©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Yeast Proprotein Convertase Encoded by YAP3 Is a Glycophosphatidylinositol-anchored Protein That Localizes to the Plasma Membrane (*)

(Received for publication, April 26, 1995; and in revised form, June 15, 1995)

Josée Ash (1) Michel Dominguez (2) John J. M. Bergeron (2) David Y. Thomas (1) (2) (3) Yves Bourbonnais (1) (4)(§)

From the  (1)Eukaryotic Genetics Group, National Research Council of Canada, Biotechnology Research Institute, Montréal, Québec, Canada H4P 2R2, the (2)Department of Anatomy and Cell Biology and the (3)Department of Biology, McGill University, Montréal, Québec, Canada H3A 2B2, and the (4)Département de biochimie, Pavillon Charles-Eugène-Marchand, Université Laval, Québec, Canada G1K 7P4

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The yeast YAP3 gene encodes an aspartyl endoprotease that cleaves precursor proteins at selected pairs of basic amino acids and after single arginine residues. Biosynthetic studies of this proprotein processing enzyme indicate that Yap3 is predominantly cell-associated and migrates as a 160-kDa protein on SDS-polyacrylamide gel electrophoresis. Nearly equal amounts of Yap3 are immunodetected in a-haploid, alpha-haploid, and a/alpha-diploid yeast, demonstrating that the expression of YAP3 is not mating type-specific. As shown by endoglycosidase H treatment, which drastically reduces both the estimated molecular mass and the heterogeneity of the protein on SDS-polyacrylamide gel electrophoresis (68 versus 160 kDa), the oligosaccharides N-linked to the protein are subjected to extensive outer chain mannosylation. Outer chain sugar mannosylation takes place in the Golgi apparatus and is commonly found on yeast secreted glycoproteins and/or cell wall mannoproteins. Treatment of the total yeast membranes with chemical agents known to disrupt protein-protein and protein-lipid interactions reveal that Yap3 is membrane-associated. Based upon the release of the membrane-bound form by bacterial phosphatidylinositol phospholipase C digestion and metabolic labeling of the protein with myo-[^3H]inositol, Yap3 owes its association with the membrane to the addition of a glycophosphatidylinositol anchor. The cellular localization of Yap3 has been addressed by subcellular fractionation studies. In both differential centrifugation of intracellular organelles and sucrose density gradients, the bulk of Yap3 at steady state co-localizes with the plasma membrane azide-insensitive ATPase. Furthermore, consistent with the transport of Yap3 to the plasma membrane, the endoprotease sediments with secretory vesicles which accumulate at restrictive temperature in the late secretory mutant sec1-1. We therefore conclude that the endoprotease encoded by YAP3 is a glycophosphatidylinositol-anchored protein, which can process substrates both intracellularly and at the cell surface.


INTRODUCTION

Most peptide hormones and neuropeptides and a large number of cell surface receptors, growth factors, and viral antigens are initially synthesized as larger inactive precursors that must undergo limited endoproteolysis in the secretory pathway (Douglass et al., 1984). The yeast Saccharomyces cerevisiae proved to be a particularly useful organism for the identification of the processing enzymes involved in the maturation of precursor proteins. The alpha-haploid yeast secretes a 13-residue peptide (alpha-pheromone) which is also synthesized from a larger molecular weight precursor requiring endoproteolysis at Lys-Arg basic pairs (Kurjan and Herskowitz, 1982). Genetic complementation of yeast mutants affecting the maturation of the alpha-pheromone precursor has led to the isolation of the genes encoding a Lys-Arg-specific endoprotease related to the subtilisin protease family (KEX2), a carboxypeptidase involved in the trimming of the COOH-terminal Lys-Arg dipeptide (KEX1) and a dipeptidyl aminopeptidase that removes the Glu-Ala and Asp-Ala dipeptides present on the NH(2)-terminal of the immature alpha-pheromone peptides (STE13) (see Fuller et al. (1988), Bussey(1988), and Bourbonnais et al. (1991a) for reviews). All three processing enzymes are transmembrane proteins that are retained in the yeast Golgi apparatus by their cytoplasmic tail (Redding et al., 1991; Cooper and Bussey, 1992; Roberts et al., 1992; Wilcox et al., 1992).

More recently, the heterologous expression of somatostatin precursors in yeast identified a novel endoproteolytic activity capable of cleaving at monobasic processing sites (single arginine residue) to release mature somatostatin-28 (Bourbonnais et al., 1991b). Genetic complementation of a mutant strain (sex1-1) unable to release the alpha-pheromone peptide when synthesized from a prosomatostatin/alpha-pheromone chimeric protein (monobasic processing site) led to the cloning of a non-essential gene coding for an endoprotease homologous to the aspartyl protease family (Bourbonnais et al., 1993). This gene (YAP3) was also independently cloned based upon its ability, when overexpressed, to partially correct the processing defect of a kex2 null mutant (Egel-Mitani et al., 1990). The KEX2 gene product is the prototype endoprotease of a processing enzyme family known as ``prohormone convertase'' recently identified in higher eukaryote based upon their structural similarity with the yeast enzyme (Hutton, 1990; Seidah et al., 1991; Steiner et al., 1992). By analogy, the isolation of higher eukaryote candidate processing enzymes characterized as aspartyl proteases (Loh et al., 1985; Mackin et al., 1991) suggests that the yeast YAP3 gene product may constitute the prototype of a novel family of proprotein processing enzymes.

