Biochimica et BiophysicaActa, 1093 (1991) 135-143
135
© 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 0167488991001920 BBAMCR 12964
The GTP-binding Sarl protein is localized to the early compartment of the yeast secretory pathway Shuh-ichi Nishikawa and Akihiko Nakano Department of Biology, Facultyof Science, Universityof Tokyo, Tokyo (Japan) (Received 10 December 1990) (Revised manuscript received 25 March 1991)
Key words: Vesicular transport; Endoplasmic reticulum; GTP-bindingprotein; SAR1; (Saccharomyces cerevisiae)
SARI, the yeast gene which encodes a novel type of small GTP-binding protein,'has been shown to be required for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To further the understanding of the function of its product, a lacZ-SARI hybrid gene was constructed and a polycional antibody was raised against the hybrid protein. This antibody specifically recognizes the SARI gene product (Sarlp) as a 23-kDa protein in the yeast cell lysate. We examined the subcellular localization of Sarlp using this antibody. In wild-type cells, Sarlp was predominantly recovered in a rapidly sedimenting membrane fraction that includes the ER. The soluble form of Sarlp was also detected when the protein was overproduced. Immunofluorescence microscopy with the anti-Sarlp antibody showed perinuclear staining that was exaggerated in the ER-accumulating secl8 mutant. Membrane association of Sarlp was shown to be very tight. Sarlp was not extracted from the membrane by treatment with alkaline sodium carbonate, and only 1% deoxycholic acid solubilized Sarlp completely. From these results, we suggest that Sarlp is firmly located on the ER membrane where it regulates the ER-Golgi traffic.
Introduction
In the secretory pathway, newly synthesized proteins are transported from the endoplasmic reticulum (ER) to the cell surface via several distinct organelles. Transport of proteins between these subcellular compartments is mediated by carrier vesicles that bud from a donor membrane and fuse with an acceptor membrane. Molecular genetics in yeast Saccharomyces cerevisiae has been a powerfukapproach to study the mechanisms of this vesicular transport, and a number of genes are currently pursued for their roles in the pathway. Recently, involvement of GTP-binding proteins in such
Abbreviations: ER, endoplasmic reticulum; PMSF, phenylmethylsulfonyl fluoride; LSP, low-speed pellet; LSS, low-speed supernatant; HSP, high-speed pellet; HSS, high-speed supernatant; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; YP, a rich medium containing 1% yeast extract and 2% polypeptone; YPD, YP+ 2% glucose; YPGS, YP+ 5% galactose +0.2% sucrose; MV, Wickerham's minimum medium; MVD, MV+2% glucose. Correspondence: A. Nakano, Department of Biology, Faculty of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
processes was unveiled by several lines of study on the yeast secretory pathway. (1) SEC4, which is essential for the discharge of secretory vesicles, encodes a raslike GTP-binding protein [1]. (2) YPT1 codes for another ras-related protein and is required for the early stages of the protein secretion in vivo [2,3] and in vitro [4,5]. (3) We have reported that a GTP-binding protein encoded by SAR1 is involved in the ER-Golgi transport [6]. (4) Analysis of a null mutation of the ARF1 gene which encodes a yeast homolog of ADP-ribosylation factor has suggested its role in secretion [7]. Furthermore, a group of GTP-binding proteins have also been implied to function in protein transport in mammalian cells. Synthetic peptides of the putative effecter domain of Rab proteins inhibited ER-Golgi and intraGolgi transport in vitro [8]. Localization of some Rab proteins to exocytic and endocytic compartments have also been demonstrated [9,10]. Involvement of GTP-binding proteins in secretion was also revealed by striking inhibitory effects of a nonhydrolyzable GTP analog, guanosine-5'-O-(3-thiotriphosphate), in several cell-free protein transport systems. Requirement of GTP hydrolysis in vesicular transport was first demonstrated by Melan~on et al. using in vitro transport assays between the Golgi cister-
136 nae [11]. Similarly, GTP hydrolysis was shown to be necessary for in vitro ER-Golgi transport in both yeast [12,13] and mammalian [14] systems. By analogy to the polypeptide chain elongation factors [15], the general role of the GTP-binding proteins in the secretory pathway is suggested to drive unidirectional reactions of protein transpo~ by using the energy of GTP [6,16]. SAR1 was first isolated as a multicopy suppressor of a secl2 mutation in the course of cloning of the SEC12 gene which is essential for the ER-Golgi transport [17]. SAR1 also suppresses on a multicopy plasmid another ER-Golgi transport mutant, secl6. DNA sequence analysis predicts that SAR1 encodes a 21-kDa GTP-binding protein which is distinct from other known rasrelated proteins [6]. SAR1 is an essential gene, and in a conditional-lethal, galactose-dependent sarl mutant, ER-form precursors of a-mating factor and carbozypeptidase Y accumulate under the .restrictive condition [6], indicating that SAR1 is involved in the ER-Golgi transport. To understand further its role in this earliest vesicular transport step, we have raised an antibody against the SARI gene product which enables us to examine the localization of Sarlp in yeast cells.
