From the Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania 17033
Received for publication, October 15, 2002 , and in revised form, May 1, 2003.
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ABSTRACT |
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INTRODUCTION |
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Protein degradation by the macroautophagy pathway is also induced when Saccharomyces cerevisiae are starved of nitrogen (1016). Interestingly, the macroautophagy pathway overlaps with the cytoplasm to vacuole (Cvt)1 pathway that directs aminopeptidase I from the cytoplasm to the vacuole (1726). Aminopeptidase I is targeted to the vacuole by Cvt vesicles during growth of cells in rich medium. However, when cells are starved of nitrogen, aminopeptidase I is targeted to the vacuole by an autophagosome-mediated process. The Cvt pathway also shares components with the pexophagy pathway that degrades peroxisomes in response to glucose (2730).
The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded rapidly when glucose-starved S. cerevisiae are replenished with fresh glucose (3135). After a glucose shift, FBPase is first targeted into Vid vesicles, and these vesicles then traffic to the vacuole. Vid vesicles appear to be distinct from other types of vesicle (34). Although the origin of Vid vesicles has not been established, their formation requires the ubiquitin-conjugating enzyme Ubc1p (36).
The import of FBPase into Vid vesicles has been reconstituted utilizing both a semi-intact cellular assay (35) and an isolated Vid vesicle assay (37). Via these approaches, we have identified several proteins that participate in this first trafficking step. These include the molecular chaperone Ssa2p (37), cyclophilin A (38), and Vid22p (39). The molecular mechanisms for the trafficking of Vid vesicle to the vacuole are less well characterized. Although FBPase delivery to the vacuole has been reconstituted in a semi-intact cellular assay, this assay has not allowed us to identify specific molecules involved in the trafficking of Vid vesicles to the vacuole. To address this issue, we have developed an assay to quantitate the Vid vesicle to vacuole trafficking process. FBPase was fused with a form of alkaline phosphatase that lacks the N-terminal 60 amino acids. Under in vivo conditions, the resultant fusion protein was delivered to the vacuole after a glucose shift, and alkaline phosphatase was activated in a Pep4p- and Vid24p-dependent manner. An in vitro Vid vesicle-vacuole assay was developed which reproduces the activation of alkaline phosphatase, and this activation was also dependent on Pep4p and Vid24p. Using this assay, we have identified the small GTPase Ypt7p as being required for Vid vesicle trafficking. Likewise, members of the SNARE and homotypic fusion vacuole protein sorting (HOPS) families of proteins are necessary components in this step of the FBPase trafficking pathway.
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EXPERIMENTAL PROCEDURES |
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The FBPase-60Pho8p Fusion ProteinTo produce
60Pho8p, the PHO8 gene was amplified by PCR using a forward
primer GCGGCCGCACGTTCTGCATCACACAAG and a reverse primer
CCGCGGGTTGGTCAACTCATGGTA. The FBP1 gene was PCR amplified with a
forward primer GGTACCATGGGTCCAACTCTAGTAAATGGA and a reverse primer
GCGGCCGCCTGTGACTTGCCAATATG. These PCR products were cloned into a pYES2.1 TOPO
TA plasmid (Invitrogen), and the orientation of the inserts was confirmed by
PCRs. The PHO8 gene was linearized with NotI and
XbaI and ligated into NotI and XbaI sites of the
FBP1 plasmid. The fusion construct was either transformed into
fbp1
pho8 strains or subcloned into an
integration vector and integrated into the FBP1 locus. The expression
of FBPase-
60Pho8p was examined by Western blotting with anti-FBPase
antibodies. Similar results were obtained whether the fusion construct was
integrated or expressed on a plasmid. The fbp1 deletion construct was
produced as described previously
(36). The deletion of
fbp1 and pho8 was confirmed by Western blotting with
anti-FBPase antibodies and anti-alkaline phosphatase antibodies.
To examine the degradation of FBPase-60Pho8p, strains were grown in
medium containing low glucose. Cells were shifted to glucose-containing medium
for the indicated times, and the levels of FBPase-
60Pho8p were
determined. To examine the cellular distribution of FBPase-
60Pho8p,
cell lysates were subjected to differential centrifugation techniques as
described previously (37).
