Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668
Protein transport in eukaryotic cells requires
the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE
molecules that are associated with distinct intracellular
compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions
with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283-amino acid protein that
shares homology with the syntaxin family of t-SNARE
molecules. Polyclonal antiserum raised against Vam3p
recognized a 35-kD protein that was associated with
vacuolar membranes by subcellular fractionation. Null
mutants of vam3 exhibited defects in the maturation of
multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3tsf). Upon shifting the
vam3tsf mutant cells to nonpermissive temperature, an
immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed:
transport via both the carboxypeptidase Y pathway and
the alkaline phosphatase pathway was inhibited. In addition, vam3tsf cells also exhibited defects in autophagy.
Both the delivery of aminopeptidase I and the docking/
fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift,
vam3tsf cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member,
VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to
function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of
the endosomal t-SNARE, Pep12p, suppressed vam3
mutant phenotypes and, likewise, overexpression of
Vam3p suppressed the pep12
mutant phenotypes.
This result indicated that SNAREs alone do not define
the specificity of vesicle docking reactions.
IN eukaryotic cells, the functional properties of a particular intracellular organelle are largely defined by its
unique set of resident proteins. Maintenance of organelle identity therefore requires effective sorting and
delivery of proteins. Protein trafficking in the secretory
pathway requires a series of events including cargo selection and vesicle budding at the donor organelle, followed by transport, docking, and fusion of transport vesicles with
the appropriate target organelle.
Protein transport to the vacuole in the budding yeast
Saccharomyces cerevisiae is one system in which these processes have been extensively studied. The yeast vacuole is
an acidified compartment analogous to the lysosome of
mammalian cells, containing a variety of hydrolytic enzymes that are responsible for macromolecular degradation (Klionsky et al., 1990 Yeast and mammalian cells appear to share a highly
conserved machinery that functions in protein sorting and
membrane fusion (Bennett and Scheller, 1993 Several representatives of these conserved protein families have been identified in the vacuolar protein sorting
pathway. For example, two distinct Rab proteins, Vps21p
and Ypt7p (Wichmann et al., 1992 We describe experiments that test if Vam3p acts with
other VPS gene products to direct endosome to vacuole
transport. Both subcellular localization studies and analysis of a vam3tsf mutant indicate that the primary function
of Vam3p is at the vacuole where it accepts protein traffic
from multiple transport pathways. Genetic studies indicate
that Vam3p acts in conjunction with Vps33p, a Sec1p homologue, to execute its function. Finally, suppression studies show that Vam3p and Pep12p can partially substitute for one another, suggesting that SNARE molecules do not
constitute the only specificity factor in vesicular targeting
and fusion events.
Strains and Media
S. cerevisiae strains used for these studies are listed in Table I. Yeast
strains were grown in standard yeast extract/peptone/dextrose (YPD),1
yeast extract/peptone/fructose (YPF), or synthetic medium (YNB) containing 2% dextrose and supplemented as necessary with essential amino
acids (Sherman et al., 1979 Table I.
Saccharomyces cerevisiae Strains Used in This Study
). Resident vacuolar proteins and proteins destined for degradation are delivered to the
vacuole via biosynthetic, endocytic, and autophagic transport routes. Thus, the vacuole represents a site of convergence for these distinct pathways. To identify the protein
machinery involved in Golgi to vacuole protein transport,
several mutant screens have been developed to detect vacuolar protein sorting (vps) (Bankaitis et al., 1986
; Rothman and Stevens, 1986
; Robinson et al., 1988
; Rothman et
al., 1989
), vacuolar morphology (vam) (Wada et al., 1992
),
and vacuolar protease activity (pep) (Jones, 1977
) mutants. Through these screens, >40 mutant complementation groups have been identified.
; Ferro-Novick
and Jahn, 1994
; Rothman, 1994
). For example, members
of the syntaxin, synaptobrevin/VAMP, Sec1p, and Rab
protein families function at multiple stages of the secretory
pathway (for reviews see Rothman, 1994
; Pfeffer, 1996
).
Syntaxin (t-SNARE) and synaptobrevin (v-SNARE) family
proteins are characterized as cytoplasmically oriented integral membrane proteins containing regions predicted to
form coiled-coil domains (Bennett et al., 1993
). A predominant model for the function of these proteins postulates
that interactions between a particular syntaxin molecule
(t-SNARE) localized on the target membrane with its corresponding VAMP/synaptobrevin molecule (v-SNARE)
localized on the vesicle membrane provides the specificity
required for faithful vesicle-mediated transport (Bennett
and Scheller, 1993
; Sollner et al., 1993
). Members of the
Sec1p and Rab families of proteins are thought to regulate
the formation and/or activity of these SNARE complexes
(for reviews see Novick and Brennwald, 1993
; Rothman,
1994
).
; Horazdovsky et al.,
1994
; Singer-Kruger et al., 1994
), and two distinct Sec1p
homologues, Vps33p and Vps45p (Banta et al., 1990
;
Wada et al., 1990
; Cowles et al., 1994
; Piper et al., 1994
),
are required for Golgi to vacuole protein transport. These
pairs of proteins define the two distinct stages in Golgi to
vacuole protein transport. Pep12p, a syntaxin homologue,
Vps45p, and Vps21p have been shown to direct Golgi to
endosome protein trafficking (Becherer et al., 1996
; Burd
et al., 1997
). Recently, Vam3p was identified as a syntaxin
homologue associated with vacuolar membranes (Wada et
al., 1997
), suggesting that it may function together with
Vps33p and Ypt7p in the endosome to vacuole transport reaction.
Materials and Methods
). Standard bacterial medium (Miller, 1972
)
supplemented with 100 µg/ml ampicillin for plasmid retention was used to
propagate Escherichia coli. Yeast strains used in EM examination of autophagy were grown in synthetic nitrogen starvation medium without
amino acids and ammonium sulfate (SD-N) (Takeshige et al., 1992
).
Transformation of S. cerevisiae was done by the lithium acetate method
(Ito et al., 1983
). E. coli transformations were done as described previously (Hanahan, 1983
).
DNA Methods
Recombinant DNA manipulations were performed using standard methods (Maniatis et al., 1982). Restriction and modification enzymes were
purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN)
and New England Biolabs (Beverly, MA). A YEp13-based genomic library plasmid (p351R1) containing the VAM3 open reading frame (ORF)
and a vam3::HIS3 deletion construct were a generous gift from Yoh Wada
(University of Tokyo, Japan) (Wada et al., 1997
). Plasmids pVAM3.BS,
pVAM3.414, pVAM3.424, and pVAM3.416 were generated by subcloning
the 2.4-kb BstBI-NsiI fragment (containing the entire VAM3 coding sequence) of p351R1 into pBluescript KS(
) (Stratagene, La Jolla, CA),
pRS414, pRS424, and pRS416 (Sikorski and Hieter, 1989
), respectively. A
VAM3 deletion construct was generated by replacing the BsmI fragment
of pVAM3.BS (eliminating 75% of the VAM3 coding sequence) with the
LEU2 gene. A linear DNA fragment containing the deletion construct
was generated by PCR with primers complementary to the VAM3 sequence. Transformation of wild-type cells with the deletion construct resulted in homologous recombination and insertion of the auxotrophic
marker at the chromosomal VAM3 locus. Transformants were selected by
amino acid prototrophy and deletions were confirmed by PCR analysis of
the chromosomal DNA. Plasmid pVAM3-6.414 containing a temperature-conditional allele of vam3 (vam3.6) was constructed by gapped plasmid
repair (Muhlrad et al., 1992
). The BamHI-KpnI fragment of pVAM3-6.414 containing vam3.6 was then subcloned into pRS416 (Sikorski and
Hieter, 1989
) to generate pVAM3-6.416. Plasmid pVPS33-8.415 containing a temperature-conditional allele of vps33 (vps33.8) was generated by
gapped plasmid repair. The SalI-NotI fragment of pVPS33-8.415 containing the vps33.8 allele was subsequently subcloned into pRS416 (Sikorski
and Hieter, 1989
) to generate pVPS33-8.416. Plasmids pCYI50, pCB31,
and pLB221 were described previously (Johnson et al., 1987
; Banta et al., 1990
; Burd et al., 1997
).