Experimental procedures Strains and culture conditions Escherichia coli strains used in this study were SE10 (F-A[iac-pro] ara rpsL thi pyrF74::Tn5[680dlacZ AM15]) [18] for DNA manipulations and JM103 ( Alacpro supE thi strA sbcBl5 endA hspR4 F' traD36 proAB laclCZ AM15] [19] for preparation of the lacZSARI fusion gene product. Yeast strains ANY1-7D (MATa ura3 leu2 gal2) [17], ANY21 (MATa ura3 leu2 trpl his gal2) and ANY26 ( ~ T a sarl::URA3 ura3 leu2 trpl his gal2 [pGALISARI TRPI])[6] have been described previously. For immunofluorescence microscopy, HMSFI76 (MATa seclS.l gal2) (Yeast Genetic Stock Center, University of California, Berkeley, CA) and MBY12.6D (MATa seclS-I ura3 ieu2 trpl his gal2) (M. Bernstein, University of California, Berkeley, CA) were mated to construct SNSI8, a secl8 diploid strain. The strain which overproduces Kex2p on galactose medium, BFYI25[pBM-KX22] ( MATa/MATa kex2 A2::HIS3-A / kex2 A2::TRPI-S ade2/ ade2 canl / canl his3/ his3 leu2 / leu2 trpl / trpl ura3 / ura3 [pGAL1-KEX2 URA3]), was a gift from Robert S: Fuller (Stanford University, Stanford, CA). Yeast strains were usually grown at 30°C in YP medium (I% (w/v) Bacto-yeast extract (Difco, Detroit, MI) and 2% (w/v) polypeptone (Nihon Seiyaku, Tokyo)) containing 2% (w/v) glucose (YPD) or in Wickerham's minimal (MV) medium [20] containing 2% (w/v) glucose (MVD). MVD was supplemented with amino acids and nucleic acids as described by
Sherman et al. [21]. In labeling experiments, sulfate salts in MV media were replaced by chlorides, secl8 strain was cultured at 24°C and incubated at 37°C for restrictive experiments. For derepression of the GALl promoter, YP medium was supplemented with 5% (w/v) galactose and 0.2% (w/v) sucrose (YPGS) as described by Nakano and Muramatsu [6]. Plasmids and DNA manipulations E. coli plasmid pUR288 [22] and yeast multicopy plasmid pSEY8 [18] have been described elsewhere. A yeast multicopy plasmid, pYO324 (a gift from Y. Ohya, University of Tokyo, Tokyo), has been constructed by inserting the 2.2 kb EcoRI fragment of pMK9 [23] containing the replication determinant of the yeast 2 micron circle plasmid at the Aatll site of pRS304 [24]. pANY1-9 [17], pANY2-7 (YEpSAR1) and pANY2-9 (YCpSAR1) [6] were described previously, pSHYI-1 was constructed by inserting the 1.5 kb HindIII-Sall fragment of pAHY2-7 containing the SAR1 gene at the polylinker site of pYO324. DNA manipulations including restriction enzyme digestions, ligations, plasmid isolation and E. coli transformation were carried out by standard methods [25]. Yeast transformation was performed by the quick method using lithium thiocyanate [26]. Purification of DNA fragments from agarose gel pieces was performed using the DNA PREP kit (Asahi Glass Co., Tokyo). Materials Sources of materials used in this work were as follows. Zymolyase 100T was from Seikagaku Kogyo (Tokyo). Protein A-Sepharose and proteinase inhibitors, phenylmethylsulfonyl fluoride, leupeptin, antipain, chymostatin and pepstatin A, were from Sigma (St. Louis, MO). Alkaline phosphatase-conjugated goat anti-rabbit IgG and goat anti-rabbit IgG were from Jackson lmmunoresearch Laboratory (West Grove, PA). Fluorescein isothiocyanate-conjugated anti-goat IgG and fluorescein isothiocyanate-conjugated antirabbit IgG were from Cappel (West Chester, PA). TranaSS-label, which is a mixture of L-[aSS]methionine and L.[35S]cysteine and 125I-protein A were from ICN Biomedicals Inc. (Costa Mesa, CA). Amplify and UDPN-acetyI-D-[U-14C]glucosamine were from Amersham Japan Limited (Tokyo). Enzymes for recombinant DNA methods were from Takara Shuzo (Kyoto). Rabbit antiSec12p antiserum was obtained as described [17]. Affinity purified rabbit anti-Kex2p antibody was a gift from Robert S. Fuller (Stanford University, Stanford, CA). Anti-Sarlp antibody The Nde-Sall fragment from pANY2-7 was inserted into pUR288 to make an in-frame gene fusion of lacZ
137 and SAR1. The resultant plasmid, pANF31, was introduced into the E. coli strain JM103 and the transformant was induced by isopropyl-/~-D-thiogalactopyranoside to express the IacZ-SARI fusion gene product. This hybrid protein was purified as described by Nakano et al. [17] and used to immunize rabbits. Primary injection contained 100 /xg of the protein in complete Freund's adjuvant. The first booster injection was given after 30 days. Boosts of 50/xg in incomplete Freund's adjuvant were given at 7 day intervals. After 7 boosts, blood was collected. Affinity-purified anti-Sarlp antibody was prepared for experiments of immunoblotting and immunofluorescence microscopy. The fl-galactosidase column was prepared by coupling 5 mg /3-galactosidase (from E. coil, Boehringer Mannheim Yamanouchi, Tokyo) with 2-ml packed volume of CNBr-activated Sepharose 4B (Pharmacia LKB Biotechnology, Tokyo). The LacZSarl hybrid protein column was prepared by coupling 1 mg of the purified hybrid protein with 1 ml of the same resin. Coupling was performed as described by the manufacturer. Affinity purification was carried out as described by Hadow and Lane [27]. Briefly, crude IgG fraction prepared by ammonium sulfate precipitation was first passed through the /3-galactosidase column and the flow through was applied on the LacZ-Sarl hybrid protein column. Affinity-purified anti-Sarlp antibody was eluted by 100 mM glycine-HCl (pH 2.5) and 1% (w/v) bovine serum albumin. The eluted fraction was neutralized by the addition of 1 M Hepes-KOH (pH 7.2) and concentrated using Centricon-30 (Amicon, Danvers, MA).