The Yap3 protein has been purified from an overexpressing yeast strain as a 68-kDa glycoprotein (Azarian et al., 1993). Its sensitivity to pepstatin A and optimal pH of activity (4.0) confirmed that it is an aspartyl protease. Although additional work is clearly needed (namely the measure of precise kinetic parameters) to firmly establish the specificity of Yap3, studies performed in vivo (Egel-Mitani et al., 1990; Bourbonnais et al., 1993) and in vitro (Azarian et al., 1993; Cawley et al., 1993; Bourbonnais et al., 1994) indicates that Yap3 can cleave both at single arginine and paired basic residues normally used as processing sites in proproteins. Also, although overlapping with that of Kex2, the specificity of Yap3 appears to be distinct (Bourbonnais et al., 1994). The COOH-terminal domain of Yap3 is predicted to anchor the protein to membranes and its deletion completely abolished the Yap3-dependent release of the mature alpha-pheromone peptide observed in a kex2 null mutant (Egel-Mitani et al., 1990). A possible explanation provided by these authors is that in the absence of this COOH-terminal domain, Yap3 is mislocalized. Experimental data demonstrating that Yap3 is a membrane-bound endoprotease are however lacking, and although several lines of evidence suggested that the Yap3-dependent processing of the alpha-pheromone (EgelMitani et al., 1990) and somatostatin (Bourbonnais et al., 1991b, 1993) precursors is occurring in an intracellular compartment, the intracellular localization of Yap3 has not been determined.

As a first step toward elucidating the physiological function of Yap3, we have, in this present report, studied its biosynthesis and intracellular localization.


MATERIALS AND METHODS

Strains, Plasmids, Growth Conditions, and Yeast Transformation

The yeast strains used were DS7 (MATalpha, mfalpha1::LEU2, mfalpha2::LEU2, ade2, leu2, his3, trp1, ura3; Bourbonnais et al., 1993), YBAD1 (MATalpha, yap3Delta::HIS3, mfalpha1::LEU2, mfalpha2::LEU2, ade2, leu2, his3, trp1, ura3; Bourbonnais et al., 1993), which is derived from the parental strain DS7, and Y1-1A (MATalpha, sec1-1, leu2, trp1, ura3; kindly provided by M. Whiteway, Biotechnology Research Institute, Montreal, Canada) which is a segregant resulting from the cross of strains SEY 5018 (MATalpha, sec1-1, leu2, ura3; kindly provided by R. Schekman, University of California, Berkeley, CA) with W303-1a (MATalpha, ade2, leu2, his3, trp1, ura3). Plasmids YEp24 and p9A242 (which has the plasmid YEp24 backbone but contains the entire YAP3 gene) have been described previously (Bourbonnais et al., 1993). Cells were grown in rich medium (YPD) containing 1% Bacto Yeast extract, 2% Bacto-peptone (Difco), and 2% dextrose or in drop out synthetic complete medium containing 2% dextrose, 0.67% yeast nitrogen base without amino acids (Difco), and 0.15% of a drop out mix containing adenine and all amino acids, but lacking uracil, in the amounts described in Sherman et al.(1986). For metabolic labeling yeast were grown in synthetic minimal medium containing 2% dextrose and either 0.67% yeast nitrogen base without amino acids (labeling with TranS-label) or 1.67% vitamin-free yeast nitrogen base supplemented with all the vitamins (but lacking inositol for labeling with myo-[^3H]inositol) in the amounts recommended in the Difco manual and supplemented for auxotrophic requirements. Yeast transformation was performed according to the procedure described by Schiestl and Gietz(1989) with lithium acetate.

Production of Yap3 Antibodies

The 636-base pair EcoRI fragment of the YAP3 gene was subcloned into pGEX-3X (Pharmacia Biotech Inc.) to create a chimeric gene encoding the bacterial Glutathione S-transferase fused in frame with amino acid residues 35 to 246 of Yap3. Expression of the resulting fusion protein (47 kDa) in E. coli (strain JM 83) was performed essentially as described by the manufacturer. Under these conditions the GST-Yap3 chimeric protein was quantitatively recovered in inclusion bodies and purification was achieved by SDS-polyacrylamide gel electrophoresis (PAGE) (^1)separation of the solubilized (by boiling in SDS-PAGE sample buffer) protein aggregate. After staining the polyacrylamide gel with 0.3 M CuCl(2), the prominent 47-kDa band was excised and electroeluted with Tris-glycine buffer for 3 h at 20 mA (Bio-Rad Electro-Eluter). Samples were then dialyzed overnight in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, lyophilized, and resuspended in 50 µl of the same buffer. For immunization, 100 µg of the fusion protein in complete (first injection) or incomplete (boost injections) Freund's adjuvant was injected intramuscularly (50 µg/thigh) to rabbits according to the following immunization protocol: day 0, first injection; day 21, 42 and 70 boost injections and; day 31, 52 and 80 bleedings (10-15-ml blood samples taken by the ear artery). The Yap3 antiserum used in this study was from the second bleeding. For immunoprecipitations, the Yap3 antiserum was affinity-purified. To deplete the antiserum of glutathione S-transferase-specific antibodies, it was first loaded on a column of Sepharose beads coupled with a nonrelated GST-Yap3 fusion protein (containing the amino acids 246-530 of Yap3). The unbound material was then incubated at 4 °C for 3 h with a specific resin (Sepharose beads coupled to the fusion protein used as the immunogen), the column washed successively with 10 volumes of phosphate-buffered saline (PBS), and PBS containing 2 M KCl, respectively, and the affinity-purified antibodies eluted with 10 volumes of 0.2 M glycine, pH 2.4. Fractions containing proteins (as measured by the absorbance at 280 nm) were pooled, dialyzed extensively (40 h at 4 °C) against PBS, and the antibody solution was concentrated 3-fold by filtration/centrifugation in a Centricon-30 device (Amicon).

