From the University of Michigan, Department of
Biology, Ann Arbor, Michigan 48109 and the § Section of
Microbiology, University of California, Davis, California 95616
Received for publication, October 18, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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The vacuole/lysosome serves an essential role in
allowing cellular components to be degraded and recycled under
starvation conditions. Vacuolar hydrolases are key proteins in this
process. In Saccharyomces cerevisiae, some resident
vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting
(Cvt) pathway, which shares mechanistic features with autophagy.
Autophagy is a degradative pathway that is used to degrade and recycle
cellular components under starvation conditions. Both the Cvt pathway
and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole. As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles. We have identified a protein, Cvt17, which is essential for this membrane lytic
event. Cvt17 is a membrane glycoprotein that contains a motif conserved
in esterases and lipases. The active-site serine of this motif is
required for subvacuolar vesicle lysis. This is the first
characterization of a putative lipase implicated in vacuolar function
in yeast.
One fundamental role of the yeast vacuole is in the recycling of
biological macromolecules. The vacuole, like the lysosome in animal
cells, is the primary site of degradation. Our understanding of the
hydrolytic vacuolar enzymes that serve in protein turnover is well
advanced. Progress has also been made in elucidating mechanisms that
deliver the substrates of these vacuolar hydrolases. While research has
focused on the biosynthesis and function of the vacuolar proteases,
little is known about how lipids are recycled in this organelle, and a
lipase that functions in membrane recycling has not been identified.
Nearly all vacuolar/lysosomal delivery pathways involve packaging of
cargo within membrane-enclosed transport compartments. Because the
vacuole/lysosome serves as the final destination for these numerous
vesicle-mediated transport pathways, the issue of how membranes
reaching the vacuole are recycled is an important one. Macroautophagy
is the major degradative process in eukaryotes and is essential during
starvation conditions (1). In yeast, autophagy overlaps with a
biosynthetic process, the Cvt pathway, that delivers the hydrolase
aminopeptidase I (API1; Ref.
2) from its site of synthesis in the cytoplasm to the vacuolar lumen.
Cvt and autophagy employ many of the same molecular components and are
mechanistically related (3-6). Both pathways involve the formation of
double-membrane cytosolic vesicles, sequestering either precursor
aminopeptidase I (prAPI) specifically, or in the case of autophagy,
also enveloping bulk cytosol in a nonselective manner. Fusion of these
vesicles with the vacuole results in the release of single-membrane
subvacuolar vesicles within the lumen. These pathways require a
mechanism for specific lysis of the internalized vesicles, so that
vesicle cargo can be released into the vacuole lumen, and further
require a mechanism for degradation of vesicle lipids.
To understand the molecular basis of these import and degradation
pathways, we carried out a genetic screen for mutants defective in
delivery of prAPI to the vacuole. We isolated a series of mutants, cvt, which accumulate the precursor form of API. The
cvt mutants overlap with mutants defective in autophagy. The
majority of these mutants are blocked at a stage involving formation of
the sequestering vesicle (reviewed in Ref. 1). One mutant,
cvt17, was found to be blocked in the breakdown of
subvacuolar vesicles, suggesting that Cvt17 acts at a late stage of the
import process (6). We report in this paper the cloning of the gene
encoding CVT17. Immunological and biochemical studies
demonstrate that Cvt17 is a glycosylated, integral membrane protein
that transits through the secretory pathway. Cvt17 contains a domain
that is conserved among lipases of the Strains and Media--
Wild type yeast strain SEY6210, was
described previously (7), as were the mutant strains THY32
(cvt17-1; Ref. 3), MGY101 (apg5 Materials--
We followed standard procedures to prepare
antiserum to Cvt17, using synthetic peptides corresponding to amino
acids 131-152 and 295-312 (Multiple Peptide Systems, San Diego, CA).
Additional antisera were described previously (13-15). The
copper-inducible promoter-based plasmid pCu426 was from Dr. Dennis J. Thiele (University of Michigan; Ref. 16). Other reagents are identical
to those described previously (12, 17).