Metabolic Labeling and Immunoprecipitation
To examine the biosynthetic transport of vacuolar proteins, cells were
grown at 26°C in synthetic medium supplemented with amino acids to an
OD600 of 0.5-1.0. Cells were harvested and converted to spheroplasts as
described previously (Paravicini et al., 1992). Spheroplasts were resuspended at a concentration of 3 OD600/ml in synthetic medium containing
amino acids and supplemented with 100 µg/ml
2-macroglobulin and 1 µg/ml BSA to stabilize secreted proteins. Cultures were preincubated at
the appropriate experimental temperature for 5 min, and then labeled
with 60 µCi [35S]cysteine/methionine per ml of cell suspension. After labeling, cultures were chased with the addition of methionine, cysteine, yeast extract, and glucose to final concentrations of 5 mM, 1 mM, 0.4%,
and 0.2%, respectively. After appropriate chase periods, samples were
harvested into an equal volume of 2× energy poison buffer (40 mM sodium azide, 40 mM sodium fluoride, 50 mM Tris, pH 7.5, and 1 M sorbitol)
and stored on ice for 2 min. The spheroplasts were then spun at 13,000 g
for 2 min and separated into intracellular and extracellular fractions. The
resulting samples were precipitated by the addition of TCA to a final concentration of 10%. For analysis of carboxypeptidase Y (CPY) sorting in
vps33-8 cells, spheroplasting was performed after labeling and chase. After the desired chase period, the cultures were harvested into 2× spheroplast buffer (40 mM sodium azide, 40 mM sodium fluoride, 50 mM Tris,
pH 7.5, and 2 M sorbitol, 20 mM DTT) and held on ice for 10 min. Zymolase was added to a concentration of 5 µg/OD600 and the cells were incubated at 30°C for 30 min. The resulting spheroplasts were spun at 13,000 g
for 2 min and separated into I and E fractions. Proteins were precipitated
by the addition of TCA to a final concentration of 10%. Analysis of aminopeptidase I (API) and CPY performed in whole cells was done as previously described (Gaynor et al., 1994
). Proteins were immunoprecipitated
with specific antibodies as previously described (Rieder et al., 1996
). Antibodies to API were a generous gift from Dan Klionsky (University of California, Davis) (Klionsky et al., 1992
). Antibodies to CPY, proteinase A
(PrA), alkaline phosphatase (ALP), carboxypeptidase S (CPS), Pep12p,
and Vps10p have been previously characterized (Klionsky et al., 1988
;
Klionsky and Emr, 1989
; Marcusson et al., 1994
; Becherer et al., 1996
; Cowles et al., 1997
).
Preparation of Antisera against Vam3p
A hybrid protein fusing the amino-terminal 202 amino acids of Vam3p to
glutathione-S-transferase (GST) was expressed in E. coli XL1Blue
[supE44 thi-1 lac endA1 gyrA96 hsdR17 relA1 FproAB lacIqZ
M15].
GST-Vam3p fusion protein was isolated by affinity binding to glutathione-coupled Sepharose and further purified by SDS-PAGE. Purified
protein was used to immunize New Zealand White rabbits as previously
described (Horazdovsky and Emr, 1993
). The crude antiserum was affinity purified by binding to cyanogen bromide-coupled GST-Vam3p (Harlow and Lane, 1988
). Vam3p was detected in yeast cell lysates by immunoblotting and enhanced chemiluminescence (ECL) detection as previously
described (Babst et al., 1997
).
Subcellular Fractionation and Gradient Analysis
For intracellular localization of Vam3p, spheroplasts were made from
wild-type cells and lysed in hypoosmotic lysis buffer as previously described (Gaynor et al., 1994). The crude lysate was centrifuged at 300 g to
remove any unlysed spheroplasts. The 300 g supernatant was sequentially
centrifuged at 13,000 g (15 min) and 100,000 g (60 min) to generate both
low and high speed pellet and supernatant fractions. Resulting samples
were precipitated in 10% TCA and analyzed by immunoblotting and ECL
detection as described previously (Babst et al., 1997
). Gradient Accudenz
(Accurate Chemical and Scientific Corp., Westbury, NY) solutions were
prepared (wt/vol) in hypoosmotic lysis buffer. The gradient was generated using the following Accudenz concentration steps from bottom to top: 0.5 ml 60%, 1 ml 50%, 1 ml 43%, 1 ml 37%, 1 ml 31%, 1 ml 27%, 1 ml 23%, 1 ml 20%, 1 ml 17%, 1 ml 13%, and 1 ml 7%. Gradient analysis was performed on 15 OD600 equivalents of spheroplast lysate (300 g supernatant
fraction) in a vol of 3 ml loaded on top of the gradient. The gradient was
subjected to centrifugation at 4°C in an SW41 rotor (Beckman Instruments, Inc., Fullerton, CA) at 170,000 g for 18 h. 12 fractions were harvested manually from the top of the gradient, and proteins were TCA precipitated and analyzed by immunoblotting. mAbs to ALP were purchased
from Molecular Probes, Inc. (Eugene, OR). Quantitation of proteins on
gels was done by densitometry using NIH Image.
Construction of VAM3 and VPS33 Mutant Alleles
A temperature-conditional allele of VAM3 was constructed by PCR-based mutagenesis (Muhlrad et al., 1992). Primers complementary to
chromosomal sequences immediately adjacent to the start and stop
codons of VAM3 were used to amplify a 900-bp fragment (containing the
entirety of the VAM3 ORF) under limiting dATP conditions (20 µM). A
gapped plasmid was generated by digesting pVAM3.414 with BsmI and by
isolating the vector by gel purification. The mutagenized PCR product
and gapped plasmid were cotransformed into CJY1 cells, and transformants in which homologous recombination resulted in integration of mutagenized PCR product were selected by amino acid prototrophy. In much
the same manner, a temperature-conditional allele of VPS33 was also constructed. Primers complementary to the sequence including both the start
and stop codons of VPS33 were used to amplify a 2.1-kb PCR fragment
under limiting dATP (20 µM) concentrations. Gapped plasmid was generated by digesting pVPS33.415 with AocI and SmaI and by isolating the
vector by gel purification. The resulting PCR product was cotransformed
with gapped plasmid into LBY317 harboring the pCYI50 plasmid, and
transformants were selected for amino acid prototrophy. In both mutant
selections, transformants were replica plated onto YPF, grown at 26°C
overnight, and tested by colorimetric invertase assay (Horazdovsky et al., 1994
) for temperature-conditional vps phenotype (CPY-invertase secretion) at 26°C and after 6 h of temperature shift to 38°C. Presumptive conditional mutant colonies were picked and retested, and plasmid linkage of
the missorting phenotype was confirmed by retransformation of isolated
plasmids into either TDY1 or LBY317 cells, respectively.
Fluorescence Microscopy and EM
To examine vacuolar morphology, [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridium dibromide] (FM4-64) labeling
of yeast cell cultures was done as previously described (Vida and Emr,
1995), except the labeling was done at a concentration of 32 µM FM4-64
(Molecular Probes, Inc.) in YPD for 15 min at 30°C. For vacuolar inheritance assays, the time of labeling was increased to 45 min at 26°C and then
cultures were chased for an additional hour at 26°C. Labeled cells were
harvested, resuspended in fresh medium, and split into two equal aliquots
that were further incubated at either 26°C or 38°C for 2 h. For conventional EM morphology studies, cells were grown at 26°C to mid-log phase. A portion of the cells were shifted to a nonpermissive temperature of 38°C
for either 1 or 3 h. The cultures were processed for EM as previously described (Rieder et al., 1996
). For the examination of autophagy by EM,
cells were grown in YNB at 26°C to mid-log phase, harvested, washed
once in SD-N medium, and resuspended at 1 OD600 per ml in SD-N medium to induce autophagy (Takeshige et al., 1992
). The cultures were split
and shifted to either 26°C or 38°C for 2.5 h and then processed for EM.
