals its direct involvement in yeast protein translocation.

Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 1999, 274

(16), pp.11296-11302. �10.1074/jbc.274.16.11296�. �hal-02693994�

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Disruption of YHC8, a Member of the TSR1 Gene Family, Reveals Its

Direct Involvement in Yeast Protein Translocation*

(Received for publication, December 29, 1998, and in revised form, February 3, 1999)

Choukri Ben Mamoun‡§¶, Jean-Marie Beckerich‡, Claude Gaillardin‡, and Francois Kepesi

From the ‡Laboratoire de Geńe´tique Molećulaire et Cellulaire, INRA-CNRS, Centre de Biotechnologie Agro Industrielle, Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France, §Departments of Molecular Microbiology and Medicine, Washington University School of Medicine, St. Louis, Missouri 63110,iService de Biochimie et de Geńe´tique Molećulaire, DBCM/DSV, BAT.142, CEA/Saclay, 91191 Gif Cedex, France

Genetic studies of Saccharomyces cerevisiae have identified many components acting to deliver specific proteins to their cellular locations. Genome analysis, however, has indicated that additional genes may also participate in such protein trafficking. The product of the yeast Yarrowia lipolytica TSR1 gene promotes the signal recognition particle-dependent translocation of secretory proteins through the endoplasmic reticulum. Here we describe the identification of a new gene family of proteins that is well conserved among different yeast species. The TSR1 genes encode polypeptides that share the same protein domain distribution and, like Tsr1p, may play an important role in the early steps of the signal recognition particle-dependent translocation pathway. We have identified five homologues of the

TSR1 gene, four of them from the yeast Saccharomyces cerevisiae and the other from Hansenula polymorpha.

We generated a null mutation in the S. cerevisiae YHC8 gene, the closest homologue to Y. lipolytica TSR1, and used different soluble (carboxypeptidase Y,a-factor, in-vertase) and membrane (dipeptidyl-aminopeptidase) se-cretory proteins to study its phenotype. A large accumu-lation of soluble protein precursors was detected in the mutant strain. Immunofluorescence experiments show that Yhc8p is localized in the endoplasmic reticulum. We propose that the YHC8 gene is a new and important component of the S. cerevisiae endoplasmic reticulum membrane and that it functions in protein transloca-tion/insertion of secretory proteins through or into this compartment.

Genetic and biochemical studies have helped to elucidate major concepts of cell biology for targeting of proteins into and across membranes. Typical secreted proteins usually contain amino-terminal signal sequences that ensure their targeting to the translocation machinery. These sequences, called signal peptides, are recognized upon their emergence from ribosomes by a cytosolic ribonucleoprotein factor, the signal recognition

particle (SRP),1_{which in mammalian cells is composed of one}

molecule of SRP RNA and six polypeptides (SRP19p, -54p, -68p,

-72p, -9p, and -14p). The resulting complex is then targeted to the endoplasmic reticulum (ER) membrane by binding to an integral protein complex called the SRP receptor. Subsequently the SRP releases the signal sequence to the translocation chan-nel (1, 2). Several genes involved in protein translocation into the ER lumen in Saccharomyces cerevisiae have been identified by genetic analysis (SEC61, SEC62, SEC63, SSS1, SEC71, and SEC72) (3–10). In this yeast, post-translational translocation has been reproduced in vitro with reconstituted proteolipo-somes containing a heptameric complex consisting of the trim-eric Sec61pC complex (Sec61p, Sss1p, and Sbh1p) and the tetrameric Sec63pC complex (Sec62p, Sec63p, Sec71p, and Sec72p) (11). Mutational tests have shown that neither SEC71 nor SEC72 is essential for cell viability. However, cells lacking Sec71p lack simultaneously Sec72p, grow slowly at elevated temperature, and depletion of Sec72p leads to the accumula-tion of precursor polypeptides (10, 12). The SSS1 gene was found in genetic screen as a suppressor of a temperature-sensitive sec61 mutation (7), and the Sss1p protein can be co-immunoprecipitated in complex with Sec61p and Sbh1p (11, 12). Sbh1p has never been found in genetic screens but has been identified during purification of the Sec61p complex.