Cloning, Disruption, and Mutagenesis of CVT17--
The
CVT17 gene was cloned by transforming the cvt17-1
strain with a plasmid genomic library and screening for complementation of the starvation-sensitive phenotype on plates containing phloxine B
(18). A secondary screen for prAPI accumulation was carried out as
described previously (2). Subcloning of a complementing plasmid
identified YCR068w as the complementing CVT17 ORF. The cvt17
The rectified CVT17 sequence shifts the frame of the coding
sequence, increasing the length of the encoded protein. The longer, full-length coding sequence was amplified by PCR and subcloned into
pCu426 to generate pCuCVT17(426). For analysis of Cvt17 expression driven by the native promoter, CVT17 was amplified by PCR
and cloned into pRS416 and pRS426, with inclusion of 0.5 kilobase before the start codon and 0.432 kilobase after the
stop codon. A PCR-based overlap-extension strategy was used in
site-directed mutagenesis of CVT17. Serine 332 was converted
to alanine by changing the T at nucleotide 994 to G.
Other Procedures--
Cell fractionation, immunoprecipitation of
radiolabeled proteins, nitrogen starvation sensitivity, prAPI protease
sensitivity, and reversal in SD-N were performed as described
previously (8, 19). For endoglycosidase H (Endo H) treatment, cells
were labeled for 10 min with no chase. Immunoprecipitated proteins were
resuspended in 0.1% SDS, 50 mM sodium citrate, pH 5.5, 1 mM PMSF. After boiling for 7 min, an equal volume of 0.1%
SDS and 0.1 M Fluorescence Microscopy--
Construction of the GFPAut7 fusion
protein and FM 4-64 labeling of cells are described
elsewhere.2 Cells were
examined using a Nikon Eclipse E-800 fluorescence microscope. Images
were captured by a Hamamatsu C4742-98 digital camera.
Cvt17 Is Required for Lysis of Subvacuolar Cvt Bodies and
Autophagic Bodies--
Previous analysis of cvt17-1 by
electron microscopy suggested a defect in the breakdown of subvacuolar,
prAPI-containing vesicles, called Cvt bodies (3, 6). We isolated the
CVT17 gene and disrupted the chromosomal locus to generate a
cvt17
To determine the site of action of Cvt17, we analyzed the state of
precursor API in cvt17
To assess whether the vesicles accumulating in cvt17
While many molecular components are shared between the Cvt and
autophagy pathways, some cvt mutants are not defective in
autophagy (3, 12). To test whether cvt17
Growing yeast cells transport prAPI to the vacuole via Cvt vesicles,
while under nutrient deprivation, prAPI can be transported by
autophagosomes (4, 5). If cvt17 Putative Serine Nucleophile in a Consensus Lipase Active Site Is
Required for Cvt17 Function--
Our original analysis of
CVT17 revealed an SGD error in the coding region of this ORF
(see "Experimental Procedures"). We discovered that the predicted
coding sequence that was given in the data base at that time did not
complement the prAPI accumulation phenotype in cvt17
To determine whether the serine of the putative lipase domain is
required for the function of Cvt17, we mutated the serine at position
332 to an alanine residue. Western blot analysis of API in the
cvt17 Cvt17 Is a Glycosylated, Integral Membrane Protein--
The
potential assignment of Cvt17 as a lipase required for the degradation
of subvacuolar vesicles led us to examine its biosynthesis. To this
end, we generated polyclonal antiserum against the protein. Immunoprecipitation revealed a single predominant band of ~70 kDa.
This band was not detected in the cvt17
Analysis of the Cvt17 primary sequence indicates a stretch of
hydrophobic amino acids (residues 13-35) that might serve as a
membrane anchor for the protein. Following osmotic lysis and subcellular fractionation, Cvt17 was predominately localized to a
13,000 × g pelletable fraction (Fig. 4B).
We investigated whether this localization was due to direct interaction
with a membrane by subjecting the P13 fraction to different wash
conditions. Alkali extraction efficiently removed the peripheral
membrane protein Vma2 from the membrane but had no effect on Cvt17 or
the integral membrane protein Dpm1 (data not shown). Treatment with the
detergent Triton X-100 resulted in the solubilization of all three
proteins. These results suggest that Cvt17 is an integral membrane protein.