Vam3p Is a Membrane-bound Protein That Localizes to the Vacuole
The sequence of Vam3p predicts a protein of 283 amino
acids that, like other SNARE family members, has a transmembrane domain predicted at the carboxy terminus of
the protein. To identify the VAM3 gene product, polyclonal antiserum was raised against a fusion protein consisting of the amino terminal 202 amino acids of Vam3p
fused to GST. The resulting antiserum was affinity purified by binding to a GST-Vam3p-coupled cyanogen bromide column (Harlow and Lane, 1988). The affinity-purified antisera recognized a 35-kD protein from wild-type
cell extracts by immunoblotting (Fig. 1 A, lane 2). The 35-kD species was not recognized by preimmune sera (data
not shown) and was not present in vam3
cells (Fig. 1 A,
lane 1). Cells expressing VAM3 from a 2µ overexpression
plasmid showed a 15-20-fold increase in the abundance of
the protein (Fig. 1 A, lane 3). The molecular mass of the
protein was consistent with the 32-kD predicted molecular
mass of Vam3p. Vam3p protein levels remained stable
during a 60-min chase in cells treated with 20 µg/ml cycloheximide to inhibit new protein synthesis (data not
shown). The relative abundance of Vam3p was ~20-fold
less than CPY (>0.05% of cell protein), indicating that
Vam3p is a fairly rare protein (data not shown).
Subcellular fractionation was performed to assess the intracellular location of Vam3p. Spheroplasts were prepared
from wild-type cells and gently lysed by Dounce homogenization. The cleared cell lysate (300 g supernatant fraction) was subjected to centrifugation at 13,000 g to generate low speed pellet (P13) and supernatant (S13) fractions.
The S13 fraction was then spun at 100,000 g to generate
high speed pellet (P100) and supernatant (S100) fractions. As shown in Fig. 1 B, Vam3p and the vacuolar protein
ALP fractionated nearly identically, with the vast majority
of each protein found in the P13 fraction. Consistent with
previous observations, the endosomal t-SNARE Pep12p
was found distributed between the P13 and P100 fractions
(Becherer et al., 1996), and the majority of Vps10p, the
vacuolar hydrolase sorting receptor that is enriched in late
Golgi membranes, was observed in the P100 fraction
(Marcusson et al., 1994
).
Further support for vacuolar localization of Vam3p was
provided by fractionation of cell lysates on an Accudenz
equilibrium density gradient. Spheroplasts were prepared
from wild-type cells and lysed as described above. The
cleared lysate (300 g supernatant fraction) was loaded at
the top of an Accudenz step gradient and centrifuged to
equilibrium. Fractions were collected starting at the top of
the gradient and analyzed for the presence of Vam3p, Pep12p, and ALP (Fig. 1 C). Vam3p colocalized with the
majority of the vacuolar membrane protein ALP in the
least dense region of the gradient (fractions 1-4). In contrast, the endosomal protein Pep12p migrated to a region
of greater density (fractions 5-8) that was distinct from
both the vacuolar marker ALP and Vam3p. In these gradients, protein markers of the Golgi, ER, mitochondria, and
plasma membrane typically migrate into more dense regions of the gradient, also distinct from Vam3p and ALP
(Singer-Kruger et al., 1993). Taken together, these data
provide strong evidence that Vam3p is localized to vacuolar membranes.
VAM3 Null Mutants Exhibit Severe Vacuolar Protein Sorting and Morphology Defects
To determine the phenotypic consequences resulting from
loss of Vam3p, deletion strains were constructed. Deletion
constructs were generated by replacing ~75% of the
VAM3 open reading frame with either the HIS3 or LEU2
genes (see Materials and Methods). Cells deleted for
VAM3 were viable and grew at rates comparable to those
of wild-type cells at elevated temperatures (38°C), indicating that VAM3 is not required for growth, even at elevated
temperatures. Examination of vam3 cells by both FM4-64 vital staining and EM revealed that these mutants completely lack normal vacuolar structures. Instead, numerous
electron-transparent compartments were present in almost all cells (data not shown). In addition, many cells contained two to five vacuole-like electron-dense structures, but these organelles were significantly smaller than
wild-type vacuoles (see Figs. 6 and 9). Accumulation of
these novel electron-transparent membranous structures
is characteristic of a subset of class B vps mutants, including vam3 (Wada et al., 1997
) and vps41 (Cowles et al.,
1997
; Radisky et al., 1997
), but not vps5 and vps17 (Kohrer and Emr, 1993
; Horazdovsky et al., 1997
).
The abnormal vacuolar morphology of vam3 cells suggested that both protein transport to the vacuole and vacuolar function may be compromised in these cells. Therefore, the ability of vam3
cells to properly transport newly
synthesized resident vacuolar proteins was examined.
Spheroplasts prepared from vam3
cells were pulse labeled for 10 min with [35S]cysteine/methionine to label
newly synthesized proteins, and then were chased with unlabeled cysteine/methionine for 45 min. The spheroplasts
were separated into intracellular and extracellular fractions. CPY was immunoprecipitated from the samples and
analyzed by SDS-PAGE and autoradiography. The biosynthesis of vacuolar proteins such as CPY can be monitored by posttranslational modifications that correlate
with transport through the secretory and vacuolar protein
sorting pathways. In the case of CPY, p1CPY is generated as a result of core glycosyl modifications in the ER, which
are then elongated in the Golgi complex to generate the
slightly larger form, p2CPY. Upon delivery to the vacuole,
the precursor CPY is cleaved to generate the mature active form of the enzyme, mCPY. As shown in Fig. 2, wild-type cells properly delivered CPY to the vacuole as shown
by the presence of mature CPY in the intracellular fraction (Fig. 2, lanes 1 and 2). In vam3
cells, CPY accumulated as the Golgi-modified p2 form, ~50% of which was
secreted into the extracellular fraction (Fig. 2, lanes 3 and
4). A small portion (~10%) of the CPY that remained in
the intracellular fraction was seen as an apparently misprocessed form that migrated as a species slightly smaller
than p2CPY, which eventually was processed to mature
CPY after prolonged chase (t1/2 > 60 min). The introduction of VAM3 on a single copy, centromere (CEN)-based
plasmid complemented the CPY maturation defect of the
vam3
mutant cells (Fig. 2, lanes 5 and 6).
A vam3tsf Mutant Is Defective for Transport of Multiple Vacuolar Proteins
Because of the dramatic effects on vacuolar morphology
in vam3 cells, it is difficult to conclude whether the defects in protein sorting are a direct consequence of loss of
Vam3p function, or due to secondary pleiotropic effects
resulting from loss of vacuolar integrity in vam3
cells. To
address the primary role of Vam3p, we generated a temperature-conditional allele of VAM3 by error prone PCR-mediated mutagenesis (Muhlrad et al., 1992
; Stack et al.,
1995
). Approximately 6,000 transformants were screened for a vps phenotype (secretion of CPY-invertase fusion
protein) at 26°C and after 6 h of temperature shift to 38°C.
Several temperature-conditional alleles were obtained that
secreted a substantial amount of CPY-invertase after a
temperature shift to 38°C, but not at 26°C. One allele (vam3-6), which will be referred to here as vam3tsf, was
chosen and characterized further.