Briefly, yeast strains (25 ml) were grown in low glucose medium and then
shifted to medium containing 2% glucose for 0 and 60 min at 30 °C. Total
lysates were subjected to differential centrifugation conditions of 1,000
x g for 10 min, 13,000 x g for 20 min, and
200,000 x g for 2 h. The 200,000 x g pellet was
resuspended in 100 µl of SDS sample buffer, whereas the final 200,000
x g supernatant (S200) was precipitated with 10%
trichloroacetic acid, washed three times in ice-cold acetone, and solubilized
in 100 µl of SDS sample buffer. Proteins were resolved by SDS-PAGE, and the
distribution of FBPase-
60Pho8p was determined by Western blotting with
anti-FBPase antibodies.
Preparation of Vid Vesicles, Vacuoles, and CytosolCells (25 ml) were grown to stationary phase and shifted to glucose for 30 min. Vid vesicles and cytosolic materials were obtained using differential centrifugation techniques described above. The final 200,000 x g pellets (Vid vesicles) and supernatant (cytosol) were aliquoted and frozen at 70 °C until further use. As an additional purification step, the 200,000 x g vesicle-containing material was fractionated further on a 2050% sucrose density gradient. The location of vesicles within this gradient was verified by electron microscopy. Samples (5 µl) were adsorbed to a Formar-coated grid for 23 min. Excess material was blotted away, and the samples were negatively stained with 2% phosphotungstic acid, pH 7.0. The grids were examined using a Phillips TEM400 transmission electron microscope, and micrographs were recorded at a magnification of 46,000.
Fractions containing fusion competent vesicles (fractions 35) were
collected and used in the in vitro assays. Vacuoles were isolated
from fbp1
pho8 strains using a protocol
described previously (40).
Cells were subjected to glucose starvation and a 30-min glucose shift.
Spheroplasts were washed and resuspended in freezing buffer (1.2 M
sorbitol, 5 mM HEPES, pH 6.8, 50 mM KOAc, and 5
mM MgOAc) and then stored at 70 °C. Cells were thawed,
washed once in freezing buffer, and then broken via passage through a
Millipore 3.0-µm filter. Samples were centrifuged at 1,000 x
g for 20 min and then 13,000 x g for 20 min. The
resultant 13,000 x g pellet was resuspended in 75 µl of
freezing buffer, and this enriched vacuole material was used for the in
vitro assay. For homotypic vacuole fusion experiments, vacuoles were also
isolated as described previously
(41). Protein concentrations
were determined by Bio-Rad Dc protein assays.
In Vitro Vid Vesicle-Vacuole AssayIn a typical experiment,
the reaction mixture (100 µl) contained 20 µg of vesicle material, 20
µg of vacuolar material, 30 µg of cytosolic proteins, and an
ATP-regenerating system (0.5 mM ATP, 0.2 mg/ml creatine
phosphokinase, and 40 mM creatine phosphate). The reaction mixture
was incubated at 30 °C for 60 min, and the reaction was terminated by a
20-min 13,000 x g centrifugation step at 4 °C. The 13,000 x
g pellet was resuspended in 500 µl of alkaline phosphatase assay
buffer (250 mM Tris-HCl, pH 9.0, 10 mM MgSO4,
and 10 mM ZnSO4) and then examined for alkaline
phosphatase activity using 55 mM -naphthyl phosphate as a
substrate. Samples were incubated at 30 °C for 20 min, and the reaction
was quenched by the addition of 500 µl of 2 M glycine, pH 11.
Alkaline phosphatase activity was measured using a fluorometer (Fluoro IV,
Gilford) with an excitation at 345 nm and an emission at 472 nm. Homotypic
vacuole fusion experiments were conducted as described previously
(41).
For antibody blocking experiments, various amounts (510 µl) of polyclonal antibodies were added to the individual vesicle and vacuole components, and these were then incubated for 30 min at 25 °C. To remove unbound antibody, the Vid vesicles were reisolated using the sucrose gradient protocol described above, whereas vacuoles were reisolated by a 20-min 13,000 x g centrifugation procedure. The antibodytreated components were then mixed with other treated or untreated components and tested in the in vitro assay.