The predicted molecular mass of Cvt17 is 58.5 kDa; however, the protein
migrated with a molecular mass of 70 kDa following SDS-polyacrylamide
gel electrophoresis (Fig. 4A). The Cvt17 sequence has three
predicted N-linked glycosylation sites. To determine whether
the aberrant migration was due to glycosylation, we treated immunoprecipitated Cvt17 with Endo H. Following treatment with the deglycosylating enzyme, the molecular mass of Cvt17 shifted to
~61 kDa (Fig. 5A). This
change in molecular mass correlates well with the predicted removal of
three N-linked carbohydrate side chains. This
post-translational modification reveals that Cvt17 must reside at least
temporarily in the secretory pathway. Furthermore, the observation that
Cvt17 is glycosylated allows us to infer the membrane topology of the
protein. All three putative glycosylation sites are C-terminal to the
transmembrane domain, suggesting that Cvt17 is a type II integral
membrane protein. Upon integration into the ER membrane, the N-terminal
13 amino acids would remain exposed to the cytosol, with the majority
of the protein located in the lumen of the ER.
The phenotype of the cvt17 mutant, accumulation of
subvacuolar vesicles, coupled with its characterization as a secretory pathway protein suggests that Cvt17 might function within the vacuole
lumen. Accordingly, we attempted to localize the Cvt17 protein.
Subcellular fractionation based on velocity sedimentation indicated
that Cvt17 was predominantly localized within a 13,000 × g fraction (Fig. 4B). The P13 fraction contains
various subcellular compartments, including the vacuole. To directly
determine whether Cvt17 was located within the vacuole, we purified
vacuoles from yeast spheroplasts. However, we were unable to detect
Cvt17 in the vacuolar fraction using this technique (data not shown).
As an alternative approach, we fractionated cell lysates on an
OptiPrep density gradient. Using this technique, in the presence
of substantial amounts of protease inhibitors, we were able to detect
small amounts of Cvt17 in fractions that contained the vacuolar protein
marker alkaline phosphatase (data not shown). However, the majority of the Cvt17 was found in fractions that were distinct from the vacuole fractions. Thus, Cvt17 is found predominately in a membrane fraction residing outside the vacuole under steady-state conditions.
To examine the localization of Cvt17 in vivo, we engineered
GFP fusions with the protein at both the N and C termini. Both constructs only partially complemented the Cvt trafficking
defect of the cvt17 We have identified a protein that is required for the turnover of
membrane vesicles in the vacuole of yeast. Sequence analysis of this
protein reveals the presence of a domain found in lipases and
esterases, and we have confirmed the importance of this domain by using
site-directed mutagenesis of a putative active site serine residue
within the lipase motif. Phenotypic analysis of yeast expressing the
altered Cvt17 protein shows that the protein is inactive in the Cvt
pathway without this residue. The cvt17 The maintenance of membrane-bound vesicles in the cvt17 The reasons for maintaining a sizable population of Cvt17 outside the
vacuole are as yet obscure. It is possible that Cvt17 performs some
additional unknown function outside the vacuole. Another possibility is
that Cvt17 acts exclusively outside the vacuole and only indirectly
influences the lytic competence of subvacuolar vesicles; Cvt17 might
act as a lipase to specifically alter the lipid composition of Cvt
vesicles and autophagosomes as they form. Specific lipid composition
might be a requirement for the subsequent lysis of Cvt bodies and
autophagic bodies. Lipids could even provide the molecular basis for
recognition within the vacuole lumen, allowing an alternate vacuolar
lipase to distinguish between subvacuolar vesicles destined for
degradation and the bounding vacuolar membrane, which must remain intact.
Cvt17 function, and specifically the function of a lipase active site
domain, is required for the lysis of subvacuolar Cvt and autophagic
bodies. Characterization of this protein is an important first step in
understanding vacuolar function in the turnover of lipids and in the
terminal steps of the Cvt and autophagic pathways. Continued analysis
of Cvt17 will provide important information about membrane recycling in
the vacuole.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
-hydrolase-fold
superfamily. Mutation of the putative lipase active site abolishes
Cvt17 function in the Cvt and autophagy pathways, suggesting that
lipase activity is critical to the role of this protein in these import processes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
::LEU2; Ref. 8), TVY1
(pep4
::LEU2; Ref. 9), WSY99 (ypt7
::HIS3; Ref. 10), and NNY20
(apg1
::LEU2; Ref. 11). Strain WPHYD7
SEY6210 aut7
::LEU2 will be described
elsewhere.2 The
cvt17
strain is described below. Yeast cells were grown as described previously (12).
strain (KTYD17) was made by inserting the
LEU2 gene into the CVT17 ORF of strain SEY6210.