Metabolic Labeling, Immunoprecipitation, Immunoblotting, and Endoglycosidase H Treatment

Exponentially growing yeast were labeled for 30 min at 30 °C with either 250 µCi/ml of TranS-label (ICN) or 200 µCi/ml of myo-[^3H]inositol (Dupont NEN) essentially as described previously (Bourbonnais et al., 1991b). Cell pellets and media were separated by low speed centrifugation and the cells, resuspended in ice-cold sterile water containing 80 µg/ml of aprotinin, were broken by vortexing (6 30 s) with an equal volume of glass beads. After addition of SDS (1% final concentration) the slurry was immediately boiled for 2 min and the yeast lysate clarified by centrifugation (5 min at 10,000 g). The cell lysates were then diluted 10-fold, and both the media and cell lysates were adjusted to 1 buffer A (50 mM Tris-HCl, pH 8.3, 190 mM NaCl, 6 mM EDTA, 2.5% Triton X-100, and 1 mg/ml bovine serum albumin (BSA)) supplemented with a mixture of protease inhibitors (20 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM pepstatin) and 5 mM each of cysteine and methionine. Immunoprecipitation with the affinity-purified Yap3 antibodies (1/500 final dilution) was allowed to proceed at room temperature for 2 h. At the end of this period, protein A-Sepharose (Pharmacia) was added and the incubation continued for an additional 90 min. The protein A-Sepharose beads were then collected by low speed centrifugation, washed twice with buffer B (10 mM Tris-HCl, pH 8.3, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, and 1 mg/ml BSA) and twice with PBS. The immune complexes were released from the beads by boiling for 3 min in either SDS-PAGE sample buffer or alternatively, for samples to be subjected to endoglycosidase H (Endo H) treatment, SDS elution buffer (1 mM Tris-HCl, pH 7.5, 40 mM dithiothreitol (DTT), 0.5% SDS).

Yeast lysates used for immunoblotting with anti-Yap3 antibodies were prepared with the glass beads procedure and processed for SDS-PAGE essentially as described above. The electrophoretic transfer of proteins to nitrocellulose filters (BA85 0.45 µm; Schleicher & Schuell), blocking of nonspecific sites, incubations with primary (anti-Yap3 used at a 1/1000 dilution) and secondary antibodies conjugated to alkaline phosphatase (Bio-Rad) (1/3000 dilution), and color detection were performed essentially according to the specifications of the supplier (Bio-Rad).

For Endo H treatment, immunoprecipitates (in SDS elution buffer) were diluted 2-fold (0.25% SDS final concentration) adjusted to a final concentration of 0.2 M in ammonium acetate, pH 5.0, and incubated at 37 °C for 4 h in the presence of 5 milliunits of Endo H (Boehringer Mannheim). Membrane preparations and fractions from the sucrose density gradient (where indicated) were processed similarly (adjusted to 0.2% SDS, 0.2 M ammonium acetate, pH 5.0) but were not boiled prior to incubation at 37 °C for 4 h in the presence of Endo H (5 milliunits). The samples were then adjusted to 1 concentration in SDS-PAGE sample buffer, boiled for 3 min, and either immediately loaded on SDS-PAGE or frozen at -20 °C until used.

Preparation of Total Yeast Membranes, Chemical, and Enzymatic Treatments

Exponentially growing yeast (A/ml 1.5) were centrifuged, and the pellet washed once with sterile water and once with buffer S (1.2 M sorbitol, 2 mM potassium phosphate, pH 7.4, 5 mM DTT). The cells were then resuspended in 1/40 volume of the original culture medium in buffer S supplemented with a mixture of protease inhibitor (1 mM PMSF, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 4 µg/ml pepstatin A) and 1 mg/ml zymolyase 100-T (ICN Biomedicals) and incubated at 30 °C for 60 min with shaking. Yeast spheroplasts were then gently centrifuged at 3000 g for 5 min over a sucrose/Ficoll cushion (0.8 M sucrose, 1.5% Ficoll, 20 mM HEPES, pH 7.4), the pellet was resuspended in 1/50 volume of lysis buffer (0.1 M sorbitol, 20 mM HEPES, pH 7.4, 2 mM EDTA, 1 mM DTT) supplemented with a mixture of protease inhibitors (see above), and the spheroplasts were lysed by 10 strokes of the pestle in a 5-ml Potter-Elvehjem homogenizer (Wheaton). The lysate was centrifuged at 1000 g for 10 min, the supernatant was saved, and the pellet was homogenized again followed by centrifugation at 1000 g. The two supernatants were pooled, and the total yeast membranes were prepared by centrifugation at 4 °C of this post-nuclear lysate for 30 min at 100,000 g (rotor TLA-45; Beckman). Membranes were resuspended in TBS buffer (100 mM Tris-HCl, pH 7.5, 30 mM NaCl) supplemented with a mixture of protease inhibitors (see above) and aliquots were either incubated on ice for 30 min with an equal volume of a TBS buffer containing one of the various salts or detergents in 2 concentration (see Fig. 3and Fig. 4) or at 37 °C for 30 min with 40 units and 0.36 unit of the bacterial phosphatidylinositol phospholipase C (PI-PLC) purified from Bacillus cereus (Sigma) and Bacillus thuringiensis (ICN Biomedicals), respectively. Following these treatments, the samples were centrifuged at 100,000 g for 30 min as described above, and the supernatants and pellets were saved and submitted to Endo H treatment prior to SDS-PAGE and immunoblotting.


Figure 3: The endoprotease Yap3 is a membrane-bound protein. Equivalent amounts of total yeast membranes prepared from a YAP3-overexpressing strain (yap3Delta (p9A242)) were incubated in the presence of the indicated reagents and then centrifuged at 100,000 g for 30 min. Supernatants (S) and pellets (P) were then separated, submitted to Endo H treatment, and analyzed on SDS-PAGE. The Yap3 protein was detected by immunoblotting with Yap3 antibodies and secondary antibodies conjugated to alkaline phosphatase. The 68-kDa molecular mass marker is indicated on the left. CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.




Figure 4: The endoprotease Yap3 is a GPI-anchored protein. A, equivalent amounts of total yeast membranes prepared from a YAP3-overexpressing strain (yap3Delta (p9A242)) were incubated in the presence of the indicated reagents (see ``Materials and Methods''), processed as described in Fig. 3and subjected to Yap3-specific immunoblotting. S, 100,000 g supernatant, P, 100, 000 g pellet. The 68-kDa molecular mass marker is indicated on the left. B, yeast strains either carrying a single disrupted copy of YAP3 (yap3Delta) or overexpressing the YAP3 gene (yap3Delta [p9A242]) were metabolically labeled with myo-[^3H]inositol (see ``Materials and Methods''), and cell extracts were immunoprecipitated with anti-Yap3 antibodies. Following Endo H treatment, the labeled proteins were then analyzed by SDS-PAGE. The arrow on the right of the figure points to a major band of 68 kDa, which can only be detected in the YAP3-overexpressing strain. Molecular mass markers are indicated on the left.



