Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, 500 University Drive, Hershey, PA 17033, USA
Author for correspondence (e-mail:
hlchiang{at}psu.edu)
Accepted 30 October 2001
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Summary |
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Key words: Vid vesicles, Vacuole, Fructose-1, 6-bisphosphatase, VID genes
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Introduction |
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Extracellular material and plasma membrane proteins can be internalized and
targeted to the vacuole via endocytosis
(Hicke, 1997). Proteins can
also be delivered from the cytoplasm into the vacuole
(Klionsky et al., 1992
;
Harding et al., 1995
;
Harding et al., 1996
). Two
enzymes, aminopeptidase I (API) and
-mannosidase, are synthesized as
inactive enzymes within the cytoplasm and are transported from the cytoplasm
to the vacuole independent of the secretory pathway
(Yoshihisa and Anraku, 1990
;
Klionsky et al., 1992
;
Scott et al., 1996
). Targeting
of API to the vacuole is mediated by the Cvt pathway, a pathway that shares
common components with the macroautophagy pathway and the pexophagy pathway
(Klionsky and Ohsumi, 1999
;
Kim and Klionsky, 2000
).
A number of circumstances such as changes in nutrient conditions can induce
the trafficking of proteins and organelles to the vacuole
(Tuttle and Dunn, 1995;
Klionsky and Ohsumi, 1999
;
Kim and Klionsky, 2000
). For
example, peroxisomes are targeted to the vacuole for degradation when S.
cerevisiae are shifted from media containing oleic acid to media
containing glucose (Chiang et al.,
1996
), while autophagosomes are formed and targeted to the vacuole
when S. cerevisiae are starved of nitrogen
(Takeshige et al., 1992
;
Tsukada and Ohsumi, 1993
;
Klionsky and Ohsumi, 1999
;
Kim and Klionsky, 2000
). Yeast
cells deliver the enzyme fructose-1,6-bisphosphatase (FBPase) to the vacuole
for degradation following a shift from low to high glucose conditions
(Chiang and Schekman, 1991
;
Chiang et al., 1996
;
Shieh and Chiang, 1998
). Prior
to its entry into the vacuole, however, FBPase is targeted to a novel type of
Vid vesicle (Huang and Chiang,
1997
).
A number of genes are known to play important roles in the FBPase
degradation pathway (Hoffman and Chiang,
1996). Previously, we identified VID24 and determined
that this gene is required for the proper targeting of FBPase from Vid
vesicles to the vacuole. VID24 encodes a peripheral membrane protein
that associates with the surface of Vid vesicles. In the absence of
VID24, FBPase remains sequestered within Vid vesicles and is unable
to traffic to the vacuole (Chiang and
Chiang, 1998
). Here we describe another novel gene,
VID22, that is essential for FBPase degradation. VID22
encodes an integral membrane protein that localizes to the plasma membrane. In
comparison with wild-type cells,
vid22 degraded FBPase at a
significantly reduced rate following a glucose shift. The majority of FBPase
accumulated in the cytosol in this mutant, suggesting that Vid22p is involved
in the targeting of FBPase into Vid vesicles. By contrast, processing of CPY
or API to the vacuole was not impaired in the
vid22 mutant.
Likewise, there was no defect in either starvation-induced autophagy or
peroxisome degradation in the
vid22 mutant. Therefore,
VID22 appears to play a specific role in the FBPase trafficking
pathway.
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Materials and Methods |
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Mouse monoclonal anti-V5 antibodies were purchased from Invitrogen (Invitrogen, Carlsbad CA). Rabbit polyclonal antibodies directed against FBPase, CPY, Pma1p, Vid24p and enolase were produced by Berkeley Antibody Company (Berkeley, CA). HRP-conjugated goat anti-rabbit and HRP conjugated goat anti-mouse antibodies were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). The enhanced chemiluminescence kit was from NEN (NEN Life Sciences, Boston, MA). Antibodies were used at 1:5000 dilution for V5 and Sec21p and 1:10,000 dilution for FBPase, CPY, Pma1p, dipeptidyl aminopeptidase B (DPAP B), Mnn1p, Pep12p, enolase, HRP-conjugated goat anti-rabbit and HRP conjugated goat anti-mouse.