Statistical AnalysisStatistical significance was determined using one-way analysis of variance followed by a Student-Newman-Keuls test. Data are presented as the mean ± S.E. * indicates mean values that are significantly different from controls at p < 0.05.
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RESULTS |
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FBPase was fused with 60Pho8p and expressed in a strain in which the
endogenous PHO8 gene was deleted. The 100-kDa fusion protein was
recognized by both FBPase- and alkaline phosphatase-specific antibodies, but
it was not present in untransformed cells (data not shown). In wild type
cells, FBPase is degraded rapidly in the vacuole in a Pep4p- and
Vid24p-dependent manner after the addition of glucose. Therefore, we first
tested whether FBPase-
60Pho8p was directed to the vacuole and degraded
in a similar manner. FBPase-
60Pho8p was induced by glucose starvation,
and cells were then shifted to glucose-containing medium for various periods
of time. As is shown in Fig.
1A, the addition of glucose to wild type cells resulted
in a significant reduction in the levels of FBPase-
60Pho8p over time.
In contrast, FBPase-
60Pho8p remained at high levels in a
pep4 strain after a glucose shift. The levels of
FBPase-
60Pho8p also remained high in the
vid24 strain,
a strain that blocks the trafficking of Vid vesicles to the vacuole
(53). Therefore, these results
suggest that FBPase-
60Pho8p is degraded in a manner similar to the wild
type FBPase protein.
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Our FBPase trafficking model predicts that FBPase-60Pho8p should
reside within Vid vesicles prior to its transport to the vacuole. To verify
this localization, cells were glucose starved and then subjected to a glucose
shift for 0 or 30 min. The distribution of the FBPase-Pho8
60p fusion
protein in the high speed supernatant (enriched for cytosol) and high speed
pellet (enriched for Vid vesicles) was then determined via differential
centrifugation and Western blot analysis
(Fig. 1B).
FBPase-
60Pho8p was observed in the high speed supernatant fraction when
cells were glucose starved. However, after a 30-min glucose shift,
FBPase-
60Pho8p was found in the Vid vesicle pellet fraction
(Fig. 1B), suggesting
that it was transported into Vid vesicles. To examine whether
FBPase-
60Pho8p is imported into a membrane bound organelle, lysates
were treated with 0.8 mg/ml proteinase K for 15 min in the presence or absence
of 2% Triton X-100. Proteinase K was inactivated via the addition of 1 ml of
15% w/v trichloroacetic acid, and trichloroacetic acid precipitants were
examined for the presence of FBPase-
60Pho8p. FBPase-
60Pho8p was
sensitive to proteinase K digestion when total cell lysates were obtained from
cells that were glucose-starved (Fig.
1C). However, FBPase-
60Pho8p was resistant to
proteinase K digestion when cells were shifted to glucose for 30 min.
FBPase-
60Pho8p was imported into the lumen of a membrane-bound
organelle because FBPase-
60Pho8p was degraded by proteinase K when cell
lysates were treated with Triton X-100.
If the FBPase-60Pho8p fusion protein behaves in a manner similar to
FBPase, it should remain in the cytosol during periods of glucose starvation
and not exhibit alkaline phosphatase activity. However, after the addition of
fresh glucose, FBPase-
60Pho8p should be targeted to the vacuole, and
alkaline phosphatase should be activated by Pep4p. Wild type,
pep4, and
vid24 strains were transformed to
express FBPase-
60Pho8p. These strains were shifted to glucose, and the
activation of alkaline phosphatase was measured
(Fig. 1D). When wild
type cells were maintained in low glucose medium, alkaline phosphatase was not
activated. However, when these cells were shifted to glucose-containing medium
for 3 h, alkaline phosphatase activity increased. Therefore, FBPase was able
to direct
60Pho8p to the vacuole where it was enzymatically activated.
In contrast, alkaline phosphatase activity was low when either the
pep4 or
vid24 strain was shifted to glucose
for 3 h. These results further confirm that FBPase-
60Pho8p is targeted
to the vacuole by the Vid vesicle-mediated pathway.