At the time of this analysis, the Saccharomyces Genome
Database (SGD) sequence had shown YCR068w as a
1.289-kilobase coding sequence. We amplified this
region and subcloned it into pCu426 using PCR (all primer sequences
available upon request). This construct (pCuCVT17
C(426)) failed to
complement the prAPI defect of cvt17
. The construct was
sequenced, and comparison to the SGD sequence revealed a single
cytosine insertion in the PCR amplified gene, at nucleotide 956 of the
ORF. Sequencing of the original genomic plasmid revealed an identical
sequence to the PCR product, suggesting an error in the SGD sequence. A
recent revision of the chromosome III sequence has corrected this error.
-mercaptoethanol was added,
then boiled for 7 min. Denatured proteins were incubated overnight at
37° C in the presence of 50 mM sodium citrate, pH 5.5, 16 mM
-mercaptoethanol, 0.03% SDS, 5 mM
PMSF, and 2 units of Endo H. A second immunoprecipitation recovered the
Cvt17 proteins.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain (see "Experimental Procedures"). The
null phenotype was accumulation of prAPI (Fig.
1A), as seen previously with
the cvt17-1 mutant.
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Fig. 1.
The cvt17
strain accumulates Cvt bodies within the vacuole.
A, precursor API is enclosed within a membrane compartment
in the cvt17
strain. Osmotically lysed spheroplasts
(T) were separated into low speed supernatant (S)
and pellet (P) fractions by a 5,000 × g
centrifugation. P fractions were resuspended in lysis buffer alone or
treated with proteinase K ± Triton X-100. B, the Cvt
vesicle marker protein GFPAut7 accumulates in subvacuolar vesicles in
cvt17
. pep4
and cvt17
cells
harboring the pCuGFPAUT7(416) plasmid were grown to mid-log phase.
GFPAut7 expression was induced with 10 µM
CuSO4 for 3 h. Vacuoles were labeled with FM
4-64.
. Yeast cells were converted to spheroplasts and subjected to osmotic lysis. Addition of exogenous protease allowed us to determine whether the accumulated prAPI was
blocked at a stage prior to Cvt vesicle formation/completion (protease-sensitive) or at a point following enclosure but prior to
fusion with the vacuole or vesicle breakdown (protease-protected). As
controls, we examined prAPI protease sensitivity in two strains that
are defective in prAPI import. Apg5 is part of a novel protein conjugation complex that is required for both the Cvt and autophagy pathways (20). The apg5
strain is blocked in the
completion of Cvt vesicles and accumulates membrane-associated, but
protease-sensitive, prAPI (8). Ypt7 is a rab GTPase that is required
for fusion of Cvt vesicles and autophagosomes with the vacuole (1). The ypt7
mutant accumulates prAPI in a protease-resistant
state within completed cytosolic vesicles. Precursor API was found in a
pelletable fraction in cvt17
, as it was in the
apg5
and ypt7
mutant strains (Fig.
1A). In the apg5
strain, prAPI was sensitive
to exogenous protease, even in the absence of detergent, consistent
with a block in vesicle formation (8). In contrast, ypt7
and cvt17
cells contained prAPI that was not degraded to
the mature form in the presence of exogenously added proteinase K
unless detergent was also added, indicating that prAPI was within a
membrane enclosed compartment (Fig. 1A; Ref. 21). These
data, coupled with the vesicle accumulation phenotype of
cvt17-1, suggest that prAPI was accumulating within
subvacuolar Cvt vesicles in the cvt17
strain.
are
indeed Cvt bodies, we examined whether a Cvt vesicle marker accumulated within subvacuolar vesicles during vegetative growth conditions. Aut7
is a component that is required for Cvt vesicle formation and is itself
enclosed within Cvt vesicles (22). We have recently demonstrated the
utility of a GFPAut7 construct as a marker for following the formation
and movement of Cvt vesicles in
vivo.2 To visualize the
vacuole membrane, we used FM 4-64, a lipophilic red dye. In
cvt17
, GFPAut7 is localized to Cvt bodies, seen as punctate structures accumulating within FM 4-64-labeled vacuoles (Fig.