Sec61p, Sbh1p, and Sss1p are related to the Sec61a, Sec61b,

and Sec61g subunits of the mammalian Sec61p complex,

re-spectively. The identification of the homologues of SEC61 and SBH1 genes, SSH1 and SBH2, respectively, in S. cerevisiae have been reported recently. In contrast to Sec61p and Sss1p, Ssh1p, is not essential for cell viability but is required for fast growth. Deletion of one of the two genes SBH1 or SBH2 is not lethal, but deletion of both genes diminishes the cell growth at elevated temperature and induces defects in the translocation

of Kar2p and ofa-factor precursors (12). With these new

com-ponents two major trimeric complexes, Sec61pC and Ssh1pC, are now known. Sss1p is common to both complexes. The Sec61pC has been shown to be associated with Sec63pC to form a heptameric complex (11) and thus could be involved either in post- or co-translational translocation pathways. In contrast, no interaction of Ssh1pC with Sec63pC has been detected, suggesting that this second trimeric complex is involved exclu-sively in co-translational translocation (13).

In the yeast Yarrowia lipolytica, a thermosensitive mutation scr2.II-13 was identified in SCR2, one of two genes encoding the 7 S RNA of SRP (14), and was shown to decrease the stability of the SRP. Genetic screening for suppressor muta-tions identified, among others, the tsr1-1 mutation in the TSR1 gene (15, 16). This mutation was shown to restore the stability of an SRP crippled by the scr2.II-13 mutation and to stabilize its binding to the ribosome at or near the translocation site (15, * This work was supported by EEC Grant BIO2 CT 930470, the

Institut National de la Recherche Agronomique, and the Center Na-tional de la Recherche Scientifique. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¶Present address and to whom correspondence should be addressed:

Depts. of Molecular Microbiology and Medicine, Washington University School of Medicine, 660 S. Euclid Ave., Box 8230, St. Louis, MO 63110. Tel.: 314-362-4780; Fax: 314-362-1232; E-mail: choukri@borcim.wustl. edu.

1_{The abbreviations used are: SRP, signal recognition particle; ER,}

endoplasmic reticulum; PCR, polymerase chain reaction; HA, hemag-glutinin; CPY, carboxypeptidase Y; DPAP, dipeptidyl-aminopeptidase.

This paper is available on line at http://www.jbc.org

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at INRA Institut National de la Recherche Agronomique, on September 16, 2010

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16). The TSR1 gene codes for a serine-rich protein of 50 kDa spanning the ER membrane. Genetic and biochemical studies implicated Tsr1p in the stability of SRP in the early steps of the SRP-dependent translocation pathway of secretory proteins. Cross-linking studies revealed that Tsr1p interacted with the ribosome-SRP complex and with BiP (16). Following the se-quencing of the S. cerevisiae genome, we identified four homo-logues of TSR1 in this unrelated fungal species, and we suggest here that they define a new gene family to be called the TSR1 gene family. To assess whether the closest of the TSR1 homo-logues YHC8 is also critically involved in protein translocation, we constructed a null mutant allele of this gene and examined the effect of its loss of function in S. cerevisiae.

EXPERIMENTAL PROCEDURES

Strains and Plasmids—Strains and plasmids used in this study are

listed in Table I.

Media—YPG medium contained 1% yeast extract, 1% Bacto-Peptone,

and 2% glucose. YPinv medium contained 1% yeast extract, 1% Bacto-Peptone, and 0.1% glucose. The selective minimal medium contained 1% glucose, 0.17% yeast nitrogen base without ammonium sulfate (Difco), and 0.1% proline as nitrogen source and was supplemented with appropriate nutrients.