Autophagic processes often result in the nonselective transport of
cytosolic proteins to the vacuole for degradation
(23). To determine whether
FBPase-60Pho8p could be targeted to vacuoles via the autophagy pathway,
we subjected cells to a period of nitrogen starvation and then examined for
the activation of alkaline phosphatase. A wild type strain expressing
60Pho8p was used as a control because
60Pho8p is targeted to the
vacuole via the autophagy pathway after nitrogen starvation
(13,
15). As expected, low levels
of alkaline phosphatase activity were observed when the
60Pho8p strain
was grown under normal conditions. However, a high level of alkaline
phosphatase activity was observed when these same cells were subjected to
nitrogen starvation (Fig.
1E). In contrast, when FBPase was fused with
60Pho8p, alkaline phosphatase activity was low either before or after
nitrogen starvation. Therefore, it appears that the FBPase-
60Pho8p
fusion protein is targeted to the vacuole via the Vid-dependent pathway, but
not via the autophagy pathway.
Development of an In Vitro AssayAlkaline phosphatase
activity has been used as an indicator of in vitro homotypic vacuole
fusion. In this assay, donor vacuoles contain an inactive precursor form of
alkaline phosphatase in a pep4 genetic background, whereas
acceptor vacuoles are derived from a strain that contains the PEP4
gene but lacks the PHO8 gene. The fusion of donor and acceptor
vacuoles results in the conversion of inactive proalkaline phosphatase to
active alkaline phosphatase after the cleavage of the pro sequence by Pep4p.
Using this as our model, we devised a strategy to examine Vid vesicle to
vacuole content mixing under in vitro conditions
(Fig. 2A). A shift to
glucose-containing medium initiates the import of FBPase-
60Pho8p into
Vid vesicles (see Fig. 1).
These loaded vesicles can be isolated and incubated with vacuoles in the
presence of cytosol and an ATP-regenerating system. If the content mixing of
Vid vesicles with vacuoles occurs under in vitro conditions, alkaline
phosphatase should be activated.
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To enrich for Vid vesicles that were competent for our in vitro
assay, an additional fractionation step was performed. The high speed pellet
material was further resolved via the use of a 2050% sucrose density
gradient. Fractions were collected from the sucrose gradient and examined for
the distribution of FBPase-60Pho8p
(Fig. 2B). The
FBPase-
60Pho8p fusion protein was found in both light (fractions
35) and heavy (fractions 911) density peaks. Vesicular material
was observed as the primary component of samples taken from each of these
peaks (Fig. 2C).
To determine whether these two Vid vesicle populations were functional in our in vitro assay, small aliquots of the sucrose gradient fractions were combined with vacuoles in the presence of ATP and cytosol and examined for alkaline phosphatase activity (Fig. 2D). Interestingly, the highest alkaline phosphatase activity was found when the light density fractions were mixed with vacuoles. In contrast, fractions 911 failed to activate alkaline phosphatase when incubated with vacuoles. Consequently, all subsequent assays were conducted using the light density Vid vesicle fractions.
To determine the optimal time required for alkaline phosphatase activation, the in vitro components were mixed and incubated for various periods of time. Alkaline phosphatase activity was low at the 0 incubation time (Fig. 3A). However, this activity increased in a time-dependent manner, reaching peak levels after a 60-min incubation. Alkaline phosphatase activity also increased in a dose-dependent manner. The optimal concentrations for Vid vesicles (Fig. 3B), vacuoles (Fig. 3C), and cytosol (Fig. 3D) were 20, 12, and 32 µg, respectively. Note that vesicles, vacuoles, and cytosol are all required for in vitro activation of alkaline phosphatase activity (Fig. 3E).
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Pep4p and Vid24p Are Required for In Vitro Activation of Alkaline
PhosphataseA prime reason for the development of the in
vitro assay was to identify proteins that are involved in the Vid vesicle
to vacuole trafficking step. To verify the efficacy of this assay, we first
tested pep4 and
vid24 mutant strains because
these strains have site-specific defects. Consistent with previous data, wild
type vesicles and vacuoles exhibited high alkaline phosphatase activity when
they were combined in our in vitro assay. By contrast, alkaline
phosphatase activity was low when these components were isolated from the
pep4 strain (Fig.