Cell Fractionation and Sucrose Density Gradient

Mid-log phase yeast cultures (A/ml between 2.0 and 4.0) grown at 30 °C (strain DS7) or 24 °C and then either shifted to 37 °C or maintained at 24 °C for an additional 3 h (strain Y1-1A, sec1-1, and strain DS7, SEC1) were converted to spheroplasts and lysed as described for the preparation of total yeast membranes (see above) with the following modifications. Cell pellets were first washed with 100 mM Tris-HCl, pH 9.4, 10 mM NaN(3), incubated for 15 min at 30 °C in the same buffer supplemented with 10 mM DTT, and then converted to spheroplasts in 0.7 M sorbitol, 1.5% Bacto-peptone, 0.25% yeast extract, 0.5% glucose, 10 mM Tris-HCl pH 7.4, 1 mM DTT containing 1 mg/g of yeast cells (wet weight) of zymolyase 100-T. Lysis was performed in a motor-driven PotterElvehjem homogenizer (10 strokes) in 0.1 M sorbitol, 20 mM HEPES, pH 7.4, 50 mM potassium acetate, 0.25 mM PMSF, 0.5 µg/ml aprotinin, 2 mM DTT. During this procedure cells were maintained at a concentration of 100 A units/ml. The resulting lysate was first centrifuged at 1000 g for 5 min, the pellet was resuspended in 0.5 volume of lysis buffer, homogenized again, and then centrifuged at 500 g for 5 min to generate the nuclear(N) fraction. The post-nuclear lysate (fraction E) consisting of the pooled supernatants was further subjected to differential centrifugation in a Ti60 rotor (Beckman), first at 18,500 rpm (25,000 g) for 8 min to give a granule (ML) fraction (this step was done twice) and the resulting supernatant centrifuged at 45,000 rpm (150,000 g) for 90 min to give the microsomal (P) fraction. An overview of the cell fractionation procedure is shown in Fig. 5.


Figure 5: Outline of the cell fractionation procedure. The 2X indicates that this centrifugation step was performed twice, the second being performed with the resuspended pellet of the first spin.



Membranes from the ML and P fractions were also loaded on top of a linear sucrose density gradient (from 0.5 M to 2.0 M in 20 mM HEPES, pH 7.4) and subjected to isopycnic centrifugation (8 h at 38 000 rpm in the Beckman SW 40 rotor). Fractions (18) were collected from the top of the tubes with an Autodensi-flow II apparatus (Searle Analytic Inc.), and the density of each fraction was determined by refractometry.

Organelle Marker Assays and Quantitative Immunoblotting

Organelle marker enzymes were assayed according to published procedures for the plasma membrane azide-insensitive ATPase (Bowman and Slayman, 1979), the NADPH cytochrome c reductase (Kubota et al., 1977), and the GDPase (Abeijon et al., 1989) activities. Quantitative Yap3, Vph1, and Kex1 immunoblottings were performed with I-protein A (Amersham Corp.). The radioactive immunoblots were quantitated with a PhosphorImager instrument (Molecular Dynamics). The recovery of each organelle marker was determined using the following equation.

Purified anti-Vph1 antibodies were kindly provided by Morris Manolson, Hospital for Sick Children, Toronto, Canada, and used at a 1/2000 final dilution. Purified anti-Kex1 antibodies were generously donated by Howard Bussey, McGill University, Montreal, Canada. Total protein content was determined by the Bio-Rad protein assay using BSA as standard.


RESULTS

The Yap3 Endoprotease Is a Hyperglycosylated Protein of 160 kDa That Is Expressed in the Three Yeast Cell Types

The yeast YAP3 gene encodes a 569-residue protein, which can tentatively be separated in five distinct domains (Fig. 1A). In addition to the catalytic domain that has the greatest sequence similarity with other members of the aspartyl protease family, there is also a hydrophobic segment of 21 residues at the extreme NH(2)-terminal end of the protein that presumably acts as a signal peptide. This domain is followed by a putative proregion of 46 residues that is flanked at the COOH terminus by a Lys Arg pair, suggesting that Yap3 is initially synthesized as a zymogen form. The catalytic domain is followed at the COOH terminus by a 43-residue Ser/Thr-rich domain. Finally, the sequence of the endoprotease ends with a stretch of hydrophobic amino acids (19 residues), which are predicted to anchor the protein to membranes. Inspection of the sequence of Yap3 also reveals the presence of 10 potential N-linked glycosylation sites dispersed throughout the molecule.


Figure 1: Immunoprecipitation of Yap3 from total yeast extracts metabolically labeled with [S]methionine. A, schematic representation of Yap3. The YAP3 gene encodes a 569-residue aspartyl protease composed of a putative NH(2)-terminal signal peptide (filled in box on the left), a putative ``proregion'' (open box with wavy lines), a catalytic domain (open rectangle), a serine/threonine-rich domain (open box with thick diagonal line), a hydrophobic domain (shaded box on the right), and several N-linked glycosylation sites (small circle with vertical line). The two asterisks below the structure of Yap3 point to the aspartyl residues forming the catalytic site of the enzyme. The bar below indicates the region of the Yap3 which was used, as a fusion protein, to immunize rabbits. B, total cell extracts were prepared from yap3-disrupted (yap3Delta), wild type (YAP3), and YAP3-overexpressing (yap3Delta (p9A242)) yeast metabolically labeled with TransS-label. They were subjected to immunoprecipitation with anti-Yap3 antibodies, and the proteins were treated with Endo H prior to analysis by SDS-PAGE (see ``Materials and Methods''). The arrow points to a major 68-kDa band immunoprecipitated by Yap3 antibodies. Molecular mass markers are indicated on the left. C, total cell extracts were prepared from metabolically labeled YAP3-overexpressing yeast, and the labeled proteins were immunoprecipitated with anti-Yap3 antibodies and either incubated with (+Endo H) or without (-Endo H) Endo H prior to analysis by SDS-PAGE. The asterisk points to the diffuse pattern of hyperglycosylated Yap3, and the arrow shows the position of migration of Yap3 after Endo H treatment. Molecular mass markers are indicated on the left.