lmmunoblotting and immunoprecipitation Crude cell lysates or subcellular fractions were electrophoresed on 12.5% (w/v) SDS-polyacrylamide gels using Laemmli's buffer system [28]. In the case of immunoblotting with anti-Secl2p antibody, /3-mercaptoethanol was omitted from the sampling buffer and proteins were resolved on 7.5% (w/v) polyacrylamide gels. Immunoblotting was performed as described by Towbin et al. [29] with some modifications [30]. In some experiments, 1251-labeled Protein A was used in place of alkaline phosphatase-conjugated antirabbit lgG antibody [6]. Crude cell lysates were prepared as follows. Logphase yeast cells ((1-4)" 108 cells) were harvested in a 2-ml fiat-bottom microfuge tube and washed once with distilled water. Cells were resuspended in 200 /xl of 62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (w/v) glycerol, 1 mM PMSF and 5/zg/ml each of leupeptin, chymostatin, antipain and pepstatin A. To the suspension, 0.2 g of glass beads (0.4-mm diameter) were added and the mixture was vortexed in four bursts of
30 s with 30-s intervals on ice and then boiled for 5 min. For immunoprecipitation of Sarlp, cells were grown to (0.4-1)-107/ml in MVD containing 100 ttM (NH4)2SO4 and 0.5% (w/v) casamino acids for vitamin assay (Difco). Cells (2-10 7) w e r e harvested, washed once with distilled water and resuspended in 0.4 ml of MVD with no sulfate. After preincubation at 30°C for 30 rain, cells were labeled with 4 MBq of Tran35S-label for 15 min. An equal volume (0.4 ml) of MVD containing 6 mM (NH4)2SO4, 0.5 mM cysteine and 0.54 mM methionine was added and incubation continued for 45 min. Metabolic labeling was terminated by the addition of 0.8 ml of ice-cold 20 mM NaN 3 and the cell extracts were prepared as described by Nishikawa et al. [30]. To the cell extracts equivalent to 1.10 7 ~,ells, 5 /zl of preimmune or anti-Sarlp serum was added. lmmunoprecipitation and analysis by SDS polyacrylamide gel electrophoresis (PAGE) and fluorography were performed as described previously [30].
Subcellular fractionation Yeast cells were grown to 1 • 107/ml in 500 ml of MVD containing 0.5% (w/v) casamino acids. Cells (5" 109) were converted to spheroplasts as described previously [30]. Spheroplasts were resuspended in 10 ml of ice-chilled lysis buffer (10 mM triethanolamine acetate (pH 7.5), 300 mM mannitol, 100 mM KCI and 1 mM EGTA) and immediately homogenized with a morter-driven 30-ml Potter-EIvehjem homogenizer (Wheaton) five times for 1 min with l-min intervals on ice. The lysate was centrifuged at 150 × g for 5 min to remove unbroken spheroplasts. The supernatant was subjected to centrifugation at 10000 x g for 10 min at 4°C in an RPRS-14 rotor (Hitachi) to yield a low-speed pellet (LSP) and low-speed supernatant (LSS) fractions. The LSS fraction was further centrifuged at 100000 x g for 60 rain at 4°C in an RP65T rotor (Hitachi) to give high-speed pellet (HSP) and highspeed supernatant (HSS). In both centrifugations, particulate fractions were collected on a 1-ml cushion of 80% (w/v) sucrose. LSP was suspended in 80% (w/v) sucrose and placed at the bottom of a 14 × 89 mm Ultra-Clear tube (Beckman). 10 mi of 35-60% (w/v) sucrose linear gradient was overlaid on the LSP suspension and the tube was centrifuged at 151000 × g for 17 h at 4°C in an SW41-Ti rotor (Beckman). The tube was punctured at the bottom, and fractions of 0.5 ml were collected. Protein was determined by a method using BCA Protein Assay Reagent (Pierce, Rockford, IL) with bovine serum albumin as a standard. Assays of NADPH-eytoehrome c reductase [31], chitin synthetase [32], Kex2 proteinase [33], guanosine diphos-
138 phatase [34] and a-mannosidase [35] have been described elsewhere.
Extraction of Sarlp Yeast cell homogenate was prepared as described above. To 200/zl of the homogenate, 300 pl of lysis buffer and 500/~! of lysis buffer containing a chemical reagent (2 M NaCI, 4 M urea, 2% (w/v) Triton X-100, 2% (w/v) sodium deoxycholic acid, or 200 mM Na2CO~, pH 11.5)were added. The mixture was placed on ice for 30 min and centrifuged at 436 000 X g for 30 min in an RP100AT rotor (Hitachi) at 4°C. Treatment by hydroxylamine was carried out by adding 500/~1 of freshly prepared 1 M NH2OH (pH 7.0) and 300/~1 of lysis buffer to 200 ~1 of the homogenate; the mixture was incubated for 60 min at 23°C and centrifuged as described above. The pellet was gently rinsed with ice-cold distilled water and dissolved in the SDS gel sampling buffer. Proteins released into the supernatant were precipitated by the addition of trichloroacetic acid as described by Nishikawa et al. [30]. Proteins from both the pellet and supernatant fractions were subjected to SDS-PAGE and analyzed by immunoblotting. Immunofluorescence microscopy secl8 (SNS18) cells were grown to 1.107 cells/ml at 24°C in YPD medium, shifted to 37°C and incubated for 2 h. Kex2p.overproducing yeast cells (BFY125 [pBM-KX22]) were grown at 30°C in MV medium containing 0.5% (w/v) casamino acids, 5% (w/v) galactose and 0.2% (w/v) sucrose with appropriate supplements. Overproduction of Kex2p did not appear to alter its subcellular localization [36]. Formaldehyde solution (37%, w/v) and 1 M potassium phosphate (pH 6.5), were added directly to the growth medium to a final concentration of 4% (w/v) formaldehyde and 100 mM potassium phosphate and cells were fixed first at YPC for 5 min and then at room temperature for 2 h. Fixed cells were collected by centrifugation, washed with I00 mM Hepes-KOH (pH 7.2), 1 M sorbitol and 5 mM NaN 3 (spheroplasting buffer), resuspended in spheroplasting buffer containing 100 ~ g / m l Zymolyase 100T at a concentration of 1. 109/ml and incubated for 30 min at 30°C. Spheroplasts were collected by centrifugation, resuspended in spheroplasting buffer at a concentration of 1. 107/ml and attached to polylysine-coated cover slips. Permeabilization of yeast cells by methanol/acetone treatment was performed by the method of Kilmartin and Adams [37] followed by incubation in phosphate-buffered saline (PBS) [38] containing 1% (w/v) bovine serum albumin. Antibody incubation was carried out in the same buffer for 1 h at room temperature. After washing ten times with PBS containing 0.1% (w/v) bovine serum albumin, cells on the cover slip were incubated for another 1 h at room
temperature with 1 /~g/ml of anti-rabbit IgG goat antibody in PBS containing 1% (w/v) bovine serum albumin. In the experiment 9f Kex2p immunofluorescence, fluorescein isothiocyanate-conjugated antirabbit antibody (1:100 dilution) was used as the second antibody. Cells were again washed as described above and further incubated in 1 : 200 dilution of fluorescein isothiocyanate-conjugated anti-goat antibody for 1 h at room temperature. After the final (10 x ) wash with PBS containing 0.1% (w/v) bovine serum albumin, cells were observed and photographed using an Olympus BH-2 photomicroscope and T-MAX 400 film (Eastman Kodak, Rochester, NY) processed at ASA 3200 with developer (T-MAX, Eastman Kodak, Rochester, NY).