1B, top panels). Some GFPAut7 punctate staining
was also observed outside the vacuole. Mutants in the PEP4
gene, encoding vacuolar proteinase A, accumulate subvacuolar vesicles,
including Cvt bodies by electron microscopy (5). Consistent with these previous observations, GFPAut7-labeled Cvt bodies also accumulated in
the vacuoles of a pep4
strain (Fig. 1B,
bottom panels) but did not accumulate in the vacuoles of
wild type yeast (data not shown). The FM 4-64 vacuolar staining in
cvt17
differed from that in other strains, including
pep4
; we observed a relative increase in the number of FM
4-64-labeled compartments per cell in the cvt17
strain
(Fig. 1B). While we do not know the molecular basis for the
change in vacuolar morphology, the microscopy data provide direct
evidence that cvt17
accumulates Cvt bodies within the vacuole.
has a defect in
autophagy, we examined whether the strain can survive growth in
nitrogen-depleted media. Because transport to the vacuole by autophagy
is the primary mode for degradation of cytoplasmic constituents under
starvation conditions, the process is essential for viability during
nutrient limitation. Wild type cells can withstand nutrient deprivation for long periods of time, while autophagy mutants such as
apg1
or protease-deficient strains such as
pep4
show reduced viability following a shift from growth
in nutrient-rich medium to nitrogen-deficient medium (Fig.
2A). Like apg1
and pep4
, the cvt17
strain was starvation-sensitive.
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Fig. 2.
The cvt17
strain is defective in autophagy. A, log phase
cultures grown in 1% yeast extract, 2% peptone, 2% glucose (YPD)
were shifted to medium lacking nitrogen (SD-N). Viability was
determined by plating aliquots on YPD plates. B, indicated
strains were shifted to SD-N as in A, with aliquots taken at
indicated times. Crude lysates were subjected to immunoblot analysis
with anti-API antiserum.
is defective in lysis of
autophagic bodies, as its starvation sensitivity suggests, we should
see a block in prAPI maturation not only in rich media but also under
starvation conditions. Indeed, both the Cvt and autophagic routes of
delivery are blocked, as indicated by analysis of API following a shift
to SD-N medium (Fig. 2B). Autophagy mutants display
differential blocks in API import under nutrient-rich versus
starvation conditions. For example, mutants such as aut7
that do not have a complete defect in autophagy accumulate prAPI in
nutrient-rich media but are able to mature prAPI under starvation conditions (Ref. 19; Fig. 2B). In contrast, strains that
have more severe autophagy defects, including apg1
,
accumulate prAPI even after prolonged growth in SD-N. The
cvt17
strain did not exhibit a rescue of the prAPI
processing defect when cells were shifted to SD-N, indicating that it
is defective in autophagy as well as the Cvt pathway (Fig.
2B). From these results we conclude that Cvt17 is involved
in the lysis of autophagic bodies, as well as Cvt bodies.
(Fig. 3A,
C-term.
2 µ). Sequencing revealed the presence of an extra cytosine base
in the gene, at nucleotide 956 of the ORF. This insertion shifts the
predicted reading frame, resulting in a protein with a predicted
molecular mass of 58.5 kDa, rather than the 49.9 kDa predicted in the
original data base. The full-length gene is able to complement the
cvt17
strain, as shown by the accumulation of mAPI (Fig.