DNA Techniques—All enzyme reactions and DNA preparations were

performed as described by Maniatis et al. (17). Oligonucleotides used for PCR disruption of YHC8 gene were as follows: oligonucleotide 1, CCC

CGG CGC GCC CCC CAT CGA ACG GTT GCT ACT G;

oligonucleo-tide 2, GCA TAT AAC GCT ACA TAC TAG CC; oligonucleooligonucleo-tide 3, TCC AGG AGG GTT CTG CG; oligonucleotide 4, GGG GGG CGC GCC

GGG GTT ATA GAC GGT GAC TCT TAT G. Bold characters indicate

the sticky end 16-base extension used to construct the AscI site (18). PCR reactions were developed as described by Maftahi and col-leagues (18). The first PCR reactions utilized primers 1 and 2 or 3 and 4. A second PCR using the products of the first PCR as a template was performed using oligonucleotides 2 and 3. The 1-kilobase pair amplified fragment was tested for the presence of the newly synthesized AscI site and blunt-ended using the T4 DNA polymerase. The plasmid pSC10 was obtained by integration of the blunt-ended fragment obtained from the second PCR at the PvuII site of pINA-KAN (18).

Protein Immunoblotting—Yeast cultures were grown overnight to

early log phase and 2 A6003 ml were collected. The cells were washed

in 10 mMNaN3and resuspended in 100ml of 23 SDS-polyacrylamide

gel electrophoresis sample buffer containing 1 mM

phenylmethylsulfo-nyl fluoride. Cells were lysed by vortexing with glass beads (0.5 mm diameter), heated for 10 min at 95 °C, separated on 10% SDS-polyacryl-amide gel electrophoresis, and transferred to nitrocellulose filters. Pre-incubation, antibody incubations, and washes were conducted in TBST

buffer (10 mMTris-Cl, pH 8, 150 mMsodium chloride, 0.05% Tween 20)

and 5% of skim milk. A chemiluminescence kit (ECL, Amersham Phar-macia Biotech, France) followed by autoradiography was used to detect the protein of interest.

Immunofluoresence Experiment—Yhc8 open reading frame was

am-plified using two oligonucleotides carrying flanking BamHI and NotI restriction sites (CGG GAT CCG CAA AAA CGC ATG CAG ACG and CCC GCG GCC GCC GTT CAT TAG, respectively) and cloned into

pYEF2 designed by C. Cullin (19). This construction put yhc8 under the control of the GAL10 promoter with the HA tag at the carboxyl-terminal end. In order to exchange the HA tag for the protein A tag, a

NotI-Bsu36I fragment was amplified from the pE9 plasmid and inserted in

the corresponding NotI-Bsu36I. Expression of the tagged Yhc8p under

the control of GAL1 promoter was monitored by growing cells to A6005

1 in synthetic media containing 2% raffinose. Galactose was added to a final concentration of 2%. Cells were collected at 0, 30, 60, and 90 min (20). Fixation and antibody decoration procedures were adapted from Pringle and collaborators (21). The primary antibody was an anti-protein A raised in rabbit (Sigma reference number P-3775) used at a dilution 1:300 and the fluorescent secondary antibody was a Cy3-con-jugated anti-rabbit IgG from donkey (reference number 711-165-152, Jackson ImmunoResearch, West Grove, PA) at a dilution 1:300. Obser-vations were performed on a Leitz Laborlux S microscope.

Transformation of S. cerevisiae—Yeast cells were transformed with

linear DNA fragments using the lithium acetate method (22). Cells were grown at 30 °C in YPD for 4 –5 h and spread on YPD plates containing 200 mg/liter G418. Resistant clones were verified by South-ern or PCR analyses for disruption of the YHC8 locus.