Results Identification of the SAR1 gene product To identify and characterize the SAR1 gene product, we prepared an antibody against the IacZ-SAR1 fusion gene product. The fusion protein overproduced in E. coli was purified on polyacrylamide gels and used to immunize a rabbit. The resulting antiserum showed titer against both E. coli /3-galactosidase and yeast Sarlp epitopes. Except for immunoprecipitation experiments, the anti-Sarlp antibody was used after affinity purification. Sarlp was detected in yeast cell lysates by two methods. First, wild-type yeast cells were lysed and analyzed by SDS-PAGE and immunoblotting. As shown in Fig. 1A, the antibody recognized a single band with an apparent molecular mass of 23 kDa. When the copy number of the SAR1 gene was increased by introducing a single-copy or multicopy plasmid containing the SAR1 gene, the amount of the 23-kDa protein increased according to the gene dosage of SARI (compare lanes 1-3). In the galactose-dependent sarl mutant which expresses the SAR1 gene under control of the GALl promoter [6], the 23-kDa protein was produced in a large amount only when the SAR1 gene was derepressed in galactose medium (lane 4). When SAR1 was repressed for 15.5 h in glucose medium, the 23-kDa protein could barely be detected (lane 5). From these results, we concluded that this 23-kDa protein was the SAR1 gene product, Sarlp. Sarlp was also detected by immunoprecipitation from extracts of radiolabeled cells (Fig. 1B). Wild-type yeast cells containing a multicopy plasmid with or without the SAR1 gene insert was labeled with Tran35S-label. Anti-Sarlp antiserum and Protein ASepharose precipitated from the extracts the single species of 23-kDa Sarlp (lanes 7 and 9). The amount of immunoprecipitated Sarlp increased in the cells harboring the SAR1 multicopy plasmid (lane 9). Sarlp was not precipitated by preimmune serum (lanes 6 and
139 TABLE I
A
0 (U
Distribution of marker enzymes in wild-type yeast
m
0 C
Im
The fractions analyzed were the same as those described in Fig. 2A.
o
Q.
0
ltl
Marker enzyme
Localization
% Total
, i
o
kDa m
200
-"
92.5 ._.
--"
'P I P I ......
69
LSP HSP HSS Total protein Chitin synthetase NADPH-cyt-c reductase Kex2 protease guanosine diphosphatase a-mannosidase
plasma membrane ER Golgi Golgi Vacuole
17 41 45 19 21 40
47 61 31 55 50 49
47 < 0.! 18 42 44 22
46
""
30
21.5 - - " 14.3 ~._ 1 2 3 4 5
.......
6 7 ~
Fig. ]. Identification of the SARI gene product, Sarlp. (A) Immunoblotting analysis of yeast extracts using the affinity purified anti-Sarlp antibody. Cells of ANY2]/pSEY8 (vector alone, lane 1), ANY21/ pANY2-9 (YCpSAR1, lane 2) and ANY2]/pANY2-7 (YEpSAR], lane 3) were grown overnight at 30°C in MVD medium containing 0.5% (w/v) casamino acids and 20 /tg/ml tWl,to~han. "Iile galactose-dependent sarl mutant (ANY26) was i.~cubated for 15.5 h in YPGS (Gel, lane 4) or YPD (Glc, lane 5) medium at 30°C. Extracts were prepared by agitation with glass beads in the SDS gel sampling buffer. Proteins (100/~g) were separated on an SDS-polyacrylamide gel and Sarlp was detected by immunoblc.tting. (B) Sarlp was also detected by immunoprecipitation. A wild-type strain (ANY1-7D) with a multicopy plasmid containing the SARI gene (YEpSARI, lanes 8 and 9) or vector alone (lanes 6 and 7) was pulse-labeled with Tran35S-label for 15 rain and chased for 45 min at 30°C. Extracts were prepared from the labeled cells and subjected to immunoprecipitation with the preimmune (P) or immune anti-Sarlp (I) serum in combination with protein A-Sepharose. The immunoprecipitates were analyzed by SDS-PAGE and fluorograpby.