4A). When Vid vesicles from the
pep4
strain were combined with wild type vacuoles, alkaline phosphatase was
activated. By contrast, when vacuoles from the
pep4 strain
were incubated with Vid vesicles from the wild type strain, alkaline
phosphatase activity was low. Therefore, the
pep4 strain
contains competent Vid vesicles but defective vacuoles that failed to activate
alkaline phosphatase.
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The vid24 strain exhibits a defect in the trafficking of
Vid vesicles to the vacuole under in vivo conditions
(53). Likewise, the
combination of
vid24 vesicles and vacuoles was also defective
in the in vitro assay (Fig.
4B). When wild type vesicles were incubated with vacuoles
from the
vid24 strain, alkaline phosphatase activity was high
(Fig. 4B). By
contrast, alkaline phosphatase activity was low when
vid24 Vid
vesicles were incubated with wild type vacuoles. Therefore, the
vid24 strain contains defective Vid vesicles but competent
vacuoles. Because Vid24p normally resides on the surface of Vid vesicles,
these results further support the notion that Vid24p is a required component
on the Vid vesicle membrane.
The GTPase Ypt7p Is Required for the Trafficking of Vid Vesicles to the
VacuoleGTPases are known to play an important role in a number of
protein trafficking events. Therefore, we suspected that GTPases may be
involved in the trafficking of Vid vesicles to the vacuole. To test this idea,
an in vitro Vid vesicle-vacuole reaction experiment was conducted in
which wild type components were incubated in the presence or absence of the
GTPase inhibitor GTPS. In the absence of GTP
S, high alkaline
phosphatase activity was observed when Vid vesicles were incubated with
vacuoles under the in vitro conditions
(Fig. 5A). However,
alkaline phosphatase activity was significantly reduced when GTP
S was
added to the wild type reaction mixture, indicating that the in vitro
Vid vesicle-vacuole reaction requires the hydrolysis of GTP.
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Ypt7p is a GTPase that is involved in the trafficking of late endosomes to
the vacuole (54,
55), targeting of Cvt vesicles
or autophagosomes to the vacuole
(22,
23) and homotypic vacuole
fusion (56,
57,
59). Ypt7p binds and
hydrolyzes GTP and thus cycles between active (GTP-bound) and inactive
(GDP-bound) states
(5659).
To determine whether Ypt7p is required for FBPase trafficking, we first
examined the degradation of FBPase in a ypt7 strain. The
ypt7 strain exhibited a defect in the degradation of FBPase
compared with a wild type strain (Fig.
5B). This suggests that Ypt7p plays some role in the
FBPase degradation pathway and that it is necessary for the delivery of FBPase
to the vacuole. To verify this, we next performed our in vivo
alkaline phosphatase assay using a
ypt7 strain expressing
FBPase-
60Pho8p. Wild type and
ypt7 strains were
subjected to glucose starvation and refeeding protocols, and cellular lysates
were examined for the in vivo activation of alkaline phosphatase.
Wild type cells exhibited an increased alkaline phosphatase activity in
response to glucose. In contrast, the
ypt7 strain failed to
activate alkaline phosphatase after a glucose shift
(Fig. 5C). Thus, Ypt7p
is clearly required for one of the steps in the FBPase trafficking
pathway.
To identify the site of the ypt7 defect, cells were
subjected to a glucose shift at 0 or 60 min, and the distribution of FBPase in
the cytosol (high speed supernatant) and Vid vesicles (high speed pellet) was
examined. As controls, we also examined the localization of FBPase in
vid22 and
vid24 strains. For the
ypt7 strain, a significant portion of FBPase was observed in
the Vid vesicle containing fraction following a glucose shift at 60 min
(Fig. 5D). This was
similar to the results obtained with the
vid24 strain, a
strain that is defective in the Vid vesicle to vacuole trafficking step. By
contrast, the
vid22 strain exhibits a defect in FBPase import
into Vid vesicles (39) and
therefore accumulated FBPase in the cytosol. These results suggest that the
ypt7 and
vid24 strains block FBPase
degradation in the same trafficking step.
