(Received for publication, April 26, 1995; and in revised form, June 15, 1995)
From the
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,
-haploid, and a/
-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-[
H]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.
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 -haploid yeast secretes a 13-residue peptide (
-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
-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
-terminal
of the immature
-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 -pheromone peptide when synthesized from a
prosomatostatin/
-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
-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
-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.
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.
Figure 3:
The endoprotease Yap3 is a
membrane-bound protein. Equivalent amounts of total yeast membranes
prepared from a YAP3-overexpressing strain (yap3 (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 (yap3 (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 (yap3
) or overexpressing
the YAP3 gene (yap3
[p9A242]) were
metabolically labeled with myo-[
H]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.
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.
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.
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
-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 (yap3
), wild type (YAP3), and YAP3-overexpressing (yap3
(p9A242)) yeast
metabolically labeled with Trans
S-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; yap3::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
haploid, and MAT a/
-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/), DS7 MAT
(
), 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.
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.
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).
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
-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
-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), ()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-[
H]inositol and
[
H]palmitic acid (Conzelmann et al.,
1988). More recently, precursor forms of the cell wall
-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/
-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 -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
-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. (
)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
-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. ()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.
This paper is dedicated to the memory of Simone Bourbonnais.