cytochrome-c reductase, was preferentially distributed in the LSP fraction and other markers such as Kex2p for Golgi behaved differently. That the yeast ER membrane sediments rapidly unlike mammalian cell microsomes has been already reported [17,39,40], perhaps because the yeast ER membrane is mostly continuous with the nuclear envelope [41]. Each fraction was analyzed for Sarlp and Sec12p by SDS-PAGE and immunoblotting (Fig. 2). Secl2p was
A
B >~
135
© 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 0167488991001920 BBAMCR 12964
The GTP-binding Sarl protein is localized to the early compartment of the yeast secretory pathway Shuh-ichi Nishikawa and Akihiko Nakano Department of Biology, Facultyof Science, Universityof Tokyo, Tokyo (Japan) (Received 10 December 1990) (Revised manuscript received 25 March 1991)
Key words: Vesicular transport; Endoplasmic reticulum; GTP-bindingprotein; SAR1; (Saccharomyces cerevisiae)
SARI, the yeast gene which encodes a novel type of small GTP-binding protein,'has been shown to be required for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To further the understanding of the function of its product, a lacZ-SARI hybrid gene was constructed and a polycional antibody was raised against the hybrid protein. This antibody specifically recognizes the SARI gene product (Sarlp) as a 23-kDa protein in the yeast cell lysate. We examined the subcellular localization of Sarlp using this antibody. In wild-type cells, Sarlp was predominantly recovered in a rapidly sedimenting membrane fraction that includes the ER. The soluble form of Sarlp was also detected when the protein was overproduced. Immunofluorescence microscopy with the anti-Sarlp antibody showed perinuclear staining that was exaggerated in the ER-accumulating secl8 mutant. Membrane association of Sarlp was shown to be very tight. Sarlp was not extracted from the membrane by treatment with alkaline sodium carbonate, and only 1% deoxycholic acid solubilized Sarlp completely. From these results, we suggest that Sarlp is firmly located on the ER membrane where it regulates the ER-Golgi traffic.
Introduction
In the secretory pathway, newly synthesized proteins are transported from the endoplasmic reticulum (ER) to the cell surface via several distinct organelles. Transport of proteins between these subcellular compartments is mediated by carrier vesicles that bud from a donor membrane and fuse with an acceptor membrane. Molecular genetics in yeast Saccharomyces cerevisiae has been a powerfukapproach to study the mechanisms of this vesicular transport, and a number of genes are currently pursued for their roles in the pathway. Recently, involvement of GTP-binding proteins in such
Abbreviations: ER, endoplasmic reticulum; PMSF, phenylmethylsulfonyl fluoride; LSP, low-speed pellet; LSS, low-speed supernatant; HSP, high-speed pellet; HSS, high-speed supernatant; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; YP, a rich medium containing 1% yeast extract and 2% polypeptone; YPD, YP+ 2% glucose; YPGS, YP+ 5% galactose +0.2% sucrose; MV, Wickerham's minimum medium; MVD, MV+2% glucose. Correspondence: A. Nakano, Department of Biology, Faculty of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
processes was unveiled by several lines of study on the yeast secretory pathway. (1) SEC4, which is essential for the discharge of secretory vesicles, encodes a raslike GTP-binding protein [1]. (2) YPT1 codes for another ras-related protein and is required for the early stages of the protein secretion in vivo [2,3] and in vitro [4,5]. (3) We have reported that a GTP-binding protein encoded by SAR1 is involved in the ER-Golgi transport [6]. (4) Analysis of a null mutation of the ARF1 gene which encodes a yeast homolog of ADP-ribosylation factor has suggested its role in secretion [7]. Furthermore, a group of GTP-binding proteins have also been implied to function in protein transport in mammalian cells. Synthetic peptides of the putative effecter domain of Rab proteins inhibited ER-Golgi and intraGolgi transport in vitro [8]. Localization of some Rab proteins to exocytic and endocytic compartments have also been demonstrated [9,10]. Involvement of GTP-binding proteins in secretion was also revealed by striking inhibitory effects of a nonhydrolyzable GTP analog, guanosine-5'-O-(3-thiotriphosphate), in several cell-free protein transport systems. Requirement of GTP hydrolysis in vesicular transport was first demonstrated by Melan~on et al. using in vitro transport assays between the Golgi cister-
136 nae [11]. Similarly, GTP hydrolysis was shown to be necessary for in vitro ER-Golgi transport in both yeast [12,13] and mammalian [14] systems. By analogy to the polypeptide chain elongation factors [15], the general role of the GTP-binding proteins in the secretory pathway is suggested to drive unidirectional reactions of protein transpo~ by using the energy of GTP [6,16]. SAR1 was first isolated as a multicopy suppressor of a secl2 mutation in the course of cloning of the SEC12 gene which is essential for the ER-Golgi transport [17]. SAR1 also suppresses on a multicopy plasmid another ER-Golgi transport mutant, secl6. DNA sequence analysis predicts that SAR1 encodes a 21-kDa GTP-binding protein which is distinct from other known rasrelated proteins [6]. SAR1 is an essential gene, and in a conditional-lethal, galactose-dependent sarl mutant, ER-form precursors of a-mating factor and carbozypeptidase Y accumulate under the .restrictive condition [6], indicating that SAR1 is involved in the ER-Golgi transport. To understand further its role in this earliest vesicular transport step, we have raised an antibody against the SARI gene product which enables us to examine the localization of Sarlp in yeast cells.