Isolation of the vid22-1 mutant
The gene encoding Vid22p was identified using a Tn-lacz/Leu2-mutagenized
library provided by Michael Snyder (Yale University, New Haven, CT). This
library was amplified in E. Coli, purified, and restriction digested
with NotI. Excised DNA fragments were purified, transformed into
HLY223 using a lithium acetate protocol, and plated onto leucine drop-out
plates for 5-7 days at 22°C. Mutants were replica plated (colony-blotted)
onto nitrocellulose membranes, which were then immersed in SD (with 5%
dextrose) for 3 hours at 37°C. Cells were lysed and blotted with affinity
purified anti-FBPase antibodies (1:1000 dilution). Membranes were incubated
with alkaline phosphatase-conjugated goat anti-rabbit antibodies (1:5000
dilution), and proteins were visualized using color development reagents
(Bio-Rad, Richmond, CA). FBPase degradation deficient mutants were identified
as dark purple colonies and were selected for further characterization.
Sequencing and cloning of the VID22 gene
The Tn disrupted genes were identified with a vectorette PCR protocol. A 10
ml culture of the vid22-1 strain was harvested and chromosomal DNA
was isolated, digested with DraI (10 U), and ligated to annealed DNA
bubble primer
3'-GAGAGGGAAGAGAGCAGGCAAGGAATGGAAGCTGTCTGTCGCAGGAGAGGAAG-5' and
5'-GACTCTCCCTTCTCGAATCGTAACCGTTCGTACGAGAATCGC TGTCCTCTCCTTC-3'.
The primers (2-4 µM) were annealed by heating at 65°C for 5 minutes
followed by the addition of MgCl2 to 1-2 mM at room temperature.
The ligation reaction (30 µl) contained digested chromosomal DNA, 1 µl
annealed anchor bubble primers, 1 µl (400 U) ligase and 50 µM ATP. This
mixture was incubated at 16°C for 9-24 hours.
The mutated gene was amplified via PCR using primers: 5'-CGAATCGTAACCGTTCGTACGAGAATCGCT-3' and 5'-CGCCAGGGTTTTCCCAGTCACGAC-3' and sequenced in the Penn State College of Medicine Core Facility. The flanking sequences of the transposon exhibited exact identity to open reading frame YLR373C using the BLAST search. This gene, VID22, was cloned by PCR with primers: 5'-AGCGGCCGCGGGATGGGGAGAGCGATGGACACACAG-3' and 5'-GAGGCGGCCGCCTGGAAGATACTGACTTGC-3'. The PCR product was ligated into pyes2.1/V5 His-TOPO plasmid and transformed into TOP10F' cells (Invitrogen, Carlsbad, CA). The VID22 gene was fused in-frame with a sequence encoding a V5 epitope as confirmed via PCR. The fusion gene was excised from the plasmid with PvuII and XbaI and ligated into the SmaI/XbaI site of a digested YIP352 plasmid. The construct was cloned, linearized with sali and integrated into HLY223 using the lithium acetate protocol. This integration resulted in the expression of the Vid22p-V5 fusion protein under the control of the endogenous VID22 promoter. The second unfused gene has no promoter and should not be expressed. The integration was confirmed by PCR reactions and the expression of Vid22p-V5 was examined by western blotting of total lysates with anti-V5 antibodies.
Characterization of Vid22p
Vid22p-V5 wild-type cells were grown for 2 days in media containing low
glucose and then shifted to media containing 2% glucose for the indicated
times. Cells were homogenized as described
(Chiang and Chiang, 1998).
Total extracts were centrifuged at 1000 g and resuspended in
either 50 µl TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), 50 µl TE with
0.1M Na2CO3 (pH 11.5) or 2% Triton X-100. Suspensions
were incubated at 4°C for 30 minutes and centrifuged at 13,000
g for 10 minutes. The distribution of Vid22p and Pma1p was
examined by western blotting of fractions with antibodies directed against
these proteins. For endo H treatments, the 1000 g cell pellet
was resuspended in endo H buffer (50 mM sodium citrate, pH 5.5, 10 mM
NaN3) and incubated at 75°C for 30 minutes. Samples were
treated with 5 mU of endo H enzyme and incubated for 16 hours at 37°C. All
samples were boiled, resolved by SDS-PAGE, and Vid22p-V5 was detected using
western blots with antibodies directed against the V5 epitope. For CON A
binding experiments, cells were lysed in CON A buffer (100 mM KPO4,
pH 6.8, 200 mM NaCl, 1 mM MnCl2, 1 mM CaCl2) containing
1% Triton X-100. The lysates were centrifuged at 13,000 g for
10 minutes and Con A beads were added to the resultant supernatants. Following
a 60 minute incubation, the beads were pelleted and the unbound material
(supernatant) was subjected to TCA precipitation. The CON A beads were washed
with CON A buffer and the bound material was eluted using SDS sample
buffer.