RESULTS

TSR1 Gene Family—We have previously described the in vivo evidence for the role of the ER membrane protein, Tsr1p, in the translocation pathway of the yeast Y. lipolytica (15). A first homology search had identified two homologues, YHC8 from S. cerevisiae and YLU2 from Hansenula polymorpha (23) of the TSR1 gene. A new search through the entire S. cerevisiae genome sequence data base led to the identification, in addition to YHC8, of three other homologues called Hre556, Scynl283, and UNF378. All of these genes encode putative proteins which, like Tsr1p and Ylu2p, contain an amino-terminal signal sequence and share a highly conserved distribution of 5 do-mains as follows: cysteine-rich (Cys-rich), serine/threonine-rich (Ser/Thr-rich), intermediary, transmembrane, and cytoplasmic (Fig. 1, A and B) (15). The topology of this last domain was previously established for Tsr1p (16). Fig. 1 summarizes the features of this gene family that we called the TSR1 family. In addition to the structural conservation (Fig. 1A), the similarity of the sequences of the four predicted proteins with Tsr1p

increases significantly toward the NH2-terminal cysteine-rich

domain (results not shown). Comparison of the putative cyto-solic domain of the six proteins shows high conservation be-tween Scynl283p and Hre556p, and Tsr1p and Yhc8p (Fig. 1, C and D). Based on the fact that the Tsr1p was more similar to Yhc8p than to any of the other homologues (Fig. 1, C and D), we focused our study on the YHC8 gene of S. cerevisiae, and we tested its possible involvement in the early steps of the secre-tory pathway.

The nucleotide sequence of the YHC8 gene revealed an open reading frame of 1815 base pairs. RNA hybridization experi-ments confirmed that this gene was expressed and produced a

TABLE I

Strains and plasmids used in this study

Strains/plasmids Description Source or Ref.

E. coli

DH5a :supE44DlacU169(f801acZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 42

S. cerevisiae

RDM15–5B :Mata,sec61–2, ura3–52, leu2–3,112, ade2–1, pep4–3 3

RDM50–94C :Mata, sec62–1, ura3–52, leu2–3,112, his4 3

RDM47–11C :Mata, sec62–1, pep4–3 3

JRM 151 :Mata, sec63–1, ura3–52, leu2–3,112, pep4–3 5

FY1679 :Mata/Mata; ura3–52/ura3–52; trp1-D63/1; leu2D1/1; his3D200/1 43

Dyp :Mata, ura3–52, leu2–3,112, ade2–1, pep4–3,Dyhc8 This work

Dypts :Mata, sec63–1, ura3–52, leu2–3,112, ade2–1, pep4–3,Dyhc8 This work

T1sp1 :Mata, ura3–52,Dyhc8, trp1-D63 This work

ABM1 :Mata/Mata; Dyhc8/1; 1/sec61–2; 1/ade2–1; ura3–52/ura3–52; trp1-D63/1; 1/leu2–3,112; 1/pep4–3 This work ABM2 :Mata/Mata; Dyhc8/1; 1/sec62–1; ura3–52/ura3–52; trp1-D63/1; 1/leu2–3,112; 1/his4 This work ABM3 :Mata/Mata; Dyhc8/1; 1/sec63–1; ura3–52/ura3–52; trp1-D63/1; 1/leu2–3,112; 1/pep4–3 This work Plasmids

pSC10 :Kanr_{-based-plasmid used for disruption of the YHC8 gene} _{This work}

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single transcript of approximately 2 kilobase pairs (data not shown). The YHC8 gene is predicted to encode a protein of 605 amino acids residues with a molecular mass of approximately 64 kDa. This protein shows an amino-terminal signal peptide and a predicted signal cleavage site between amino acids 26 and 27 (Fig. 1, A and B). The last cytoplasmic domain (170 amino acids) is strongly conserved between Yhc8p and Tsr1p and was predicted to play an important role in Tsr1p interac-tion with SRP and/or ribosomes (16).