3A, 2µ and CEN). Importantly, the
frameshift occurred just 5' to a consensus site that is conserved in
lipases and esterases (EC 3.1.1.3). The presence of this motif was
revealed by a BLOCK search using the full-length, correct Cvt17
sequence. The sequence from amino acid 324 to 338 matches the
consensus pattern of serine-active lipases (PROSITE reference PS00120;
[LIV]-X-[LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC];
Fig. 3B). A BLAST search identified related proteins in
several distinct fungi, including Candida albicans
(GenBankTM accession number AL033391; 43%
identity), as well as the fission yeast Schizosaccharomyces
pombe (GenBankTM accession number Z99753; 48%
identity) and the tomato pathogen Cladosporium fulvum
(GenBankTM accession number Y14554; 38% identity
(23)). Alignments of the partial sequences over an area of significant
homology are shown in Fig. 3B. Cvt17 is most similar to the
subfamily of fungal lipases within the larger
/
-hydrolase-fold
superfamily (24, 25). Triglcyeride lipases are lipolytic enzymes that hydrolyze the ester bond of triglycerides. In addition to the conserved
serine within the active site consensus motif, a histidine residue and
an aspartic acid residue act in hydrolysis in a charge-relay system
(26). We found two well conserved aspartic acid residues in Cvt17 and
other related fungal genes as well as a conserved histidine (Fig.
3B).
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Fig. 3.
The nucleophilic serine of a putative lipase
domain is required for Cvt17 function. A, wild
type yeast or strain cvt17 with the indicated
CVT17-containing plasmids were grown to mid-log stage.
Western blots with anti-API antiserum are shown. Plasmids:
library, complementing library plasmid;
C-term.
2µ, truncated CVT17 gene with the last 87 residues
deleted cloned in pCu426; 2µ and CEN,
full-length CVT17 gene in pRS426 and pRS416, respectively;
S332A 2µ and S332A CEN, Cvt17 with Ser to
Ala mutation in the putative catalytic site cloned in pRS426 and
pRS416, respectively. B, sequence alignments of Cvt17 and
related fungal proteins (see "Results" for details). Gaps in
the alignment (-), fully conserved residues (*), and residues with
strong (:) and weaker (.) similarity are
indicated. Putative catalytic triad residues (S-D-H) are indicated by
outlined boxes; only one of the conserved Asp residues is
expected to be active. The gray box indicates the sequence
that matches the consensus lipase pattern. ClustalX software was used
to align sequences.
strain that contained the mutated gene
(cvt17S332A) showed accumulation of the precursor form of API, even
when the mutant gene was expressed from a high copy plasmid (Fig.
3A). In contrast, the wild type gene allowed for prAPI
maturation on both high and low copy plasmids. Thus, Cvt17 cannot
function to break down subvacuolar vesicles and allow prAPI to be
released into the vacuole without this serine within the
G-X-S-X-G lipase motif.
strain,
and expression of CVT17 from a high copy plasmid greatly
increased the abundance of this protein (Fig.
4A, 2µ
lanes), suggesting that the serum was specific for Cvt17. We
also immunoprecipitated Cvt17 from the strain harboring the mutant
protein, which lacked the putative catalytic serine (cvt17S332A 2µ).
The altered protein was recovered in equivalent amounts to the wild
type protein, indicating that the single point mutation did not affect
the stability of the protein.
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Fig. 4.
Cvt17-specific antiserum recognizes a 70-kDa
membrane protein. A, cells from cvt17 ,
wild type and cvt17
cells harboring 2µ wild type
CVT17 and S332A mutant plasmids were radiolabeled
for 5 min followed by a 10-min chase. Crude extracts were
immunoprecipitated with antiserum to Cvt17. B, spheroplasts
of wild type cells were osmotically lysed, and the resultant lysate
(T) was centrifuged at 13,000 × g to yield
supernatant (S13) and pellet (P13) fractions. S13
was further separated into high speed S100 and P100 fractions by
centrifugation at 100,000 × g. Samples were subjected
to immunoblot analysis with antisera to Cvt17, the vacuolar
membrane-associated protein Vac8, and the cytosolic protein
phosphoglycerate kinase (PGK).
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Fig. 5.
Cvt17 is a short-lived glycoprotein.