Polyclonal antibodies were raised against a portion of Yap3 comprising amino acids 35-246, and these were used to immunoprecipitate the endoprotease from metabolically labeled yeast (Fig. 1B). When the intracellular extracts were first submitted to Endo H treatment prior to resolution by SDS-PAGE, a band of approximately 68 kDa was detected in yeast containing the genomic copy of YAP3 (DS7), but not in yap3-disrupted yeast (YBAD1; yap3Delta::HIS3). A few smaller molecular weight species were also observed. However, they are not related to Yap3, as they were present in the lysates of both the wild type and yap3-disrupted strains. That the 68-kDa protein corresponds to the YAP3 gene product was further confirmed by the increase in intensity of this band when the YAP3 gene was expressed from a multicopy plasmid (YBAD1 (p9A242)). Expressing YAP3 from a 2-µm plasmid yielded a 8-fold increase in the production of the protein based on densitometric scanning of the fluorogram. In the absence of Endo H treatment, Yap3 migrated on SDS-PAGE as a diffuse band with an average molecular mass of approximately 160 kDa which was best observed when YAP3 was overexpressed (Fig. 1C). This smear replaced the 68-kDa protein detected after Endo H treatment. The diffuse migration of yeast glycoproteins on SDS-PAGE is typical of a post-translational modification known as hyperglycosylation. It consists of the addition of outer chain mannose residues (up to 50 residues/oligosaccharyl side chain) in the Golgi apparatus (Kukuruzinska et al., 1987). Nearly identical amounts of Yap3 could be immunodetected in extracts prepared from MAT a haploid, MAT alpha haploid, and MAT a/alpha-diploid yeast (Fig. 2). We therefore conclude that the endoprotease encoded by YAP3 is a hyperglycosylated protein that is expressed in all three yeast cell types.


Figure 2: The Yap3 protein is expressed in all three yeast cell types. Total intracellular proteins was prepared from a set of isogenic strains differing by their mating types: DS7 diploid (a/alpha), DS7 MAT alpha (alpha), and DS7 MAT a (a). An equivalent amount was analyzed by SDS-PAGE, and the Endo H-treated Yap3 protein was detected by immunoblotting with anti-Yap3 antibodies and I-protein A. This figure presents the corresponding autoradiogram (16-h exposure) on a Kodak AR X-Omat film. Molecular mass markers are indicated on the left.



The Yap3 Endoprotease Behaves as an Integral Membrane Protein

In a previous study we showed that the Yap3 endoproteolytic activity was recovered in the yeast membrane pellet (Bourbonnais et al., 1994). In addition we found no Yap3-immunoreactive material in the culture medium of a Yap3-overexpressing strain (YBAD1 (p9A242)) pulse-labeled for 30 min (not shown). These results suggest that Yap3 is either soluble but maintained in intracellular organelles or a membrane-bound protein. The possible interaction of Yap3 with membranes was investigated more thoroughly by subjecting the membrane fraction (100,000 g pellet) to various chemical agents that differentially release peripheral and integral membrane proteins. Following these treatments, the membranes were spun at 100,000 g and the presence of Yap3 in the supernatants and pellets was detected by immunoblotting (Fig. 3). In the absence (mock-treated) or presence of reagents that disrupt protein-protein interaction or protein-lipid interaction and therefore release peripheral membrane proteins and/or soluble proteins in sealed compartments (urea, sodium chloride, and sodium bicarbonate), Yap3 was always quantitatively recovered in the membrane fraction. In contrast, and as would be expected for an integral membrane protein, Yap3 was found predominantly in the soluble fraction after treatment with any of the detergents used. In these experiments the molecular mass of Yap3 associated with the membrane pellets was consistently 4 kDa higher than the 68-kDa species observed in the soluble fraction. We believe that this simply reflects a differential deglycosylation of the protein by Endo H. This probably results from the limited access of one or two of the oligosaccharide side chains when the protein is associated to membranes. In support of this hypothesis, when samples were first boiled in SDS before Endo H treatment (immunoprecipitated samples; Fig. 1and 4B), the upper molecular mass species, if detected, was only a very minor species. Under milder denaturing conditions, however, the glycosidase treatment led instead to variable ratios of the 72- and 68-kDa species ( Fig. 3and Fig. 4A).

The Yap3 Endoprotease Is Modified by a Glycophosphatidylinositol (GPI) Group

The domain of Yap3, which is predicted to anchor the protein to the membrane, is located at the very COOH-terminal end of the molecule (Fig. 1). The absence of any putative cytosolic tail is not typical of most type I integral proteins and rather suggests that the protein is anchored to the membrane by a GPI group. The presence of a GPI anchor in Yap3 was therefore investigated. A common way to diagnose the presence of this structure in a membrane-bound protein is to incubate the membrane fraction with bacterial PI-PLC which, by releasing the fatty acids from the anchor, breaks the association of the protein with the membrane. When the membrane fraction was incubated with the PI-PLC purified from B. cereus, Yap3 remained associated with the pellet fraction following a 100,000 g spin (Fig. 4A, B. cereus). However, nearly 50% of Yap3 was released in the soluble fraction upon treatment of the membranes with the PI-PLC purified from B. thuringiensis (Fig. 4A, B. thuringiensis). That this partial release was specifically mediated by PI-PLC was confirmed by control incubations of the membrane fraction with either the buffer alone or sodium bicarbonate, in each case resulting in the quantitative recovery of Yap3 in the subsequent 100,000 g pellet, or deoxycholate, which in contrast led to the complete solubilization of Yap3. To establish that Yap3 is indeed a GPI-anchored protein, metabolic labeling of Yap3 with a radioactive precursor of the GPI core structure was attempted (Fig. 4B). A labeled protein of 68 kDa (after Endo H treatment) could be specifically immunoprecipitated from a YAP3-overexpressing strain (YBAD1 (p9A242)). However, no radioactive material could be immunoprecipitated with anti-Yap3 from the yap3-null mutant (YBAD1 (YEp24)) pulse incubated for 30 min with myo-[^3H]inositol. We therefore conclude that Yap3 is anchored to the membrane by virtue of a GPI group that is post-translationally transferred to the protein.