Experimental procedures Strains and culture conditions Escherichia coli strains used in this study were SE10 (F-A[iac-pro] ara rpsL thi pyrF74::Tn5[680dlacZ AM15]) [18] for DNA manipulations and JM103 ( Alacpro supE thi strA sbcBl5 endA hspR4 F' traD36 proAB laclCZ AM15] [19] for preparation of the lacZSARI fusion gene product. Yeast strains ANY1-7D (MATa ura3 leu2 gal2) [17], ANY21 (MATa ura3 leu2 trpl his gal2) and ANY26 ( ~ T a sarl::URA3 ura3 leu2 trpl his gal2 [pGALISARI TRPI])[6] have been described previously. For immunofluorescence microscopy, HMSFI76 (MATa seclS.l gal2) (Yeast Genetic Stock Center, University of California, Berkeley, CA) and MBY12.6D (MATa seclS-I ura3 ieu2 trpl his gal2) (M. Bernstein, University of California, Berkeley, CA) were mated to construct SNSI8, a secl8 diploid strain. The strain which overproduces Kex2p on galactose medium, BFYI25[pBM-KX22] ( MATa/MATa kex2 A2::HIS3-A / kex2 A2::TRPI-S ade2/ ade2 canl / canl his3/ his3 leu2 / leu2 trpl / trpl ura3 / ura3 [pGAL1-KEX2 URA3]), was a gift from Robert S: Fuller (Stanford University, Stanford, CA). Yeast strains were usually grown at 30°C in YP medium (I% (w/v) Bacto-yeast extract (Difco, Detroit, MI) and 2% (w/v) polypeptone (Nihon Seiyaku, Tokyo)) containing 2% (w/v) glucose (YPD) or in Wickerham's minimal (MV) medium [20] containing 2% (w/v) glucose (MVD). MVD was supplemented with amino acids and nucleic acids as described by
Sherman et al. [21]. In labeling experiments, sulfate salts in MV media were replaced by chlorides, secl8 strain was cultured at 24°C and incubated at 37°C for restrictive experiments. For derepression of the GALl promoter, YP medium was supplemented with 5% (w/v) galactose and 0.2% (w/v) sucrose (YPGS) as described by Nakano and Muramatsu [6]. Plasmids and DNA manipulations E. coli plasmid pUR288 [22] and yeast multicopy plasmid pSEY8 [18] have been described elsewhere. A yeast multicopy plasmid, pYO324 (a gift from Y. Ohya, University of Tokyo, Tokyo), has been constructed by inserting the 2.2 kb EcoRI fragment of pMK9 [23] containing the replication determinant of the yeast 2 micron circle plasmid at the Aatll site of pRS304 [24]. pANY1-9 [17], pANY2-7 (YEpSAR1) and pANY2-9 (YCpSAR1) [6] were described previously, pSHYI-1 was constructed by inserting the 1.5 kb HindIII-Sall fragment of pAHY2-7 containing the SAR1 gene at the polylinker site of pYO324. DNA manipulations including restriction enzyme digestions, ligations, plasmid isolation and E. coli transformation were carried out by standard methods [25]. Yeast transformation was performed by the quick method using lithium thiocyanate [26]. Purification of DNA fragments from agarose gel pieces was performed using the DNA PREP kit (Asahi Glass Co., Tokyo). Materials Sources of materials used in this work were as follows. Zymolyase 100T was from Seikagaku Kogyo (Tokyo). Protein A-Sepharose and proteinase inhibitors, phenylmethylsulfonyl fluoride, leupeptin, antipain, chymostatin and pepstatin A, were from Sigma (St. Louis, MO). Alkaline phosphatase-conjugated goat anti-rabbit IgG and goat anti-rabbit IgG were from Jackson lmmunoresearch Laboratory (West Grove, PA). Fluorescein isothiocyanate-conjugated anti-goat IgG and fluorescein isothiocyanate-conjugated antirabbit IgG were from Cappel (West Chester, PA). TranaSS-label, which is a mixture of L-[aSS]methionine and L.[35S]cysteine and 125I-protein A were from ICN Biomedicals Inc. (Costa Mesa, CA). Amplify and UDPN-acetyI-D-[U-14C]glucosamine were from Amersham Japan Limited (Tokyo). Enzymes for recombinant DNA methods were from Takara Shuzo (Kyoto). Rabbit antiSec12p antiserum was obtained as described [17]. Affinity purified rabbit anti-Kex2p antibody was a gift from Robert S. Fuller (Stanford University, Stanford, CA). Anti-Sarlp antibody The Nde-Sall fragment from pANY2-7 was inserted into pUR288 to make an in-frame gene fusion of lacZ
137 and SAR1. The resultant plasmid, pANF31, was introduced into the E. coli strain JM103 and the transformant was induced by isopropyl-/~-D-thiogalactopyranoside to express the IacZ-SARI fusion gene product. This hybrid protein was purified as described by Nakano et al. [17] and used to immunize rabbits. Primary injection contained 100 /xg of the protein in complete Freund's adjuvant. The first booster injection was given after 30 days. Boosts of 50/xg in incomplete Freund's adjuvant were given at 7 day intervals. After 7 boosts, blood was collected. Affinity-purified anti-Sarlp antibody was prepared for experiments of immunoblotting and immunofluorescence microscopy. The fl-galactosidase column was prepared by coupling 5 mg /3-galactosidase (from E. coil, Boehringer Mannheim Yamanouchi, Tokyo) with 2-ml packed volume of CNBr-activated Sepharose 4B (Pharmacia LKB Biotechnology, Tokyo). The LacZSarl hybrid protein column was prepared by coupling 1 mg of the purified hybrid protein with 1 ml of the same resin. Coupling was performed as described by the manufacturer. Affinity purification was carried out as described by Hadow and Lane [27]. Briefly, crude IgG fraction prepared by ammonium sulfate precipitation was first passed through the /3-galactosidase column and the flow through was applied on the LacZ-Sarl hybrid protein column. Affinity-purified anti-Sarlp antibody was eluted by 100 mM glycine-HCl (pH 2.5) and 1% (w/v) bovine serum albumin. The eluted fraction was neutralized by the addition of 1 M Hepes-KOH (pH 7.2) and concentrated using Centricon-30 (Amicon, Danvers, MA).