A, cells were labeled for 10 min, followed by
immunoprecipitation with antiserum to Cvt17. Immunoprecipitated
proteins were mock treated or treated with Endo H. B, wild
type cells were labeled for 10 min. Crude extracts from samples
collected at the indicated chase times were immunoprecipitated with
Cvt17-specific antiserum.
strain, as assessed by API
Western blotting (data not shown). Furthermore, we were unable to
visualize strong fluorescent signals anywhere in cells containing these
GFP fusion constructs. An explanation for this lack of signal might be
an inherent instability of Cvt17. To examine stability of Cvt17
directly, we carried out a pulse-chase analysis. Yeast cells were
labeled with [35S]methionine/cysteine and subjected to a
nonradioactive chase. At each time point, samples were removed and
immunoprecipitated. We found that Cvt17 was unstable in wild type
cells; the protein was degraded with a half-life of ~45 min (Fig.
5B). The inherent instability of this protein made its
localization problematic as much of it was degraded during
fractionation procedures.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant is
unable to degrade both Cvt bodies and autophagic bodies and is
extremely sensitive to starvation (Fig. 2A). The ability to degrade membranes that are released into the vacuole lumen is required
for starvation survival. In the absence of this degradative capacity,
the cell is unable to break down lipid and, as a result, is unable to
access cargo contained within autophagic bodies. The occurrence of
genes with similarity to CVT17 in other fungi suggests that this may be a conserved protein in the kingdom.
strain suggests a role for the protein in degrading a phospholipid bilayer. Sequence comparisons, however, place the protein in a class of
hydrolytic enzymes that act primarily on triglycerides, not
phospholipids. It will be necessary to perform biochemical studies with
the purified enzyme to investigate its substrate specificity. To
function properly, lipases must be targeted to the appropriate
substrates. The membrane source of Cvt vesicles and autophagosomes is
not known. If Cvt17 is incorporated into Cvt vesicles and
autophagosomes, its topology would place the Cvt17 active site on the
outer surface of the Cvt and autophagic bodies. To prevent membrane
degradation during transit, it is possible that Cvt17 becomes active
only in the vacuole. If this is the case, the lytic activity of this
protein must be suppressed during transit. Some aspect of the vacuolar
milieu may then activate Cvt17 as is thought to be the case for
proteinase A. If Cvt17 acts directly to degrade subvacuolar vesicles,
it should reside within the vacuole. Subcellular fractionation did
reveal a small population of vacuolar Cvt17 that may play a primary
role in directly degrading Cvt and autophagic bodies. Most of the
protein, however, did not cofractionate with this organelle. In
agreement with these results, the rapid Cvt17 degradation that we
observed (Fig. 5B) was independent of the major vacuole
proteases (data not shown).
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ACKNOWLEDGEMENTS |
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We thank Jacob Teter for technical assistance, Bob Fuller for use of the fluorescence microscope, and E. J. Brace for help with microscopy. We thank Maria Hutchins for comments on the manuscript and for technical assistance.
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FOOTNOTES |
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* This work was supported by Public Health Service Grant GM53396 from the National Institutes of Health (to D. J. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 734-615-6556; Fax: 734-647-0884; E-mail: klionsky@umich.edu.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.C000739200
2 Kim, J., Huang, W.-P., and Klionsky, D. J. (2001) J. Cell Biol. 152, in press
3 J. Kim, W.-P. Huang, and D. J. Klionsky, submitted for publication.
4 J. Kim, W.-P. Huang, and D. J. Klionsky, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: API, aminopeptidase I; prAPI, precursor API; Cvt, cytoplasm to vacuole targeting; Endo H, endoglycosidase H; ER, endoplasmic reticulum; GFP, green fluorescent protein; PMSF, phenylmethylsulfonyl fluoride; SD-N, synthetic minimal medium lacking nitrogen; ORF, open reading frame; SGD, Saccharomyces Genome Database; PCR, polymerase chain reaction.
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1. | Kim, J., and Klionsky, D. J. (2000) Annu. Rev. Biochem. 69, 303-342[CrossRef][Medline] [Order article via Infotrieve] |
2. | Harding, T. M., Morano, K. A., Scott, S. V., and Klionsky, D. J. (1995) J. Cell Biol. 131, 591-602[Abstract] |
3. |
Harding, T. M.,
Hefner-Gravink, A.,
Thumm, M.,
and Klionsky, D. J.