The Yap3 Endoprotease Localizes with a Marker of the Plasma Membrane in Subcellular Fractionation Studies

The cellular localization of Yap3 was studied by subcellular fractionation according to the scheme shown in Fig. 5. First, the distribution of Yap3 was compared with that of the various organelle markers using differential centrifugation to separate components approximately according to their size (de Duve, 1975). In wild type yeast (strain DS7) lysate the bulk of the total protein was found in the soluble fraction (fraction S; 55.9%) with 13.0, 10.8, and 20.3% of protein in the fractions N, ML, and P, respectively. The vacuolar marker Vph1 (a subunit of the vacuolar membrane-bound ATPase) (Manolson et al., 1992) sedimented predominantly in the N fraction (43.4%; 3.3-fold enrichment), though a significant amount (33.3%; 3.1-fold enrichment) was also found in the ML fraction, whereas nearly 50% (49.1%; 2.4-fold enrichment) of the GDPase activity (a Golgi marker) was associated with the P fraction (Fig. 6). This is in sharp contrast with the distribution of both the endoplasmic reticulum (ER) marker (NADPH cytochrome c reductase; 41.0 and 54.8% in the N and ML fraction, respectively) and the plasma membrane azide-insensitive ATPase activity (57.4% in fraction ML). The relative abundance of Yap3 in these fractions (30.7, 58.4, and 10.8% for fractions N, ML, and P, respectively) was clearly distinct from both the vacuole and Golgi markers and approximated the distribution of the plasma membrane and/or the ER markers. These were enriched 5.3- and 5.1-fold, respectively, in the ML fraction compared with 5.4-fold for Yap3. To distinguish between these compartments the intracellular localization of Yap3 was further investigated by submitting the ML and P fractions to equilibrium sucrose density gradient.


Figure 6: Distribution of organelle markers in the various differential centrifugation fractions. The distribution of the organelle markers is expressed as a de Duve plot (Parlati et al., 1995). The nuclear (N), large granule (ML), microsomal (P), and cytosolic (S) fractions are indicated. The recovery of each organelle marker calculated as described under ``Materials and Methods'' is shown in brackets.



When the ML fraction was loaded on top of a linear sucrose density gradient and submitted to isopycnic centrifugation, the distribution of Yap3 was similar to that of the plasma membrane ATPase activity, with median densities of 1.1716 and 1.1633 g/ml respectively (Fig. 7, left panel). This median density of Yap3 was similar to that of the ER marker (NADPH cytochrome c reductase; median density 1.1710 g/ml), although the distribution of the ER marker appeared different, the main peak of activity being shifted toward the denser fractions (around 1.1850 g/ml). The distribution of Yap3 was distinct from that of the vacuolar marker Vph1 (median density, 1.1256 g/ml). The total membrane-bound proteins of the ML fraction were distributed across the gradient very similarly to the ER marker. The distribution of the very small amount of Yap3 (<11%) present in the P fraction was also compared with that of the GDPase activity and the Kex1 protein (Fig. 7, right panel). Both GDPase activity and Kex1 revealed similar distributions and median densities (1.1228 and 1.1296 g/ml, respectively), which were distinct from that of Yap3. The distribution of Yap3 was similar to that observed in the ML fraction (main peak centred at 1.1600-1.1700 g/ml), although the median densities (1.1531 g/ml compared with 1.1716 g/ml in the ML fraction) were slightly different. This difference in median densities might possibly result from the transient accumulation of Yap3 in the ER and the Golgi apparatus at steady state. The total protein content of fraction P was, compared with that observed for the ML fraction, shifted toward the light fractions of the gradient. Hence, at steady state, Yap3 co-localized with a plasma membrane marker in both differential centrifugation fractions and in linear sucrose density gradients.


Figure 7: Sucrose density gradient analysis of the distribution of Yap3 and organelle markers in the parent ML and P fractions. The distribution of Yap3 from the parent ML fraction (left side) was compared with organelle markers for the plasma membrane (ATPase), the ER (NADPH cytochrome c reductase (NADPH cyt c)), the vacuole (Vph1), and to total proteins. The small amount of Yap3 recovered in the parent P fraction (right side) was also compared with that of two Golgi markers (GDPase and Kex1 protein) and to total proteins. The quantitative distribution of Yap3 and organelle markers was evaluated as described under ``Materials and Methods.'' The median densities for the distribution of the respective constituents are indicated.



The Cellular Distribution of Yap3 Is Altered in the Secretory Mutant sec1-1

The yeast sec1-1 mutant accumulates secretory vesicles at the nonpermissive temperature (Novick et al., 1981). These vesicles require a high centrifugal force (i.e. 100,000 g) to sediment (Goud et al., 1988) and would therefore be expected to accumulate in fraction P by differential centrifugation. We therefore tested the distribution of Yap3 in the sec1-1 mutant to evaluate if Yap3 might also accumulate in secretory vesicles. When grown at the permissive temperature, the distribution of Yap3 in the sec1-1 mutant was very similar to that observed previously in wild type yeast (strain DS7, Fig. 6) with 32.8, 59.4, and 5.9% of the proteins in fractions N, ML, and P, respectively (Fig. 8). However, upon shifting the strain to the restrictive temperature for 3 h, the relative amount of Yap3 in the ML fraction was drastically reduced (59.4 versus 18.7%), and a significant increase of the protein in the P fraction was observed (from 5.0 to 32.3%). Control experiments revealed that the temperature shift itself increased the expression of Yap3 by 12.1-fold as evaluated by quantitative immunoblotting followed by densitometry (not shown). However, the temperature shift alone did not markedly affect the distribution of Yap3 in a SEC1 strain (strain DS7) except for a slight increase in Yap3 immunoreactivity in the P fraction (8.7% at 24 °C as compared with 13.3% at 37 °C). Hence, as would be predicted for a plasma membrane targetted protein, Yap3 accumulated in vesicles sedimenting in the P fraction in the sec1-1 mutant.