If Vam3p is directly required for vacuolar protein transport, then inactivation of the protein (i.e., by temperature
shift) would be expected to result in an immediate block in
vacuolar protein processing. We tested vam3tsf cells for
sorting of vacuolar hydrolases after a short incubation at
nonpermissive temperature. Spheroplasts were prepared
from both vam3tsf and wild-type cells. The cultures were
split and half was incubated at 26°C while the other half
was shifted to 38°C for 5 min. Each culture was then pulse
labeled for 10 min with [35S]cysteine/methionine and chased
with unlabeled cysteine/methionine for 45 min. Samples
were harvested and separated into intracellular and extracellular fractions. Vacuolar proteins were immunoprecipitated with specific antibodies and analyzed by SDS-PAGE. As shown in Fig. 3, at the permissive temperature
of 26°C, vam3tsf cells matured CPY in a manner indistinguishable from wild-type cells (Fig. 3, lanes 1-4). However, at 38°C, a rapid block in the maturation of CPY was
observed (Fig. 3, lanes 5 and 6). Approximately 60% of
CPY accumulated as the Golgi-modified p2 precursor
form. The remaining 40% of CPY was processed aberrantly
and accumulated intracellularly. Less than 5% of the
newly synthesized CPY was secreted to the extracellular
media fraction. Similar results were observed for another
soluble vacuolar hydrolase, PrA. We also examined the
processing of two vacuolar membrane proteins, ALP and
CPS. In vam3tsf cells at permissive temperature, the processing of both ALP and CPS occurred in a manner identical to wild-type cells (Fig. 3, lanes 1-4). However, in vam3tsf
cells after a 5-min incubation at nonpermissive temperature, both proteins were completely blocked as the Golgi-modified precursor forms (Fig. 3, lanes 5 and 6). Vam3p
thus appears to play an essential role in vacuolar protein
transport of both soluble and integral membrane proteins.
Vam3p Function Is Required for Cytoplasm to Vacuole Delivery of API
Some newly synthesized vacuolar proteins do not reach
the vacuole via the secretory pathway but instead follow a
direct cytosol to vacuole delivery pathway (Klionsky et al.,
1992). The precursor form of API is synthesized in the cytoplasm and transported directly into the vacuole where
the amino-terminal precursor is cleaved, producing the
mature form of the enzyme (Klionsky et al., 1992
). Many
mutants defective for the processing of API (cvt mutants) are also defective in autophagy (aut and apg mutants), indicating that delivery of API to the vacuole may be mediated by an autophagic mechanism (i.e., macroautophagy)
(Harding et al., 1996
; Scott et al., 1996
). The processing of
API was examined in vam3tsf cells to determine if Vam3p
is required for delivery of API to the vacuole. Wild-type
and vam3tsf cells were shifted to nonpermissive temperature for 5 min and then labeled for 10 min. Samples were
harvested after 0, 30, 60, 90, and 120 min of chase. The
maturation of API was analyzed by immunoprecipitation,
SDS-PAGE, and autoradiography. As shown in Fig. 4, cells
containing the wild-type copy of VAM3 matured API with
normal kinetics; essentially all API was processed to the mature vacuolar form after 120 min of chase. However,
vam3tsf cells at nonpermissive temperature (38°C) accumulated newly synthesized API in its precursor form, with
virtually no maturation of the protein, even after 120 min
of chase. In vam3tsf cells at the permissive temperature of
26°C, API was also matured with kinetics comparable to
that of wild-type cells (data not shown). The defect in API
processing observed in vam3tsf cells at nonpermissive temperature indicates that Vam3p is directly required for the
delivery of API to the vacuole.
Vam3p Is Required for the Docking and/or Fusion of Autophagosomes with the Vacuole
The block of API maturation in vam3tsf cells suggested
that targeting of autophagosomes to the vacuole may be
disrupted. Autophagosomes appear by EM as membrane-enclosed structures between 400 and 600 nm in diam
(Baba et al., 1994). The compartments are primarily composed of cytoplasmic material, as they direct bulk uptake
and vacuolar transport of cytoplasmic constituents, and
thus stain with an electron density similar to the cytoplasm
(Baba et al., 1994
). To further examine possible autophagic
defects associated with the loss of Vam3p function, we induced the formation of autophagic intermediates by nitrogen starvation and examined their uptake by the vacuole
in both wild-type and vam3tsf cells. vam3tsf cells and complemented vam3
cells, both of which were also deleted for the PEP4 gene to prevent vacuolar degradation of autophagic bodies, were grown at 26°C to mid-log phase in
YNB medium. The cells were then harvested and resuspended in SD-N. The cultures were split and incubated at
either 26°C or 38°C for 2.5 h. Cells were harvested, fixed,
and processed for EM. Wild-type cells starved at both 26°C
and 38°C, as well as vam3tsf cells that were starved at 26°C,
accumulated numerous autophagic bodies in the vacuoles
(Fig. 5 A). In contrast, vam3tsf cells starved at 38°C for 2.5 h
accumulated multiple autophagosomes in the cytoplasm,
with no detectable accumulation of autophagic bodies in the
vacuole (Fig. 5 B). The double membranes surrounding the cytoplasmic autophagosomes appear to be very fragile
and easily disrupted as a result of preparation for EM.
Thus, the perimeter of the autophagosomes was often seen
as the electron-transparent space that the membrane previously occupied (Fig. 5 C). The temperature-conditional block of autophagosome delivery to the vacuole suggests
that this process is dependent on the function of Vam3p.
Morphological Analysis of vam3tsf Cells Reveals Accumulation of Aberrant Membrane Compartments
Due to the dramatic accumulation of novel membrane
compartments in vam3 cells, we also were interested in
the onset, severity, and characteristics of morphological
defects associated with the vam3tsf allele. vam3tsf cells that
had been grown at permissive temperature (26°C) or shifted to nonpermissive temperature (38°C) for either 1 or 3 h were characterized by EM. At 26°C, vam3tsf cells
displayed typical wild-type morphology, containing prominent, intact, electron-dense vacuoles (Fig. 6 A). Although
the majority of vam3tsf cells that had been shifted to 38°C
for both 1 and 3 h maintained intact vacuoles, an accumulation of 300-500-nm novel membrane-enclosed compartments was also observed (Fig. 6 B). The accumulation of these aberrant compartments increased with respect to
time of incubation at 38°C. The compartments were heterogeneous in both shape and content as indicated by electron-dense or -transparent staining (Fig. 6, B and C). In
addition, vam3tsf cells at high temperature displayed an accumulation of multivesicular membrane compartments
(Fig. 6 C). These data are consistent with a requirement
for Vam3p in the docking/fusion of multiple transport intermediates with the vacuole.
Vacuolar Inheritance Is Unaffected in vam3tsf Cells
Since multiple fusion events with the vacuole are disrupted because of the inactivation of Vam3p, we were interested in investigating the effect of Vam3p on vacuolar
inheritance, a process thought to involve homotypic vacuole-vacuole fusion (Conradt et al., 1992). Therefore, we
examined the ability of vam3tsf cells to properly segregate
vacuoles into the daughter bud. The vacuoles of both wild-type and vam3tsf cells were labeled with FM4-64 for 45 min
at 26°C. The cells then were chased in YPD for 1 h at 26°C
and split into two aliquots. One aliquot was maintained at
26°C and the other was shifted to 38°C for 2 h. Cells were
harvested and viewed by fluorescence microscopy for the
presence of vacuolar segregation structures. At either
26°C or 38°C, vam3tsf cells formed segregation structures in
a manner indistinguishable from wild type; in both cases at
least 96% of budding cells (n = 200 cells) had vacuoles in
both the mother and daughter cells (Fig. 7). Furthermore,
vam3tsf cells, even after 2 h at nonpermissive temperature,
extended continuous segregation structures into the daughter bud and maintained intact vacuoles. Therefore, the
function of Vam3p does not appear to be required for
proper vacuolar segregation during mitosis.
Vam3p Genetically Interacts with Vps33p, a Sec1p Homologue
Distinct protein complexes are thought to mediate vesicle
targeting and fusion at discrete steps in the secretory and
vacuolar protein sorting pathways. For example, members
of the Sec1 family of proteins are thought to regulate vesicle targeting and fusion through specific interactions with
t-SNARE proteins (Garcia et al., 1994; Pevsner et al.,
1994
). Thus, just as Pep12p has been shown to interact
with the Sec1p homologue Vps45p to mediate protein sorting from the Golgi to the endosome (Burd et al., 1997
),
Vam3p is also likely to interact with regulatory proteins to
execute its function. A strong candidate for such an interaction with Vam3p is Vps33p, a Sec1p homologue that
may act at a late step in the vacuolar protein sorting pathway (Banta et al., 1990
; Wada et al., 1990
). Therefore, genetic interactions between VAM3 and VPS33 were examined. First, temperature-conditional alleles of VPS33 were
generated by PCR-based mutagenesis in a manner similar to that described for VAM3 (see Materials and Methods).