We next performed in vitro experiments to clarify further the
function of Ypt7p in Vid vesicle-vacuole trafficking. When
ypt7 Vid vesicles and vacuoles were combined in the in
vitro assay, alkaline phosphatase activity was low
(Fig. 6A). When
ypt7 vesicles were combined with wild type vacuoles, alkaline
phosphatase was not activated. However, when wild type vesicles were combined
with
ypt7 vacuoles, high levels of alkaline phosphatase
activity were observed (Fig.
6A). Thus, it appears that Ypt7p is a required component
for the proper function of Vid vesicles.
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The preceding data suggest that Ypt7p exerts its effect at the Vid vesicle,
and thus it may be a Vid vesicle resident protein. To test this possibility,
the distribution of Ypt7p was examined in various cellular fractions
(Fig. 6B). As a
control, we also examined the localization of FBPase-60Pho8p and CPY
because these proteins reside in the Vid vesicles and vacuoles, respectively.
In cells that were shifted to glucose for 30 min, the majority of
FBPase-
60Pho8p was detected in the Vid vesicle fraction, whereas CPY
was found primarily in the vacuole fraction. In contrast, Ypt7p was
distributed in both the vacuole- and Vid vesicle-containing fractions
(Fig. 6B). Taken
together, these results indicate that a portion of Ypt7p resides on Vid
vesicles. Therefore, Ypt7p appears to play a direct role in the trafficking of
Vid vesicles to the vacuole.
t-SNAREs, v-SNAREs, and the HOPS Complex Are Required for the
Activation of the FBPase-60Pho8p Fusion Protein
The transport of cargo from one cellular compartment to another often requires
the participation of SNARE proteins. v-SNARES are on the donor membranes,
whereas t-SNAREs are required on the target membranes. Unique combinations of
SNAREs are often utilized in particular trafficking events
(60). For example, v-SNAREs
(Nyv1p, Vti1p,Ykt6p), t-SNAREs (Vam3p, Vam7p), and HOPS complex family members
(Vps39p, Vps41p) participate in homotypic vacuolar fusion
(42). However, the function of
individual SNAREs is not restricted to one cellular compartment, and there can
be considerable overlap between different trafficking pathways. For example,
many of these same proteins are also involved in heterotypic fusion events in
which two dissimilar membrane-bound compartments (e.g. vesicles and
vacuoles) fuse together. Because FBPase traffics to the vacuole via Vid
vesicles, it is possible that the final step in this transport also involves
the fusion of Vid vesicles with the vacuole. If so, then one would also
predict that a particular subset of SNARE proteins may participate in this
process.
In preliminary experiments, we determined that each of the aforementioned
SNARE proteins associates with Vid vesicle membranes (data not shown). To
determine whether these SNAREs play an essential role in FBPase trafficking,
we tested various deletion strains for their ability to degrade FBPase. Nyv1p
is a v-SNARE that is required for heterotypic vesicle fusion
(47,
61) and homotypic vacuolar
fusion (61,
62). The nyv1
strain exhibited a defect in FBPase degradation compared with a wild type
strain (Fig. 7A),
indicating that some step in the FBPase degradation pathway was compromised in
this strain. To identify the site of the defect, we performed fractionation
experiments and determined that FBPase was imported into Vid vesicles in the
wild type strain (data not shown). This suggested that the Vid vesicle to
vacuole trafficking step might be defective in this strain. To verify this, we
next performed our in vitro alkaline phosphatase activity assay. As
expected, there were low levels of alkaline phosphatase activity when Vid
vesicles and vacuoles were obtained from the
nyv1 strain
(Fig. 7B). In further
experiments, we were able to identify the Vid vesicles as the defective in
vitro component because alkaline phosphatase activity was observed when
wild type vesicles were mixed with
nyv1 vacuoles but not when
nyv1 vesicles were mixed with wild type vacuoles
(Fig. 7B).