To assess the YHC8 null phenotype, a sticky-end polymerase (SEP) disruption strategy was adopted (18). The promoter and terminator regions of the YHC8 gene were amplified separately and served to construct the disrupting plasmid pSC10 carrying the kanamycin resistance cassette. The linearized plasmid was then targeted for integration into the YHC8 genomic locus of

the diploid strain FY1679, and Kanr_{clones were selected.}

Dis-ruption of one of the YHC8 alleles was confirmed by Southern blot on several transformants (results not shown). One of the heterozygous disrupted diploids was sporulated, and tetrads were dissected (Fig. 2A). Each of the 20 dissected tetrads

yielded four viable spores, two of which were Kanr _implying

that they were disrupted for YHC8, which was confirmed by

PCR (data not shown). We tested the growth ofDyhc8 at 28 and

38 °C and compared it with the wild type and with the

sec61-3-thermosensitive mutant. The growth of Dyhc8 was slightly

affected compared with the wild type strain but not blocked as is the case for Sec61-3 at 38 °C (Fig. 2B). This result shows that the YHC8 gene is not essential in S. cerevisiae under standard

laboratory conditions.

Deletion of YHC8 Induces Defects in the Translocation of Soluble Secretory Proteins—To determine whether YHC8 is important for protein translocation, we examined the fate of

several well characterized secreted proteins in a yhc8::Kanr

strain by immunoblotting of whole cell extracts.

Carboxypeptidase Y (CPY) is a vacuolar protease that is synthesized as a 59-kDa inactive precursor (prepro-CPY). Re-moval of its amino-terminal signal sequence in the ER lumen gives rise to the 57-kDa pro-CPY. ER glycosylation of pro-CPY yields the 67-kDa p1 form (24), which is then transported to the Golgi apparatus where it is glycosylated to generate the 69-kDa p2 form. Upon arrival in the vacuole, an amino-terminal se-quence of approximately 8 kDa is removed from the p2 form, yielding the 61-kDa mature CPY, active form (m-CPY). This maturation of p2 requires the PEP4 gene product (Ref. 25; see

Fig. 3A). Fig. 3A shows the fate of CPY inDyhc8, wild type

(SEC1), and sec mutant cells. To discriminate between the

prepro and m-CPY forms all the strains used in the CPY immunoblotting experiments carried the pep4-3 mutation so that no maturation of the p2 could occur in the vacuole. In addition, we used the ER-to-Golgi blocked sec18-1 mutant (Fig. 3A, 6th lane) to detect the ER p1 form. Results in Fig. 3 show that for sec61, -62, and -63 mutants primarily prepro-CPY and

p2 forms were detected and for_{Dyhc8 mutants primarily}

pre-pro-CPY and pre-pro-CPY forms were detected.

The secretory invertase is synthesized as a precursor of 61 kDa with a 19-residue hydrophobic signal sequence (absent from the cytosolic form) which ensures its targeting to the ER membrane. Upon translocation to the ER, this precursor un-dergoes signal peptide cleavage and core glycosylation (26, 27). Transported to the Golgi apparatus, it is subjected to further mannosylations before reaching its periplasmic location (28). To test the effect of the absence of the YHC8 product on the translocation and transit of the invertase precursors, we

com-pared the secretory phenotype of the_{Dyhc8 mutant with that of}

wild type and sec mutant cells (Fig. 3B). In the wild type cells primarily highly glycosylated forms were detected (5th lane). For sec mutants primarily pre-invertase and the highly

glyco-sylated forms (1st to 3rd lanes) were detected;Dyhc8 mutant

cells, however, accumulated preinvertase (4th lane), and only a small level of highly glycosylated forms can be detected.

Thea-factor mating pheromone is a 13-amino acid peptide

that is secreted into the culture medium by MATa cells (29). It

is synthesized as a precursor polypeptide of 21 kDa (pp-aF)

that contains a prepro-leader sequence of 83 amino acids. Cleavage of the signal sequence after translocation into the ER

gives rise to the pro-a-factor. This is then decorated with three

core oligosaccharides during its translocation across the ER membrane, yielding a 26-kDa ER form (30 –32). Directed to the Golgi apparatus, this form undergoes outer chain glycosylation and proteolytic maturation (33). The processing is then com-pleted within the secretory vesicles by Kex2p and

dipeptidyl-aminopeptidase A (DPAP A) (34). ForDyhc8 and like sec

mu-tants (Fig. 3C, 1st to 3rd lanes) the cells accumulate the prepro-a-factor (4th lane), which indicates a significant defect in the translocation of this molecule into the ER.