One temperature-conditional mutant (vps33-8), which will
be referred here as vps33tsf, was chosen for further characterization. After a 5-min incubation at either permissive
(26°C) or nonpermissive (38°C) temperature, vps33tsf cells
were pulse labeled for 10 min and then chased for 30 min. After chase, the cells were spheroplasted and separated
into intracellular and extracellular fractions. The processing of CPY was analyzed in each fraction by immunoprecipitation, SDS-PAGE, and autoradiography (Fig. 8 A).
At 26°C, vps33tsf cells properly processed the majority of
CPY to the mature vacuolar form, with only a minor
(~10%) portion delayed intracellularly in the p2 form
(Fig. 8 A, lanes 5 and 6). However, in vps33tsf cells after a
5-min incubation at 38°C, CPY was blocked intracellularly as the Golgi-modified p2 form of CPY. Similar to vam3tsf
cells, only a small percentage (<5%) of p2CPY was secreted into the extracellular fraction and a small portion of
the p2CPY was misprocessed (Fig. 8 A, lanes 7 and 8).
Wild-type cells properly matured CPY at both temperatures (Fig. 8 A, lanes 1-4). A defect in the processing of
the vacuolar membrane protein ALP also was observed,
with a subtle processing defect at the permissive temperature and a complete block in ALP maturation at the nonpermissive temperature (data not shown). Thus, like vam3tsf
cells, vps33tsf mutants exhibited a rapid defect in the transport of both CPY and ALP to the vacuole at nonpermissive temperature.
To determine whether Vam3p and Vps33p might functionally interact, we examined a vam3tsfvps33tsf double mutant for synthetic vacuolar protein sorting defects. A haploid double mutant strain was constructed and CPY maturation was assayed by metabolic labeling of whole cells. Cells were pulse labeled for 10 min and then chased for an additional 30 min at 26°C. After chase, the cells were lysed, and proteins were immunoprecipitated, separated on SDS-PAGE, and visualized by autoradiography (Fig. 8 B). Under these conditions, both vam3tsf and vps33tsf single mutant cells matured >90% of CPY (Fig. 8 B, lanes 1 and 2). However, in vam3tsfvps33tsf double mutant cells, a moderate synthetic sorting defect was observed; ~50% of newly synthesized CPY remained blocked in the p2 precursor form (Fig. 8 B, lane 3). The synthetic sorting defect of the double mutant strain is not as severe as that of the single mutant strains at nonpermissive temperature, indicating that transport to the vacuole is not completely disrupted in the double mutant cells. However, the increased sorting defect suggests that the two proteins function together to mediate protein sorting to the vacuole.
Pep12p and Vam3p Can Partially Substitute for One Another in the Vacuolar Protein Transport Pathway
Although Vam3p shares significant homology with many
members of the syntaxin family, it is most similar to
Pep12p, an endosomal t-SNARE. To address whether
these SNARE proteins can functionally substitute for one
another, we overexpressed each SNARE in the reciprocal
null mutant background and examined the strains for improved transport of CPY to the vacuole. Spheroplasts
were prepared from each strain, radiolabeled, and chased;
proteins were then immunoprecipitated and analyzed by
SDS-PAGE (Fig. 9 A). In both vam3 and pep12
cells,
CPY accumulated as the Golgi-modified precursor form
(Fig. 9 A, lanes 1 and 4). Introduction of CEN plasmids
harboring VAM3 or PEP12 into vam3
or pep12
cells,
respectively, complemented the CPY sorting defects of
these mutants (Fig. 9 A, lanes 3 and 6). Interestingly, the
overexpression of Pep12p in vam3
cells and Vam3p in
pep12
cells resulted in significant conversion of CPY to
the mature vacuolar form (Fig. 9 A, lanes 2 and 5). In addition to suppression of the CPY sorting defect, the ALP
maturation defects were similarly suppressed (data not shown). To further determine the requirements of VAM3
suppression of pep12
cells, we examined the effect of
VAM3 overexpression in vps45
and vps45
pep12
cells.
VPS45 gene encodes a member of the Sec1p family of proteins (Cowles et al., 1994
; Piper et al., 1994
) and is required, with Pep12p, for Golgi to endosome protein transport (Burd et al., 1997
). Cultures of each strain were
radiolabeled and chased, and proteins were immunoprecipitated and resolved by SDS-PAGE (Fig. 9 A). In both
vps45
mutant cells and vps45
pep12
double mutant
cells, CPY was blocked in the Golgi-modified precursor
form (Fig. 9 A, lanes 7 and 8). Overexpression of VAM3 was unable to suppress the CPY sorting defects of either
mutant strain (Fig. 9 A, lanes 8 and 9). These results indicate that VAM3 requires VPS45 to facilitate CPY transport in pep12
cells. In a reciprocal experiment we found
that overexpression of PEP12 does not suppress a vps33
mutant (data not shown).
Suppression of morphological defects associated with
vam3 and pep12
mutant cells was also examined by vital staining of the vacuoles with FM4-64. As shown in Fig.
9 B, wild-type cells typically contain several brightly staining vacuolar structures that corresponded to vacuolar depressions in Nomarski images (Vida and Emr, 1995
). In
contrast, vam3
cells displayed a punctate staining pattern
throughout the cytoplasm. pep12
cells stained with FM4-64 displayed a single, large, vacuolar structure, characteristic of class D vps mutants (Vida and Emr, 1995
). Overexpression of Pep12p dramatically suppressed the morphological
defects of vam3
cells, resulting in several normal appearing vacuole structures. Similarly, pep12
cells that overproduced Vam3p contained near wild-type vacuolar structures. The introduction of VAM3 or PEP12 on low copy
plasmids completely complemented the morphological defects of both the vam3
cells and pep12
cells, respectively. Overexpression of other presumptive yeast SNARE molecules (Sec22p, Bos1p, ORF #D8035.11p, ORF #YLR0936)
or other class D proteins (Vps45p, Vac1p, Vps9p, Vps8p)
did not improve vacuolar protein sorting of vam3
cells,
indicating that the suppression by Pep12p is specific.
We report on the primary function of Vam3p, a syntaxin homologue, in the docking and/or fusion of transport intermediates from multiple protein sorting pathways to the vacuole/lysosome. Subcellular fractionation and density gradient data indicated that Vam3p colocalized with vacuolar membranes. Conditional vam3tsf mutants exhibited a rapid defect in the transport of multiple resident vacuolar proteins after shifting to the nonpermissive temperature, indicating that Vam3p is a component of the vacuolar protein transport machinery. Additionally, autophagic pathway intermediates and API precursor accumulated in vam3tsf cells, indicating that Vam3p is also required for autophagy. Finally, EM analysis of these conditional mutants revealed a temperature-dependent accumulation of novel membrane structures, including multivesicular bodies, which may represent distinct transport intermediates destined for fusion with the vacuole. Thus, our data are most consistent with Vam3p residing on the vacuolar membrane where it functions as a multispecificity receptor required for docking and/or fusion of multiple prevacuolar transport intermediates.