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The v-SNAREs Vti1p and Ykt6p play important roles in multiple cellular trafficking events. For example, Vti1p is involved in Golgi to vacuole trafficking (62, 63), whereas Ykt6p is a v-SNARE that plays a role in trafficking through the Golgi (64, 65). Both of these proteins also play a role in homotypic vacuolar fusion (51). Deletion of either the VTI1 or YKT6 genes is lethal in yeast. Furthermore, strains with temperature-sensitive mutations of these genes were problematic in terms of FBPase and fusion protein expression. Therefore, we performed antibody-blocking experiments to examine the role that these proteins play in Vid vesicle trafficking. When anti-Vti1p or anti-Ykt6p antibodies were added both to Vid vesicles and vacuoles, there was a statistically significant decrease in the levels of alkaline phosphatase activity (Fig. 8). In contrast, the addition of equivalent amounts of preimmune serum had no effect on alkaline phosphatase activity. Antibodies were also added to the individual Vid vesicle or vacuole components, and these components were reisolated prior to their use in the in vitro assay. The addition of anti-Vti1p antibody to the Vid vesicle fraction resulted in a significant decrease in alkaline phosphatase activity (Fig. 8A). In contrast, the addition of anti-Vti1p antibody to the vacuole fraction did not have an inhibitory effect. Thus, it appears that Vid vesicles require the presence of functional Vti1p for the in vitro activation of the fusion protein, but vacuoles do not. Similar results were obtained when antibody-blocking experiments were performed using the anti-Ykt6p antibody. The anti-Ykt6p antibody blocked the function of Vid vesicles, but it had no effect on the function of vacuoles (Fig. 8B).
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Vam3p and Vam7p are vacuolar t-SNAREs that are part of a cis-SNARE complex
on vacuoles (51). Vam3p plays
a role in the docking process
(61), whereas Vam7p stabilizes
the interactions of other SNARE components
(66). Deletion of either of
the VAM3 or VAM7 genes resulted in an FBPase degradation
defect (Fig. 9A).
Furthermore, both of these strains exhibited a defect in alkaline phosphatase
activation, when Vid vesicles and vacuoles were obtained from the deletion
strains (Fig. 9). However, in
contrast to our results with v-SNAREs, the presence of Vam3p appeared to be
required on the vacuole fraction because vacuoles obtained from the deletion
strain were defective (Fig.
9B). Interestingly, the combination of
vam7 vesicles and wild type vacuoles, or wild type vesicles
and
vam7 vacuoles, resulted in intermediate alkaline
phosphatase activities (Fig.
9C). Thus, the presence of this protein on either
compartment may allow a partial activation of alkaline phosphatase.
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The HOPS complex is known to be required for homotypic vacuole fusion and
endosome to vacuole fusion
(42). Two members of this
complex, Vps39p and Vps41p, play an essential role in the docking step of
homotypic vacuole fusion (67).
Therefore, we tested whether these proteins also play some role in the FBPase
trafficking pathway. Both vps39 and
vps41
deletion strains exhibited a defect in FBPase degradation
(Fig. 10A). Likewise,
both of these strains contained defective in vitro components that
exhibited low alkaline phosphatase activities
(Fig. 10, B and
C). Here again, the vesicles were identified as the site
of this defect because there was low alkaline phosphatase activity when mutant
vesicles were combined with wild type vacuoles. However, neither Vps39p nor
Vps41p appears to be required on the vacuole fraction. This is in contrast to
the results observed when homotypic fusion experiments were performed with
these same strains. Isolated vacuoles from these strains exhibited low levels
of in vitro homotypic fusion activity
(Fig. 10D).
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DISCUSSION |
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Ypt7p was found to play an important role in the Vid vesicle/vacuole
trafficking step. Ypt7p is a GTPase that is essential for a number of cellular
trafficking events. Endosomes require the presence of Ypt7p to be targeted to
the vacuole (54,
55), as do Cvt vesicles or
autophagosomes (22,
23). Likewise, Ypt7p is
required for the process of homotypic vacuolar fusion
(56,
57,
59) where it mediates
tethering (48). In our
studies, FBPase accumulated in Vid vesicles in the ypt7
mutant. In vitro activation of alkaline phosphatase was defective in
this mutant, and Vid vesicles were identified as the defective component.
Therefore, Ypt7p is necessary for the proper function of Vid vesicles.
Interestingly, Ypt7p was not required for the function of vacuoles in our
assay. This is in contrast to the situation in homotypic vacuole fusion, where
Ypt7 is required on both the donor and acceptor vacuoles
(56).