lmmunoblotting and immunoprecipitation Crude cell lysates or subcellular fractions were electrophoresed on 12.5% (w/v) SDS-polyacrylamide gels using Laemmli's buffer system [28]. In the case of immunoblotting with anti-Secl2p antibody, /3-mercaptoethanol was omitted from the sampling buffer and proteins were resolved on 7.5% (w/v) polyacrylamide gels. Immunoblotting was performed as described by Towbin et al. [29] with some modifications [30]. In some experiments, 1251-labeled Protein A was used in place of alkaline phosphatase-conjugated antirabbit lgG antibody [6]. Crude cell lysates were prepared as follows. Logphase yeast cells ((1-4)" 108 cells) were harvested in a 2-ml fiat-bottom microfuge tube and washed once with distilled water. Cells were resuspended in 200 /xl of 62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (w/v) glycerol, 1 mM PMSF and 5/zg/ml each of leupeptin, chymostatin, antipain and pepstatin A. To the suspension, 0.2 g of glass beads (0.4-mm diameter) were added and the mixture was vortexed in four bursts of
30 s with 30-s intervals on ice and then boiled for 5 min. For immunoprecipitation of Sarlp, cells were grown to (0.4-1)-107/ml in MVD containing 100 ttM (NH4)2SO4 and 0.5% (w/v) casamino acids for vitamin assay (Difco). Cells (2-10 7) w e r e harvested, washed once with distilled water and resuspended in 0.4 ml of MVD with no sulfate. After preincubation at 30°C for 30 rain, cells were labeled with 4 MBq of Tran35S-label for 15 min. An equal volume (0.4 ml) of MVD containing 6 mM (NH4)2SO4, 0.5 mM cysteine and 0.54 mM methionine was added and incubation continued for 45 min. Metabolic labeling was terminated by the addition of 0.8 ml of ice-cold 20 mM NaN 3 and the cell extracts were prepared as described by Nishikawa et al. [30]. To the cell extracts equivalent to 1.10 7 ~,ells, 5 /zl of preimmune or anti-Sarlp serum was added. lmmunoprecipitation and analysis by SDS polyacrylamide gel electrophoresis (PAGE) and fluorography were performed as described previously [30].
Subcellular fractionation Yeast cells were grown to 1 • 107/ml in 500 ml of MVD containing 0.5% (w/v) casamino acids. Cells (5" 109) were converted to spheroplasts as described previously [30]. Spheroplasts were resuspended in 10 ml of ice-chilled lysis buffer (10 mM triethanolamine acetate (pH 7.5), 300 mM mannitol, 100 mM KCI and 1 mM EGTA) and immediately homogenized with a morter-driven 30-ml Potter-EIvehjem homogenizer (Wheaton) five times for 1 min with l-min intervals on ice. The lysate was centrifuged at 150 × g for 5 min to remove unbroken spheroplasts. The supernatant was subjected to centrifugation at 10000 x g for 10 min at 4°C in an RPRS-14 rotor (Hitachi) to yield a low-speed pellet (LSP) and low-speed supernatant (LSS) fractions. The LSS fraction was further centrifuged at 100000 x g for 60 rain at 4°C in an RP65T rotor (Hitachi) to give high-speed pellet (HSP) and highspeed supernatant (HSS). In both centrifugations, particulate fractions were collected on a 1-ml cushion of 80% (w/v) sucrose. LSP was suspended in 80% (w/v) sucrose and placed at the bottom of a 14 × 89 mm Ultra-Clear tube (Beckman). 10 mi of 35-60% (w/v) sucrose linear gradient was overlaid on the LSP suspension and the tube was centrifuged at 151000 × g for 17 h at 4°C in an SW41-Ti rotor (Beckman). The tube was punctured at the bottom, and fractions of 0.5 ml were collected. Protein was determined by a method using BCA Protein Assay Reagent (Pierce, Rockford, IL) with bovine serum albumin as a standard. Assays of NADPH-eytoehrome c reductase [31], chitin synthetase [32], Kex2 proteinase [33], guanosine diphos-
138 phatase [34] and a-mannosidase [35] have been described elsewhere.
Extraction of Sarlp Yeast cell homogenate was prepared as described above. To 200/zl of the homogenate, 300 pl of lysis buffer and 500/~! of lysis buffer containing a chemical reagent (2 M NaCI, 4 M urea, 2% (w/v) Triton X-100, 2% (w/v) sodium deoxycholic acid, or 200 mM Na2CO~, pH 11.5)were added. The mixture was placed on ice for 30 min and centrifuged at 436 000 X g for 30 min in an RP100AT rotor (Hitachi) at 4°C. Treatment by hydroxylamine was carried out by adding 500/~1 of freshly prepared 1 M NH2OH (pH 7.0) and 300/~1 of lysis buffer to 200 ~1 of the homogenate; the mixture was incubated for 60 min at 23°C and centrifuged as described above. The pellet was gently rinsed with ice-cold distilled water and dissolved in the SDS gel sampling buffer. Proteins released into the supernatant were precipitated by the addition of trichloroacetic acid as described by Nishikawa et al. [30]. Proteins from both the pellet and supernatant fractions were subjected to SDS-PAGE and analyzed by immunoblotting. Immunofluorescence microscopy secl8 (SNS18) cells were grown to 1.107 cells/ml at 24°C in YPD medium, shifted to 37°C and incubated for 2 h. Kex2p.overproducing yeast cells (BFY125 [pBM-KX22]) were grown at 30°C in MV medium containing 0.5% (w/v) casamino acids, 5% (w/v) galactose and 0.2% (w/v) sucrose with appropriate supplements. Overproduction of Kex2p did not appear to alter its subcellular localization [36]. Formaldehyde solution (37%, w/v) and 1 M potassium phosphate (pH 6.5), were added directly to the growth medium to a final concentration of 4% (w/v) formaldehyde and 100 mM potassium phosphate and cells were fixed first at YPC for 5 min and then at room temperature for 2 h. Fixed cells were collected by centrifugation, washed with I00 mM Hepes-KOH (pH 7.2), 1 M sorbitol and 5 mM NaN 3 (spheroplasting buffer), resuspended in spheroplasting buffer containing 100 ~ g / m l Zymolyase 100T at a concentration of 1. 109/ml and incubated for 30 min at 30°C. Spheroplasts were collected by centrifugation, resuspended in spheroplasting buffer at a concentration of 1. 107/ml and attached to polylysine-coated cover slips. Permeabilization of yeast cells by methanol/acetone treatment was performed by the method of Kilmartin and Adams [37] followed by incubation in phosphate-buffered saline (PBS) [38] containing 1% (w/v) bovine serum albumin. Antibody incubation was carried out in the same buffer for 1 h at room temperature. After washing ten times with PBS containing 0.1% (w/v) bovine serum albumin, cells on the cover slip were incubated for another 1 h at room
temperature with 1 /~g/ml of anti-rabbit IgG goat antibody in PBS containing 1% (w/v) bovine serum albumin. In the experiment 9f Kex2p immunofluorescence, fluorescein isothiocyanate-conjugated antirabbit antibody (1:100 dilution) was used as the second antibody. Cells were again washed as described above and further incubated in 1 : 200 dilution of fluorescein isothiocyanate-conjugated anti-goat antibody for 1 h at room temperature. After the final (10 x ) wash with PBS containing 0.1% (w/v) bovine serum albumin, cells were observed and photographed using an Olympus BH-2 photomicroscope and T-MAX 400 film (Eastman Kodak, Rochester, NY) processed at ASA 3200 with developer (T-MAX, Eastman Kodak, Rochester, NY).