Figure 8: Distribution of Yap3 in differential fractions obtained from the sec1-1 mutant yeast grown at 24 and 37 °C. Fractions N, ML, and P were prepared, as outlined in Fig. 5, from the sec1-1 mutant (sec1) yeast grown at the indicated temperatures and the distribution of Yap3 determined by quantitative immunoblotting. The relative distribution of Yap3 is expressed in percentage of the total amount recovered in the three subcellular fractions (N + ML + P).




DISCUSSION

The yeast YAP3 gene encodes a novel proprotein processing enzyme (Egel-Mitani et al., 1990; Bourbonnais et al., 1993), which may represent the yeast homolog of two candidate convertases purified from higher eukaryotes: a 70-kDa paired basic residue-specific aspartic protease purified from bovine pituitaries, which cleaves in vitro proopiomelanocortin to a subset of biologically active peptide (Loh et al., 1985), and a 39-kDa endoprotease purified from anglerfish pancreatic islets, which converts in vitro prosomatostatin-II to mature somatostatin-28 by cleavage at a single arginine residue (Mackin et al., 1991). Previous studies have concentrated primarily on enzymatic properties of Yap3 (Azarian et al., 1993; Cawley et al., 1994; Bourbonnais et al., 1994). To date, however, no physiological substrates have been identified for this endoprotease. The aim of the present study was to characterize biochemically and localize intracellularly the YAP3 gene product as a first step toward elucidating its functional role.

Through biosynthetic studies we have shown that Yap3 is a heterogenous glycoprotein of 160 kDa and that the N-linked sugar moieties contributed for approximately 92 kDa of this mass, suggesting that they are modified by outer chain mannose residues. This is in sharp contrast with the molecular mass of 68 kDa reported for the purified endoprotease (Azarian et al., 1993). In that study, Yap3 was purified from a Triton X-100-solubilized yeast extract submitted to affinity chromatography, first on a concanavalin A-Sepharose column and then on a pepstatin A-immobilized support. The first purification step on concanavalin A-Sepharose may explain the discrepancy between our estimated molecular mass and that reported for the purified enzyme. For example, it is possible that hyperglycosylated Yap3 was bound so tightly to the lectin that elution by 0.5 M alpha-methyl-D-mannopyranoside released only minor species of Yap3 corresponding to core-glycosylated molecules. Alternatively, the level of overexpression used by these authors may have resulted in the accumulation of significant amounts of mislocalized core-glycosylated Yap3, yet fully active molecules. The molecular mass of the Endo H-treated protein (68 kDa) is significantly larger than that predicted from the amino acid sequence of either the putative proform (51 kDa) or mature form (44 kDa; i.e. lacking the putative prosegment). This suggests that Yap3 may be submitted to O-glycosylation, presumably in the Ser/Thr-rich domain. There is no apparent consensus signals for O-glycosylation in yeast, however, the presence of a Ser/Thr-rich domain is an obvious feature of O-mannosylated yeast proteins (Orlean et al., 1991). The fact that the 68-kDa species did not migrate as a sharp band on SDS-PAGE would be consistent with heterogeneity in the addition of the O-linked sugars. Confirmation of this hypothesis, and the nature of the NH(2)-terminal end of the protein, however, awaits additional studies.

We have shown here that Yap3 is a GPI-anchored protein. A great number of proteins from different species and of diverse biological functions are known to be submitted to this post-translational modification. The unifying property of GPI-anchored proteins is that they rely entirely on the glycolipid structure for their membrane interaction (Cross, 1990). With few exceptions (some proteins of pancreatic and adrenal secretory granules), the vast majority of known GPI-anchored proteins are associated with the plasma membrane. It is unclear why some proteins are bound to membranes by this mechanism. However, it was demonstrated that GPI-anchored proteins have a greater mobility in membrane (about 10-fold) than transmembrane proteins, with diffusion coefficients comparable with lipids. They can also be regulated by specific phospholipases that have been shown to trigger their release into the extracellular milieu. This could, for instance, mediate the rapid down-regulation of receptors or the release of enzymes into the culture medium (Cross, 1990). Such a regulation by phospholipases might explain why significant amounts of Yap3 could be recovered or immunodetected in the culture medium of an overnight yeast culture (Cawley et al., 1993), (^2)whereas the presence of secreted Yap3 could not be demonstrated after a 30-min pulse labeling (not shown). In the yeast Saccharomyces cerevisiae, Yap3 is only the third characterized protein known to be GPI-anchored. A 125-kDa plasma membrane glycoprotein of unknown function, called Gas1, was shown previously to be modified by this lipid moiety based upon PI-PLC treatment, partition in Triton X-114, and metabolic labeling with myo-[^3H]inositol and [^3H]palmitic acid (Conzelmann et al., 1988). More recently, precursor forms of the cell wall alpha-agglutinin protein were also shown to contain a GPI anchor that was lost before cell wall association (Wojciechowicz et al., 1993; Lu et al., 1994). Interestingly, based upon sequence similarity between the carboxyl termini of Gas1 and Yap3, it was hypothesized that Yap3 would be modified by a GPI anchor (Nuoffer et al., 1991). Very little is known about the cellular machinery implicated in the recognition of, and the transfer of GPI anchors to, proteins destined to this post-translational modification. The identification of a yeast GPI-anchored protein (Yap3) with a readily observable biological function (Bourbonnais et al., 1993) is thus likely to be helpful. For instance, the primary defect of some of the previously isolated sex mutants (Bourbonnais et al., 1993), which perturb or prevent maturation of the prosomatostatin/alpha-factor chimera, might be in GPI anchoring. This is likely if Yap3 indeed requires removal of a putative pro region in a post-ER compartment, since previous studies suggested that failure to transfer a GPI structure to proteins normally GPI-anchored prevents their exit from the ER (Nuofer et al., 1993). We are currently testing this hypothesis.