We have also identified a role for various SNARE proteins in the trafficking and degradation of FBPase. v-SNAREs and t-SNAREs are generally found on opposing vesicle and target membranes, respectively. Under the proper circumstances, these SNAREs can assemble to form a SNARE complex, which promotes the fusion of the membranes. In our studies, the v-SNAREs Nyv1p, Vti1p, and Ykt6p were required for the trafficking of Vid vesicles to the vacuole, and they exerted their effects on the Vid vesicle fraction. The requirement for multiple v-SNAREs on Vid vesicles is similar to that reported for the homotypic fusion of vacuoles. Vacuoles utilize these same three v-SNAREs (42). The t-SNARE Vam3p was also necessary for in vitro activation in our assay, but it was functional only on the vacuoles. Interestingly, when examined under in vitro conditions, the SNAP25 homolog Vam7p exhibited a partially defective phenotype. When Vam7p was absent from both the Vid vesicles and vacuoles, there were low levels of alkaline phosphatase activity. However, if Vam7p was present on either of these fractions, there was an intermediate level of alkaline phosphatase activity observed. The explanation for this effect is not clear at the present time. However, Vam7p does not directly mediate fusion, but instead it stabilizes the interaction of other SNARE components in homotypic vacuolar fusion (66). Therefore, there may be a dosage effect whereby the presence of Vam7p on one compartment allows partial formation of these complexes.
Many of the proteins required for homotypic vacuolar fusion are present on the surface of the vacuole in the form of oligomeric complexes (42). These include Vam3p, Vam7p, Nyv1p, Vti1p, and Ykt6p. Likewise, the HOPS complex is also part of a hexameric complex that localizes to this organelle (48). We have not determined whether similar complexes exist on the Vid vesicles. However, the presence of these proteins on Vid vesicles may indicate that they function in a similar manner on this organelle. Because members of the HOPS complex were required on the Vid vesicle fraction, this suggests that the association of Vid vesicles with vacuoles appears to resemble more closely a heterotypic rather than a homotypic fusion event. This was further confirmed by our results with the t-SNARE Vam3p. Even though this protein was present on both Vid vesicles and vacuoles (data not shown), it was only required on the vacuolar fraction. This observation is in agreement with the established role of Vam3p as a t-SNARE. In homotypic vacuole fusion, transient formation of a Vam3p and Nyv1p SNARE pair between opposite membranes stabilizes vacuole docking (50). Therefore, it is possible that a similar situation exists during the association of Vid vesicles with vacuoles.
Recent data regarding the structural characteristics of Vam3p and Vti1p may shed some light on the specificity of Vid vesicle trafficking (68, 69). Conformational changes in the target membrane syntaxin may play an important role in SNARE complex formation. It has been proposed that the functions of many SNAREs can be regulated by their adoption of a closed versus an open conformation of their four-helix bundle structure. SNAREs in the closed conformation are inactive, whereas SNAREs in the open conformation are active. Both Vam3p and Vti1p, however, appear to remain in the open conformation (68, 69), suggesting that there must be some other method of regulation. This may include the presence of other proteins that regulate the conformation of the SNARE complex. One likely candidate is the protein Vps33, a member of the HOPS complex which has been shown to form an interaction with Vam3p (68).
The FBPase trafficking and degradation pathway is distinct from other known protein trafficking pathways. As such, it requires the participation of at least one protein, Vid24p, that has no other known function. Here, for the first time, we have identified components that are required both for Vid vesicle trafficking and for other well characterized protein trafficking pathways. Thus, the Vid vesicle-vacuole trafficking step requires proteins that are common to other trafficking pathways, as well as molecules specifically involved in FBPase degradation. Further experiments will be required to identify additional components that are essential for this step of the FBPase degradation pathway.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Cellular and Molecular
Physiology, Penn State College of Medicine, 500 University Dr., Hershey, PA
17033. Tel.: 717-531-0859; Fax: 717-531-0859; E-mail:
crb13{at}psu.edu.
1 The abbreviations used are: Cvt, cytoplasm to vacuole; FBPase,
fructose-1,6-bisphosphatase; GTPS, guanosine
5'-3-O-(thio)triphosphate; HOPS, homotypic fusion vacuole
protein sorting; SNARE, soluble NSF attachment protein receptor.
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ACKNOWLEDGMENTS |
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REFERENCES |
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