Members of the syntaxin family of proteins are thought
to act at specific intracellular organelles as receptors for
the selective docking of appropriate transport intermediates. For instance, in neuronal cells, syntaxin is localized to
the plasma membrane where it mediates the docking and
fusion of secretory vesicles containing neurotransmitters
(Bennett et al., 1992, 1993
). In yeast, Sso1p and Sso2p, two
redundant syntaxin homologues whose function is required for secretion, are localized to the plasma membrane (Aalto et al., 1993
; Brennwald et al., 1994
). Furthermore, Pep12p, an endosomal protein, is required for Golgi
to endosome protein transport (Becherer et al., 1996
; Burd
et al., 1997
). Thus, the membrane localization of such proteins is suggestive of their site of function. We have demonstrated through subcellular localization experiments
that the majority of Vam3p resides in vacuolar membranes. It has also been reported recently that Vam3p is
localized to vacuolar membranes by indirect immunofluorescence (Wada et al., 1997
). Together, these data provide
strong evidence that Vam3p resides on the vacuole, consistent with its function as a vacuolar t-SNARE.
Vam3p Functions as a Multispecificity t-SNARE
Our analysis of a temperature-conditional vam3 mutant
(vam3tsf) suggests a direct involvement of Vam3p in a late
step of protein transport to the vacuole. Multiple transport
pathways to the vacuole were blocked even after a short
incubation of vam3tsf cells at nonpermissive temperature.
There are at least two pathways of biosynthetic traffic to
the vacuole. CPY, PrA, and CPS transit through an endosomal compartment before final delivery to the vacuole.
ALP is delivered by an alternate Golgi-to-vacuole transport pathway, possibly bypassing the endosomal compartment (Cowles et al., 1994, 1997
; Stack et al., 1995
; Burd et
al., 1996
, 1997
; Horazdovsky et al., 1996
). vam3tsf cells displayed immediate defects in the transport of CPY, PrA, CPS, and ALP, which is indicative of a direct involvement
of Vam3p in both biosynthetic transport pathways.
Interestingly, the fate of accumulated p2CPY in vam3tsf
cells suggests that Vam3p acts at a late step in the vacuolar
protein sorting pathway. Defects in receptor-mediated
transport of CPY from the Golgi to the endosome result in
the rapid secretion of CPY (Cowles et al., 1994; Piper et
al., 1994
; Stack et al., 1995
; Burd et al., 1997
). Unlike these
early acting conditional vps mutants (pep12tsf and vps45tsf
mutants) that secrete p2CPY immediately upon inactivation, vam3tsf cells accumulated p2CPY intracellularly. The
lack of CPY secretion in vam3tsf cells at nonpermissive
temperature indicates that transport and recycling of the
Vps10p hydrolase sorting receptor between the Golgi and
the prevacuolar endosome is relatively unaffected and is
thus consistent with a block in transport at a point beyond the endosomal intermediate. Additionally, the accumulated intracellular p2CPY in vam3tsf cells at nonpermissive
temperature is slowly processed to aberrant forms. This
processing is unlikely to be occurring in the vacuole. These
cells have normal vacuoles populated with active processing proteases that would rapidly process p2CPY to its mature form. Similar aberrant processing of vacuolar protein
precursors has been observed in other vps mutants that
have defects in endosome to vacuole transport as a result
of accumulation in a proteolytically competent endosomal
compartment (Babst et al., 1997
). Extended accumulation
of CPY in a late prevacuolar compartment in vam3tsf cells
results in slow maturation of p2CPY (t1/2 of 60 min). Consistent with these observations, at nonpermissive temperature, vam3tsf cells accumulated novel membrane-enclosed
compartments that may represent these blocked transport
intermediates.
The maturation of API, a protein delivered to the vacuole directly from the cytosol, possibly by an autophagic
pathway (Klionsky et al., 1992), is also completely blocked
at nonpermissive temperature in vam3tsf cells. Furthermore, the docking/fusion of large autophagosomes with
the vacuole is disrupted in vam3tsf cells at nonpermissive
temperature. Thus, the delivery of autophagic intermediates appears to require a functional vacuolar t-SNARE. If
the mechanism of autophagic vesicle fusion uses conserved components such as SNAREs, autophagosomes
may contain complementary components on their membranes that specifically target them to the vacuole via interactions with Vam3p.
The vam3 mutant was originally identified as a vacuolar
morphology mutant (Wada et al., 1992). This phenotype is
consistent with a primary role for Vam3p in maintenance
of vacuolar morphology. However, in vam3tsf cells, the
vacuoles remain stable for >3 h at nonpermissive temperature. Instead, vam3tsf cells accumulate novel membrane
structures, which suggests that the primary defect associated with the loss of Vam3p function is impaired docking
and/or fusion of transport intermediates with the vacuole.
Thus, the complex morphology of vam3
cells appears to
result from the accumulation of these transport intermediates, rather than instability and fragmentation of the vacuoles. Recent in vitro studies analyzing extracts from vam3
cells indicated that Vam3p is involved in homotypic vacuole-vacuole fusion (Nichols et al., 1997
). While our data
do not rule out a role for Vam3p in homotypic fusion, they
suggest that these in vitro observations may correspond to
heterotypic fusion of isolated prevacuolar intermediates from vam3
cells with wild-type vacuoles. Moreover, because we observed no obvious vacuolar inheritance defects in vam3tsf cells incubated for extended time at nonpermissive temperature, Vam3p function does not appear
to play an essential role in vacuole-vacuole fusion during
the inheritance process. Similar in vitro fusion experiments analyzing vacuoles isolated from vam3tsf cells may
offer a means to determine the role, if any, of Vam3p in
homotypic vacuole fusion reactions.
The accumulation of protein precursors and membrane-bound intermediate compartments that transit to the vacuole via distinct routes is consistent with a transport block
at a site where these various pathways converge, the vacuole (Fig. 10). While we cannot rule out the possibility that
Vam3p mediates the delivery of an unstable component,
which in turn is required for the docking and fusion of
other transport intermediates, this explanation seems unlikely because other vps mutants (i.e., pep12) do not show
similar general transport defects (Burd et al., 1997; Cowles et al., 1997
). Thus, analysis of protein transport and vacuolar morphology in vam3tsf cells is consistent with Vam3p
acting as a multispecificity vacuolar t-SNARE that mediates the docking/fusion of multiple distinct late transport
intermediates with the vacuole. The early Golgi t-SNARE,
Sed5p, may also function as a multispecificity t-SNARE as it
has been shown to function in both biosynthetic ER-to-Golgi and retrograde late-to-early Golgi transport (Hardwick
and Pelham, 1992
; Banfield et al., 1994
, 1995
).
Interaction between Vam3p and Late Acting Vacuolar Transport Components
t-SNAREs are thought to act in conjunction with members of other well-conserved protein families (such as the
v-SNARE, Sec1p, and Rab GTPase families) to execute
their proposed function in targeting and fusion (for reviews see Rothman, 1994; Pfeffer, 1996
). Thus, it is likely
that Vam3p associates with accessory proteins in the same
manner as other t-SNAREs. Mutations in VPS33 and
YPT7, which encode Sec1p and Rab GTPase homologues,
respectively, result in morphological and protein sorting
defects similar to those observed in vam3 mutants (Banta
et al., 1990
; Wada et al., 1990
; Wichmann et al., 1992
), suggesting that they may mediate a common transport step.
Although vps33 null alleles tend to exhibit more severe
phenotypes than vam3 and ypt7 null mutants, these differences may be attributable to a more stringent requirement for Vps33p in late vacuolar protein transport. The observed genetic interaction between VAM3 and VPS33 indicates that these proteins do in fact function together to
direct the docking and fusion of transport intermediates
with the vacuole. These genetic data do not yet reveal the
precise molecular mechanism of this interaction; however,
several possibilities can be envisioned. For example, direct
in vitro binding of neuronal Sec1 to syntaxin has been demonstrated in mammalian systems (Hata et al., 1993
;
Garcia et al., 1994
; Pevsner et al., 1994
). Thus, Vps33p may
bind directly to Vam3p, and thereby regulate the function
of Vam3p by either preventing the formation of inappropriate interactions or by facilitating the assembly of the
correct components. Further studies investigating physical
interactions between the Vps33p and Vam3p, as well as other protein factors that may regulate these interactions,
such as Ypt7p, are in progress and should facilitate our understanding of the molecular mechanisms involved in this
process.