We have shown by subcellular fractionation studies that Yap3 localizes predominantly to the plasma membrane. Additional support for this localization comes from the observation that Yap3 is both GPI-anchored and hyperglycosylated. Hence, although hyperglycosylation is taking place in the Golgi apparatus, known yeast hyperglycosylated proteins are either secreted proteins or cell wall mannoproteins and, therefore, have reached the plasma membrane (Kukuruzinska et al., 1987). Similarly, most GPI-anchored proteins so far described are plasma membrane proteins. It thus suggests that Yap3 plays a major role at the plasma membrane, possibly in the proteolytic activation of secreted exoglucanases or in the shedding of plasma membrane receptors. However, it is very likely that its physiological function is not restricted to the plasma membrane. Previous studies indeed demonstrated that Yap3 could also activate protein precursors in intracellular compartments. For instance, the Yap3-dependent activation of prosomatostatins heterologously expressed in yeast was shown to be initiated intracellularly (Bourbonnais et al., 1991b, 1993). Furthermore, the observation that mature alpha-mating factor could be secreted by a kex2 null mutant strain (Egel-Mitani et al., 1990), presumably by the combined action of the YAP3, KEX1, and STE13 gene products, implied that endoproteolysis by Yap3 occurred before or in the same intracellular compartment as exoproteolysis by Kex1 and Ste13: two late Golgi resident proteins that could not be detected at the plasma membrane (Roberts et al., 1992; Cooper and Bussey, 1992). The observation that COOH-terminal truncations of Yap3 had a profound effect on the efficiency of alpha-mating factor precursor processing was hypothesized to result from mislocalization of the truncated forms (Egel-Mitani et al., 1990). In light of the present study showing that Yap3 is modified by the addition of a GPI anchor, the most likely explanation is that removal of the COOH-terminal 18 and 68 amino acid residues leads to secretion of Yap3 into the culture medium. In further support to this hypothesis, Yap3-specific activity from a strain carrying the COOH-terminal 18-residues truncation, but not from that harboring the full-length enzyme, could be detected at early time points in the culture medium during a time course experiment. (^3)Given that the final destination of the full-length (plasma membrane-associated) and truncated forms (secreted) of Yap3 is in both cases beyond the Kex1- and Ste13-containing compartment, it, however, suggests that the final localization of Yap3 per se is not responsible for this altered phenotype. We rather propose that removal of the putative GPI attachment site and the Ser/Thr-rich domain leads to a reduced proportion of Yap3 in intracellular compartments. Both the remodeling of GPI anchors (Sipos et al., 1994; Horvath et al., 1994) and the elongation of O-linked oligosaccharides (Orlean et al., 1991) may possibly slow down the transport of Yap3 to the plasma membrane and provide the necessary transient accumulation of the endoprotease in the Golgi apparatus. The fact that the last 68-amino acid residue truncation, lacking both the GPI anchor attachment site and the Ser/Thr-rich domain, affected the release of mature alpha-mating factor more severely than the 18-amino acid deletion (Egel-Mitani et al., 1990) would be consistent with this hypothesis.

Previous in vivo and in vitro studies showed that Yap3 and Kex2 possess distinct but overlapping specificities (Egel-Mitani et al., 1990; Bourbonnais et al., 1993; 1994; Azarian et al., 1993; Cawley et al., 1993). Like Kex2, we have shown here that Yap3 is expressed in all three yeast cell types and that although the final localization of these endoproteases is different, they are at least transiently present in the same intracellular compartments, namely the Golgi apparatus. This suggests that the two processing enzymes may have some common substrates. In further support to this hypothesis, yap3 and mkc7 (a YAP3 homologous gene) double null mutants were recently shown to put further restriction on the temperature range at which kex2 null mutant yeast can grow. (^4)The next step toward elucidating the physiological role of Yap3 will require the identification of its natural substrates, some being common substrates for other yeast endoproteases.


FOOTNOTES

*
This work was supported in part by grants from the Medical Research Council of Canada (to J. J. M. B.) and the Natural Science and Engineering Research Council of Canada (to Y. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the memory of Simone Bourbonnais.

§
To whom all correspondence should be addressed: Département de biochimie, Pavillon Charles-Eugène-Marchand, Université Laval, Québec, Canada G1K 7P4. Tel.: 418-656-7069; Fax: 418-656-7176; ybourbon{at}rsvs.ulaval.ca.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; Endo H, endoglycosidase H; DTT, dithiothreitol; PI-PLC, phosphatidylinositol phospholipase C; GPI, glycophosphatidylinositol; ER, endoplasmic reticulum.

(^2)
Y. Bourbonnais, J. Ash, and D. Y. Thomas, unpublished data.

(^3)
K. Vad and M. Egel-Mitani, personal communication.

(^4)
H. Komano and R. S. Fuller, personal communication.


ACKNOWLEDGEMENTS

We are grateful to Drs. Vad and Egel-Mitani (Novo Nordisk, Bagsvaerd, Denmark) and Drs. Komano and Fuller (University of Michigan, Ann Arbor, MI) for communicating results prior to publication. We thank Drs. Malcolm Whiteway (Biotechnology Research Institute, Montreal, Canada) and Randy Schekman (University of California, Berkeley, CA) for yeast strains and Drs. Morris Manolson (Hospital for Sick Children, Toronto, Canada) and Howard Bussey (McGill University, Montreal, Canada) for purified anti-Vph1 and anti-Kex1 antibodies, respectively. We also thank Dr. Dominick Pallotta (Université Laval, Québec, Canada) for critical reading of the manuscript.


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