(1996)
J. Biol. Chem.
271,
17621-17624 |
4. |
Scott, S. V.,
Hefner-Gravink, A.,
Morano, K. A.,
Noda, T.,
Ohsumi, Y.,
and Klionsky, D. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12304-12308 |
5. |
Baba, M.,
Osumi, M.,
Scott, S. V.,
Klionsky, D. J.,
and Ohsumi, Y.
(1997)
J. Cell Biol.
139,
1687-1695 |
6. |
Scott, S. V.,
Baba, M.,
Ohsumi, Y.,
and Klionsky, D. J.
(1997)
J. Cell Biol.
138,
37-44 |
7. | Robinson, J. S., Klionsky, D. J., Banta, L. M., and Emr, S. D. (1988) Mol. Cell. Biol. 8, 4936-4948 |
8. |
George, M. D.,
Baba, M.,
Scott, S. V.,
Mizushima, N.,
Garrison, B. S.,
Ohsumi, Y.,
and Klionsky, D. J.
(2000)
Mol. Biol. Cell
11,
969-982 |
9. | Gerhardt, B., Kordas, T. J., Thompson, C. M., Patel, P., and Vida, T. (1998) J. Biol. Chem. 19, 15818-15829 |
10. |
Wurmser, A.,
and Emr, S.
(1998)
EMBO J.
17,
4930-4942 |
11. | Matsuura, A., Tsukada, M., Wada, Y., and Ohsumi, Y. (1997) Gene (Amst.) 192, 245-250[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Scott, S. V.,
Nice, D. C., III,
Nau, J. J.,
Weisman, L. S.,
Kamada, Y.,
Keizer-Gunnink, I.,
Funakoshi, T.,
Veenhuis, M.,
Ohsumi, Y.,
and Klionsky, D. J.
(2000)
J. Biol. Chem.
275,
25840-25849 |
13. | Klionsky, D. J., Cueva, R., and Yaver, D. S. (1992) J. Cell Biol. 119, 287-299[Abstract] |
14. |
Tomashek, J. J.,
Sonnenburg, J. L.,
Artimovich, J. M.,
and Klionsky, D. J.
(1996)
J. Biol. Chem.
271,
10397-10404 |
15. |
Wang, Y. X.,
Catlett, N. L.,
and Weisman, L. S.
(1998)
J. Cell Biol.
140,
1063-1074 |
16. | Labbé, S., and Thiele, D. J. (1997) Methods Enzymol. 306, 145-153 |
17. |
Noda, T.,
Kim, J.,
Huang, W.-P.,
Baba, M.,
Tokunaga, C.,
Ohsumi, Y.,
and Klionsky, D. J.
(2000)
J. Cell Biol.
148,
465-479 |
18. | Tsukada, M., and Ohsumi, Y. (1993) FEBS Lett. 333, 169-174[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Abeliovich, H.,
Dunn, W. A., Jr.,
Kim, J.,
and Klionsky, D. J.
(2000)
J. Cell Biol.
151,
1025-1033 |
20. | Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M. D., Klionsky, D. J., Ohsumi, M., and Ohsumi, Y. (1998) Nature 395, 395-398[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Kim, J.,
Dalton, V. M.,
Eggerton, K. P.,
Scott, S. V.,
and Klionsky, D. J.
(1999)
Mol. Biol. Cell
10,
1337-1351 |
22. | Huang, W.-P., Scott, S. V., Kim, J., and Klionsky, D. J. (2000) J. Cell Biol. 275, 5845-5851 |
23. | Coleman, M., Henricot, B., Arnau, J., and Oliver, R. P. (1997) Mol. Plant Microbe Interact. 10, 1106-1109[Medline] [Order article via Infotrieve] |
24. |
Cousin, X.,
Hotelier, T.,
Giles, K.,
Toutant, J. P.,
and Chatonnet, A.
(1998)
Nucleic Acids Res.
26,
226-228 |
25. | Derewenda, Z. S., and Sharp, A. M. (1993) Trends Biochem. Sci. 18, 20-25[Medline] [Order article via Infotrieve] |
26. | Blow, D. (1990) Nature 343, 694-695[Medline] [Order article via Infotrieve] |