Some insight into the function of syntaxin and Sec1p homologues can be gained by our observations that overexpression of Vam3p partially suppresses both the vacuolar
protein sorting and morphological defects of a pep12
strain and, conversely, that overexpression of Pep12p results
in partial suppression of the sorting and morphological defects associated with a vam3
strain. Apparently, increased cellular levels of these t-SNAREs results in redistribution
of the excess protein to compartments other than their
normal resident organelles, thus allowing them to function
at inappropriate compartments. Indeed, overexpression of
Pep12p results in a substantial pool of the protein redistributing to the vacuolar membrane (data not shown). Interestingly, overexpression of VAM3 and PEP12 was not capable of suppressing deletions of VPS45 and VPS33, respectively. Therefore, it is possible that under these conditions Pep12p and Vam3p may transiently interact with inappropriate components of the docking/fusion machinery
to execute their function (i.e., Vam3p with Vps45p, and
Pep12p with Vps33p). These observations indicate that
SNARE molecules may not be the only specificity factors
necessary for protein targeting. In fact, other studies indicate additional factors are required to direct docking and fusion of transport intermediates. For example, in mammalian cells, syntaxin is ubiquitously distributed throughout the neuronal plasma membrane, yet fusion occurs primarily at the active site of the nerve terminal, indicating
that other factors are acting to direct fusion to this specific
region (Bennett et al., 1992
; Sollner et al., 1993
; Garcia et
al., 1995
). Several candidate proteins that may be involved
in this additional specificity function include members of
the Sec1 and Rab protein families.
Studies examining the physical and genetic relationships between Vam3p and other transport components, as well as in vitro reconstitution of protein transport to the vacuole, are necessary to determine the mechanistic details of this final step of protein trafficking. Through these types of investigations, significant progress will be made in elucidating the molecular mechanisms of protein and membrane transport in yeast and other eukaryotic organisms.
Received for publication 28 April 1997 and in revised form 9 June 1997.
We are very grateful to Michael McCaffery and Tammie McQuistan for outstanding EM work (Electron Microscopy Core B, Program Project grant CA58689). We thank Yoh Wada and Dan Klionsky for helpful discussions and for generously providing plasmids and antisera. We also thank Colin Jamora for assistance with initial experiments, and members of the Emr laboratory, especially Erin Gaynor, Marcus Babst, and Chris Burd, for helpful comments and for critical reading of this manuscript.This work was supported by grants GM32703 and CA58689 from the National Institutes of Health to S.D. Emr. S.D. Emr is an investigator of the Howard Hughes Medical Institute.
ALP, alkaline phosphatase; API, aminopeptidase I; CPS, carboxypeptidase S; CPY, carboxypeptidase Y; ECL, enhanced chemiluminescence; FM4-64, [N-(3-triethylammoniumpropyl)- 4-(p-diethylaminophenylhexatrienyl) pyridinum dibromide]; GST, glutathione-S-transferase; ORF, open reading frame; PrA, proteinase A; YPD, yeast extract/peptone/dextrose.
1. | Aalto, M.K., H. Ronne, and S. Keranen. 1993. Yeast syntaxins Sso1p and Sso2p belong to a family of related membrane proteins that function in vesicular transport. EMBO (Eur. Mol Biol. Organ.) J. 12: 4095-4104 [Abstract]. |
2. | Baba, M., K. Takeshige, N. Baba, and Y. Ohsumi. 1994. Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization. J. Cell Biol. 124: 903-913 [Abstract]. |
3. |
Babst, M.,
T.K. Sato,
L.M. Banta, and
S.E. Emr.
1997.
Endosomal transport
function in yeast requires a novel AAA-type ATPase, Vps4p.
EMBO (Eur.
Mol Biol. Organ.) J.
16:
1820-1831
|
4. | Banfield, D.K., M.J. Lewis, C. Rabouille, G. Warren, and H.R. Pelham. 1994. Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains. J. Cell Biol. 127: 357-371 [Abstract]. |
5. | Banfield, D.K., M.J. Lewis, and H.R. Pelham. 1995. A SNARE-like protein required for traffic through the Golgi complex. Nature (Lond.). 375: 806-809 |
6. | Bankaitis, V.A., L.M. Johnson, and S.D. Emr. 1986. Isolation of yeast mutants defective in protein targeting to the vacuole. Proc. Natl. Acad. Sci. USA. 83: 9075-9079 [Abstract]. |
7. | Banta, L.M., T.A. Vida, P.K. Herman, and S.D. Emr. 1990. Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis. Mol. Cell. Biol. 10: 4638-4649 |
8. | Becherer, K.A., S.E. Rieder, S.D. Emr, and E.W. Jones. 1996. Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast. Mol. Biol. Cell. 7: 579-594 [Abstract]. |
9. | Bennett, M.K., and R.H. Scheller. 1993. The molecular machinery for secretion is conserved from yeast to neurons. Proc. Natl. Acad. Sci. USA. 90: 2559-2563 [Abstract]. |
10. | Bennett, M.K., N. Calakos, and R.H. Scheller. 1992. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science (Wash. DC). 257: 255-259 |
11. | Bennett, M.K., J.E. Garcia-Arraras, L.A. Elferink, K. Peterson, A.M. Fleming, C.D. Hazuka, and R.H. Scheller. 1993. The syntaxin family of vesicular transport receptors. Cell. 74: 863-873 |
12. | Brennwald, P., B. Kearns, K. Champion, S. Keranen, V. Bankaitis, and P. Novick. 1994. Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis. Cell. 79: 245-258 |
13. | Burd, C.G., P.A. Mustol, P.V. Schu, and S.D. Emr. 1996. A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins. Mol. Cell. Biol. 16: 2369-2377 [Abstract]. |
14. | Burd, C.G., M. Peterson, C.R. Cowles, and S.E. Emr. 1997. A novel Sec18p/ NSF-dependent complex required for Golgi to endosome transport in yeast. Mol. Biol. Cell. 8: 1089-1104 [Abstract]. |
15. | Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae. J. Cell Biol. 119: 1469-1479 [Abstract]. |
16. |
Cowles, C.R.,
S.D. Emr, and
B.F. Horazdovsky.
1994.
Mutations in the VPS45
gene, a SEC1 homologue, result in vacuolar protein sorting defects and accumulation of membrane vesicles.
J. Cell Sci.
107:
3449-3459
|
17. |
Cowles, C.R.,
W.B. Snyder,
C.G. Burd, and
S.D. Emr.
1997.
An alternative
Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component.
EMBO (Eur. Mol Biol. Organ.)
J.
16:
2769-2782
|
18. | Ferro-Novick, S., and R. Jahn. 1994. Vesicle fusion from yeast to man. Nature (Lond.). 370: 191-193 |
19. | Garcia, E.P., E. Gatti, M. Butler, J. Burton, and P. De Camilli. 1994. A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin. Proc. Natl. Acad. Sci. USA. 91: 2003-2007 [Abstract]. |
20. | Garcia, E.P., P.S. McPherson, T.J. Chilcote, K. Takei, and P. De Camilli. 1995. rbSec1a and b colocalize with syntaxin 1 and SNAP-25 throughout the axon, but are not in a stable complex with syntaxin. J. Cell Biol. 129: 105-120 [Abstract]. |
21. | Gaynor, E.C., S. te Heesen, T.R. Graham, M. Aebi, and S.D. Emr. 1994. Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast. J. Cell Biol. 127: 653-665 [Abstract]. |
22. | Hanahan, D.. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580 |
23. |
Harding, T.M.,
A. Hefner-Gravink,
M. Thumm, and
D.J. Klionsky.
1996.
Genetic and phenotypic overlap between autophagy and the cytoplasm to vacuole protein targeting pathway.
J. Biol. Chem.
271:
17621-17624
|
24. | Hardwick, K.G., and H.R. Pelham. 1992. SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex. J. Cell Biol. 119: 513-521 [Abstract]. |
25. | Harlow, E., and D.L. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. |
26. | Hata, Y., C.A. Slaughter, and T.C. Sudhof. 1993. Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature (Lond.). 366: 347-351 |
27. |
Horazdovsky, B.F., and
S.D. Emr.