Results Identification of the SAR1 gene product To identify and characterize the SAR1 gene product, we prepared an antibody against the IacZ-SAR1 fusion gene product. The fusion protein overproduced in E. coli was purified on polyacrylamide gels and used to immunize a rabbit. The resulting antiserum showed titer against both E. coli /3-galactosidase and yeast Sarlp epitopes. Except for immunoprecipitation experiments, the anti-Sarlp antibody was used after affinity purification. Sarlp was detected in yeast cell lysates by two methods. First, wild-type yeast cells were lysed and analyzed by SDS-PAGE and immunoblotting. As shown in Fig. 1A, the antibody recognized a single band with an apparent molecular mass of 23 kDa. When the copy number of the SAR1 gene was increased by introducing a single-copy or multicopy plasmid containing the SAR1 gene, the amount of the 23-kDa protein increased according to the gene dosage of SARI (compare lanes 1-3). In the galactose-dependent sarl mutant which expresses the SAR1 gene under control of the GALl promoter [6], the 23-kDa protein was produced in a large amount only when the SAR1 gene was derepressed in galactose medium (lane 4). When SAR1 was repressed for 15.5 h in glucose medium, the 23-kDa protein could barely be detected (lane 5). From these results, we concluded that this 23-kDa protein was the SAR1 gene product, Sarlp. Sarlp was also detected by immunoprecipitation from extracts of radiolabeled cells (Fig. 1B). Wild-type yeast cells containing a multicopy plasmid with or without the SAR1 gene insert was labeled with Tran35S-label. Anti-Sarlp antiserum and Protein ASepharose precipitated from the extracts the single species of 23-kDa Sarlp (lanes 7 and 9). The amount of immunoprecipitated Sarlp increased in the cells harboring the SAR1 multicopy plasmid (lane 9). Sarlp was not precipitated by preimmune serum (lanes 6 and
139 TABLE I
A
0 (U
Distribution of marker enzymes in wild-type yeast
m
0 C
Im
The fractions analyzed were the same as those described in Fig. 2A.
o
Q.
0
ltl
Marker enzyme
Localization
% Total
, i
o
kDa m
200
-"
92.5 ._.
--"
'P I P I ......
69
LSP HSP HSS Total protein Chitin synthetase NADPH-cyt-c reductase Kex2 protease guanosine diphosphatase a-mannosidase
plasma membrane ER Golgi Golgi Vacuole
17 41 45 19 21 40
47 61 31 55 50 49
47 < 0.! 18 42 44 22
46
""
30
21.5 - - " 14.3 ~._ 1 2 3 4 5
.......
6 7 ~
Fig. ]. Identification of the SARI gene product, Sarlp. (A) Immunoblotting analysis of yeast extracts using the affinity purified anti-Sarlp antibody. Cells of ANY2]/pSEY8 (vector alone, lane 1), ANY21/ pANY2-9 (YCpSAR1, lane 2) and ANY2]/pANY2-7 (YEpSAR], lane 3) were grown overnight at 30°C in MVD medium containing 0.5% (w/v) casamino acids and 20 /tg/ml tWl,to~han. "Iile galactose-dependent sarl mutant (ANY26) was i.~cubated for 15.5 h in YPGS (Gel, lane 4) or YPD (Glc, lane 5) medium at 30°C. Extracts were prepared by agitation with glass beads in the SDS gel sampling buffer. Proteins (100/~g) were separated on an SDS-polyacrylamide gel and Sarlp was detected by immunoblc.tting. (B) Sarlp was also detected by immunoprecipitation. A wild-type strain (ANY1-7D) with a multicopy plasmid containing the SARI gene (YEpSARI, lanes 8 and 9) or vector alone (lanes 6 and 7) was pulse-labeled with Tran35S-label for 15 rain and chased for 45 min at 30°C. Extracts were prepared from the labeled cells and subjected to immunoprecipitation with the preimmune (P) or immune anti-Sarlp (I) serum in combination with protein A-Sepharose. The immunoprecipitates were analyzed by SDS-PAGE and fluorograpby.
cytochrome-c reductase, was preferentially distributed in the LSP fraction and other markers such as Kex2p for Golgi behaved differently. That the yeast ER membrane sediments rapidly unlike mammalian cell microsomes has been already reported [17,39,40], perhaps because the yeast ER membrane is mostly continuous with the nuclear envelope [41]. Each fraction was analyzed for Sarlp and Sec12p by SDS-PAGE and immunoblotting (Fig. 2). Secl2p was
A
B >~