Deletion of YHC8 Induces Defects in the Assembly of

Mem-FIG. 1. The TSR1 gene family. A, structural organization of the five putative homologous proteins encoded by the genes of the TSR1 gene

family. The percentages of identity between Tsr1p and the other five proteins of the TSR1 gene family for the cysteine-rich, serine/threonine-rich, and cytoplasmic domains are indicated. Tm, transmembrane; Cyt, cytoplasmic; S.c., S. cerevisiae; H.p., H. polymorpha; Y.l., Y. lipolytica; aa, amino acid. Tsr1p (15), Yhc8p (SwissProt accession number, P38739), Scynl238p (EMBL accession number, Z71559), Hre556 (PIR accession number,

S51892), Ylu2p (23) and Unf378p (GenBankTM_{accession number, U39481). B, comparison of the pattern of hydrophobicity of the different members}

of the TSR1 gene family. C, a tree of sequence similarities showing the Tsr1p with its homologues. The tree was generated using the Pileup program from GCG. D, alignment of the cytosolic domains of the five homologous proteins using the PILEUP program from the GCG software package with the scoring matrix of Risler et al. (40). Identities between Scynl238p, Hre556p, Ylu2p, Tsr1p, and Yhc8p are presented in bold.

FIG. 2. A, Yhc8::kanr_{disrupted strains are viable. Tetrad dissection of}

the parental diploid strain FY1679 (lane 1) and six isolated

YHC81/yhc8::Kanr

-disrupted diploids (lanes 2–7) was developed. The four spores were isolated and grown on YPD or YPD supplemented with

200mg/ml geniticin (G418). B, mild defect of yhc8::Kanr_{strain at 38 °C.}

Wild type, sec61-3, yhc8::kanr_{or a sec61-31yhc8::kan}r_{strains were}

tested for growth of YPD at 28 and 38 °C.

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brane Proteins—To test the effect of YHC8 deletion on the insertion of membrane proteins into the ER membrane, we used the dipeptidyl-aminopeptidase B (DPAP B) as a reporter

protein and compared its kinetics of insertion inDyhc8 and sec

mutant strains. DPAP B is an integral membrane glycoprotein with a carboxyl-terminal domain localized in the lumen of the ER (35). The unglycosylated pre-DPAP B can be observed at 96 kDa, and the mature vacuolar form migrates as a 120-kDa species. Contrary to the wild type or sec61-2 mutant strains where no accumulation or only a small accumulation of pre-DPAP B was detected (Fig. 3D, 1st and 5th lanes) (4),

immu-noblotting from Dyhc8 showed accumulation of pre-DPAP B

(4th lane). However, the amount is not as great as that ob-served in sec62-1 and sec63-1 mutants (2nd and 3rd lanes). These results clearly demonstrate a partial defect in the as-sembly of this integral membrane protein in the yhc8 null mutant cells.

Yhc8p Is an ER Membrane Protein—In order to localize Yhc8p inside the cell, it was tagged at its carboxyl-terminal end using the vectors developed by Cullin and Minvielle-Sebastia (19) which fused the HA tag and placed the open reading frame under the control of the GAL1 promoter. When grown on 2% galactose as carbon source, cells decorated with HA

anti-a-factor in Dyhc8-deleted strain. Cells were grown on YPD until A600

0.2. Yeast whole cell extracts were prepared from yhc8::kanr_{and wild}

type strains growing under permissive conditions and from sec mutant cells after a shift to restrictive conditions. The extracts were electro-phoresed through 12.5% SDS-polyacrylamide gel, blotted to

nitrocellu-lose, and probed with anti-a-factor serum. Bound antibodies were

visu-alized by enhanced chemiluminescence (Amersham Pharmacia

Biotech). D, translocation of dipeptidyl-aminopeptidase B in

Dyhc8-deleted strain. Yeast whole cell extracts were prepared from yhc8::kanr

growing under permissive conditions and from sec mutant cells after a shift to restrictive conditions. The extracts were electrophoresed through 7.5% SDS-polyacrylamide gel, blotted to nitrocellulose, and probed with anti-DPAP B serum. Bound antibodies were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).