1993.
The VPS16 gene product associates
with a sedimentable protein complex and is essential for vacuolar protein
sorting in yeast.
J. Biol. Chem.
268:
4953-4962
|
28. | Horazdovsky, B.F., G.R. Busch, and S.D. Emr. 1994. VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins. EMBO (Eur. Mol Biol. Organ.) J. 13: 1297-1309 [Abstract]. |
29. |
Horazdovsky, B.F.,
C.R. Cowles,
P. Mustol,
M. Holmes, and
S.D. Emr.
1996.
A
novel RING finger protein, Vps8p, functionally interacts with the small
GTPase, Vps21p, to facilitate soluble vacuolar protein localization.
J. Biol.
Chem.
271:
33607-33615
|
30. | Horazdovsky, B.F., M.N.J. Seaman, S.A. McLaughlin, S.-H. Yoon, and S.D. Emr. 1997. Vps5p, a sorting nexin homologue, forms a complex with Vps17p and is required for vacuolar protein sorting. Mol. Biol. Cell. In press. |
31. | Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163-168 |
32. | Johnson, L.M., V.A. Bankaitis, and S.D. Emr. 1987. Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. Cell. 48: 875-885 |
33. |
Jones, E.W..
1977.
Proteinase mutants of Saccharomyces cerevisiae.
Genetics.
85:
23-33
|
34. | Klionsky, D.J., and S.D. Emr. 1989. Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. EMBO (Eur. Mol Biol. Organ.) J. 8: 2241-2250 [Abstract]. |
35. | Klionsky, D.J., L.M. Banta, and S.D. Emr. 1988. Intracellular sorting and processing of a yeast vacuolar hydrolase; proteinase A propeptide contains vacuolar targeting information. Mol. Cell. Biol. 8: 2105-2116 |
36. | Klionsky, D.J., P.K. Herman, and S.D. Emr. 1990. The fungal vacuole: composition, function, and biogenesis. Microbiol. Rev. 54: 266-292 . |
37. | Klionsky, D.J., R. Cueva, and D.S. Yaver. 1992. Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independant of the secretory pathway. J. Cell Biol. 119: 287-299 [Abstract]. |
38. |
Kohrer, K., and
S.D. Emr.
1993.
The yeast VPS17 gene encodes a membrane
associated protein required for the sorting of soluble vacuolar hydrolases.
J.
Biol. Chem.
268:
559-569
|
39. | Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. |
40. | Marcusson, E.G., B.F. Horazdovsky, J.L. Cereghino, E. Gharakhanian, and S.D. Emr. 1994. The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene. Cell. 77: 579-586 |
41. | Miller, J. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. |
42. | Muhlrad, D., R. Hunter, and R. Parker. 1992. A rapid method for localized mutagenesis of yeast genes. Yeast. 8: 79-82 |
43. | Nichols, B.J., C. Ungermann, H.R.B. Pelham, W.T. Wickner, and A. Haas. 1997. Homotypic vacuolar fusion mediated by t- and v- SNAREs. Nature (Lond.). 387: 199-202 |
44. | Novick, P., and P. Brennwald. 1993. Friends and family: the role of Rab GTPases in vesicular traffic. Cell. 75: 597-601 |
45. | Paravicini, G., B.F. Horazdovsky, and S.D. Emr. 1992. Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. Mol. Biol. Cell. 3: 415-427 [Abstract]. |
46. | Pevsner, J., S.C. Hsu, and R.H. Scheller. 1994. n-Sec1: a neural-specific syntaxin-binding protein. Proc. Natl. Acad. Sci. USA. 91: 1445-1449 [Abstract]. |
47. | Pfeffer, S.R.. 1996. Transport vesicle docking: SNAREs and associates. Annu. Rev. Cell Dev. Biol. 12: 441-461 . |
48. | Piper, R.C., E.A. Whitters, and T.H. Stevens. 1994. Yeast Vps45p is a Sec1p-like protein required for the consumption of vacuole-targeted, post-Golgi transport vesicles. Eur. J. Cell Biol. 65: 305-318 |
49. |
Radisky, D.C.,
W.B. Snyder,
S.E. Emr, and
J. Kaplan.
1997.
Identification of
VPS41, a gene required for vacuolar trafficking and the assembly of the
yeast high affinity iron transport system.
Proc. Natl. Acad. Sci. USA.
94:
5662-5666
|
50. | Rieder, S.E., L.M. Banta, K. Kohrer, J.M. McCaffery, and S.D. Emr. 1996. Multilamellar endosome-like compartment accumulates in the yeast vps28. Mol. Biol. Cell. 7: 985-999 [Abstract]. |
51. | Robinson, J.S., D.J. Klionsky, L.M. Banta, and S.D. Emr. 1988. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol. Cell. Biol. 8: 4936-4948 |
52. | Rothman, J.E.. 1994. Mechanisms of intracellular protein transport. Nature (Lond.). 372: 55-63 |
53. | Rothman, J.H., and T.H. Stevens. 1986. Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell. 47: 1041-1051 |
54. | Rothman, J.H., I. Howald, and T.H. Stevens. 1989. Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae. EMBO (Eur. Mol Biol. Organ.) J. 8: 2057-2065 [Abstract]. |
55. |
Scott, S.V.,
A. Hefner-Gravink,
K.A. Morano,
T. Noda,
Y. Ohsumi, and
D.J. Klionsky.
1996.
Cytoplasm-to-vacuole targeting and autophagy employ the
same machinery to deliver proteins to the yeast vacuole.
Proc. Natl. Acad.
Sci. USA.
93:
12304-12308
|
56. | Sherman, F., G.R. Fink, and L.W. Lawrence. 1979. Methods in Yeast Genetics: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. |
57. |
Sikorski, R.S., and
P. Hieter.
1989.
A system of shuttle vectors and yeast host
strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Genetics.
122:
19-27
|
58. |
Singer-Kruger, B.,
R. Frank,
F. Crausaz, and
H. Riezman.
1993.
Partial purification and characterization of early and late endosomes from yeast.
J. Biol.
Chem.
268:
14376-14386
|
59. | Singer-Kruger, B., H. Stenmark, A. Dusterhoft, P. Philippsen, J.S. Yoo, D. Gallwitz, and M. Zerial. 1994. Role of three rab5 like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast. J. Cell Biol. 125: 283-298 [Abstract]. |
60. | Sollner, T., S.W. Whiteheart, M. Brunner, H. Erdjument-Bromage, S. Geromanos, P. Tempst, and J.E. Rothman. 1993. SNAP receptors implicated in vesicle targeting and fusion. Nature (Lond.). 362: 318-324 |
61. | Stack, J.H., D.B. DeWald, K. Takegawa, and S.D. Emr. 1995. Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast. J. Cell Biol. 129: 321-334 [Abstract]. |
62. | Takeshige, K., M. Baba, S. Tsuboi, T. Noda, and Y. Ohsumi. 1992. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J. Cell Biol. 119: 301-311 [Abstract]. |
63. | Vida, T.A., and S.D. Emr. 1995. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128: 779-792 [Abstract]. |
64. | Wada, Y., K. Kitamoto, T. Kanbe, K. Tanaka, and Y. Anraku. 1990. The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function. Mol. Cell. Biol. 10: 2214-2223 |
65. |
Wada, Y.,
Y. Ohsumi, and
Y. Anraku.
1992.
Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae.
J. Biol. Chem.
267:
18665-18670
|
66. |
Wada, Y.,
N. Nakamura,
Y. Ohsumi, and
A. Hirata.
1997.
Vam3p, a new member of syntaxin related proteins, is required for vacuolar assembly in the
yeast Saccharomyces cerevisiae.
J. Cell Sci.
110:
1299-1306
|
67. | Wichmann, H., L. Hengst, and D. Gallwitz. 1992. Endocytosis in yeast: evidence for the involvement of a small GTP binding protein (Ypt7p). Cell. 71: 1131-1142 |