FIG. 3. A, translocation of carboxypeptidase Y in a Dyhc8-deleted

strain. Yeast whole cell extracts were prepared from yhc8::Kanr_and

wild type strains growing under permissive conditions and from sec mutant cells after a 2-h shift to 38 °C. The extracts were electrophore-sed through 7.5% SDS-polyacrylamide gel, blotted to nitrocellulose, and probed with anti-CPY serum. Bound antibodies were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). B,

translocation of invertase inDyhc8-deleted strain. Cells were grown on

YPD until A6000.2 and then transferred to YPinv medium. Yeast whole

cell extracts were prepared from yhc8::kanr_{and wild type strains}

grow-ing under permissive conditions and from sec mutant cells after a shift to restrictive conditions. The extracts were electrophoresed through 7.5% SDS-polyacrylamide gel, blotted to nitrocellulose, and probed with anti-invertase serum. Bound antibodies were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). C, translocation of

FIG. 4. Yhc8p is an ER membrane protein. Localization of Yhc8p

by cells expressing a tagged fusion of YHC8 with a protein A tag. Cells were grown overnight on complete medium containing 2% raffinose and then transferred to a pre-warmed complete medium containing 1% raffinose and 2% galactose. Samples were taken at 0, 30, and 60 min after shift. The cells were fixed with 5% formaldehyde and, after proc-essing with anti-protein A antibodies, were decorated with Cy3-conju-gate anti-rabbit antibodies. Nuclear DNA was visualized with 4,6-diamidino-2-phenylindole (dapi).

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bodies displayed a strong accumulation of fluorescent material in an intracellular organelle away from the nucleus (data not shown) which was probably the vacuole. This localization could result from a mistargeting due to the HA tag and/or be a consequence of its overexpression in conditions of GAL1 induc-tion. The HA tag was exchanged for a protein A tag, and the induction conditions were changed. The cells were grown on 2%

raffinose, a non-repressible carbon source (20). At time t5 0,

the cells were transferred into a pre-warmed medium contain-ing 2% galactose, and aliquots were fixed and decorated at 0, 30, and 60 min. Under these conditions, only few cells were decorated by the anti-protein A antibodies, and this proportion did not increase with the incubation time on galactose. Results in Fig. 4 show that the cells were primarily labeled at the periphery of the nucleus and the plasma membrane. This pat-tern is characteristic of the endoplasmic reticulum location. At 60 min, the cells appeared to be more heavily decorated, and the labeling around the nucleus appeared to be more diffuse. We concluded that Yhc8p was first directed to the endoplasmic reticulum membrane as suggested by the structural features of its sequence. Upon its accumulation in this compartment, the overproduced polypeptides were then transferred to the vacu-olar compartment.

DISCUSSION

We have identified four coding sequences, YHC8, Hre556, Scynl283, and UNF378, in the genome of S. cerevisiae, as homologues (43, 33.5, 34.5, and 32.5% amino acid sequence identity, respectively) of the TSR1 gene of Y. lipolytica. Our study on Tsr1p suggested that it is localized in the ER mem-brane and is an important component of the SRP-dependent translocation pathway (15, 16). The proteins encoded by these TSR1 homologues share high homology in both the amino-terminal and cytosolic domains; these two domains were dem-onstrated to be involved in the interaction of Tsr1p with BiP and with the SRP-ribosome complex, respectively (16). We called this new family of genes, TSR1 gene family. Homology of the members of this family with Tsr1p and mutational test on YHC8 gene suggest that they may be involved in the SRP-de-pendent translocation pathway.

Here we focused on YHC8 because its putative product, Yhc8p, was most closely matched with Tsr1p. By using immu-nofluorescence experiments, we showed that Yhc8p is localized in the ER. The presence of an amino-terminal signal sequence and of a membrane-spanning domain suggested that Yhc8p, like its homologue Tsr1p, is a component of the ER membrane. We have demonstrated that deletion of one member of this family, YHC8 gene, although without effect on viability, in-duces large defects in the translocation of secretory soluble proteins, resulting in the accumulation of preinvertase,

pre-CPY, prepro-a-factor. Only a slight defect was observed on the

translocation of pre-DPAP B.

Previous studies have shown that mutations in sec61, sec62, and sec63 lead to a large accumulation of precursors of several

secretory and soluble vacuolar proteins, such asa-factor

pre-cursor, CPY, and acid phosphatase (5, 36) (see Fig. 3, A and C, 1st to 3rd lanes). However, these mutations have only marginal defects on the insertion of the integral membrane protein dipeptidyl-aminopeptidase B (DPAP B) (4) (see Figs. 3D, 1st to 3rd lanes). Other genetic screenings permitted identification of new mutants in the same genes that were defective in the insertion of integral membrane proteins (4). More recently, Pilon and colleagues (37) have characterized strains of S. cer-evisiae expressing cold-sensitive alleles of SEC61 and show that these mutants exhibit a large cytoplasmic accumulation of co- and post-translationally translocated precursors. All to-gether these data pointed to a model where Sec61p acts as the

core of the translocon, controlling both the docking step onto the receptor site and insertion/translocation, whereas Sec63p and Sec62p were implicated specifically in the SRP-independ-ent translocation pathway (1, 4, 11).

Our results are consistent with those obtained for the sec61, sec62, and sec63 mutants where the level of accumulation of precursors was dependent on the allele involved and the re-porter protein used (4) (see Fig. 3A, 1st lane, and Fig. 3C, 1st lane). Only a small accumulation of ER forms was detected in

the cases of invertase, CPY, anda-factor compared with that

observed in sec18-1 mutant. The sec18-1 mutant has been iso-lated as a thermosensitive mutant that exhibits a block in protein transport from the ER to the Golgi apparatus (38, 39), resulting from impaired targeting of the vesicles to an early

Golgi compartment (40). The results obtained with_Dyhc8

sug-gest that Yhc8p controls primarily the translocation step and has only little effect on ER glycosylation. This could explain why the translocation defect and the accumulation of ER in-termediates were more or less pronounced and dependent on the reporter protein used.

Our data show that the secretory defect inDyhc8 mutant is

pleiotropic. Why is the null phenotype of the YHC8 gene not lethal? The fact that four homologues of the TSR1 gene have been identified in this yeast suggests that the products of these remaining three genes cooperate to allow partial suppression of

the yhc8 null. Our results with theDyhc8 mutant are

reminis-cent of those with SEC71 and SEC72 mutants, which show pleiotropic defects in protein trafficking across the ER mem-brane but do not affect cell viability (10, 12). The Tsr1p gene product which was studied in more detail in Y. lipolytica was shown to interact with the SRP-ribosome complex on the cyto-plasmic side and with BiP in the ER lumen in the predominant SRP-dependent translocation pathway in this yeast (16). If Yhc8p is involved in the same process, it is difficult to

under-stand why the prepro-a-factor and CPY, which were

deter-mined to be post-translationally translocated (41), are affected

in theDyhc8 mutant cells. One explanation is that the loss of

Yhc8p could induce a large decrease in the number of sites accessible for post-translational translocation. The fact that Tsr1p interacts with the SRP ribosome complex and deletion of YHC8 gene induces a large translocation defect suggests that Yhc8p may be an intermediary between the docking site on the SRP-receptor and the SEC61 complex, allowing it to play a general role in co- and post-translational translocation.

Acknowledgments—We are indebted to Drs. S. Mauersberger, T.

Stevens, and R. Schekman for gifts of antisera to invertase, DPAP B,

CPY, and a-factor antisera, respectively. We thank Drs. Daniel E.

Goldberg and Douglas E. Berg for the critical reading of this manuscript.

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