Pulse-chase analysis of Vid22p
To investigate the processing of Vid22p, a temperature sensitive
sec18-1 strain was transformed with a plasmid containing the
VID22-V5 fusion gene. Cells were grown at 22°C to an OD of 2-3,
diluted to OD=0.4 and then re-grown to an OD of 0.8. Cells were spheroplasted
and resuspended to 5 OD/ml in media containing yeast nitrogen base (YNB)
without ammonium sulfate, without amino acids, with 2% glucose, with 1.2 M
sorbitol and supplements. Cells were labeled by the addition of 200 µCi/ml
35S-methionine and cysteine (Specific activity=10 mci/ml) for 20
minutes at 37°C. The labeling media were removed and cells were washed and
resuspended in the chase media containing excess unlabeled L-methionine and
L-cysteine (20 µg/ml). Cells were incubated for 0, 30 and 60 minutes at
either 22°C or 37°C. Cells were harvested at each time point and
solubilized in 200 µl of IP buffer (50 mM Tris, pH 7.4, 1% SDS, 5 mM EDTA).
Samples were diluted to 1 ml with IP dilution buffer (50 mM Tris, pH 7.4, 1%
Triton X-100 and 200 mM NaCl) and then centrifuged at 13,000 g
for 20 minutes. Supernatants were immunoprecipitated with 10 µl of anti-V5
antibodies or 5 µl of anti-CPY antibodies, followed by the addition of 50
µl of 50% protein G beads or 50 µl of protein A beads (Pharmacia).
Precipitated proteins were separated by SDS-PAGE using 7.5% polyacrylamide
gels. These radiolabeled proteins were visualized and quantitated with a
phosphorimager (Molecular Dynamics).
To study the effect of glucose on the half-life of Vid22p, we used a wild-type strain expressing the Vid22p-V5 protein. Cells were incubated in labeling media containing 2% ethanol plus 200 µCi/ml 35S-methionine and cysteine for 5 hours (glucose starvation conditions). Cells were washed and then incubated in chase media containing either 2% ethanol or 2% glucose. Cells were harvested at 0, 1 and 2 hours and total lysates were immunoprecipitated with anti-V5 antibodies. Immunoprecipitated material was solubilized in SDS sample buffer, boiled and separated by SDS-PAGE. Radiolabeled proteins were visualized with a phosphorimager.
Differential centrifugation
A 10 ml culture of the Vid22p-V5 strain was grown for 2 days in YPKG. Cells
were shifted to YPD for t=30 minutes, after which, samples were
pelleted at 1000 g. The pellets were resuspended in 200 µl
of FBPase lysis buffer (50 mM Hepes-NaOH, 5 mM MgSO4, 40 mM
(NH4)2SO4, 0.1 mM EDTA, 200 µg/ml
phenylmethylsufonyl fluoride) and homogenized as described
(Chiang and Chiang, 1998). The
yeast lysate was centrifuged at 1000 g (P1) for 15 minutes at
4°C. Both the low-speed pellet and supernatant were collected. The
supernatant was sequentially centrifuged; first at 13,000 g
(P13) for 20 minutes in a desktop centrifuge (American Scientific Products),
and then at 100,000 g (P100) and 200,000 g
(P200) for 2 hours using a Beckman ultracentrifuge and a Beckman TLA 100.2
rotor. Supernatant fractions were TCA precipitated and the resultant pellets
were resuspended in 100 µl SDS sample buffer. All samples were boiled,
resolved by SDS-PAGE, and examined via western blotting using ECL
reactions.
Subcellular distribution of newly synthesized Vid22p
Wild-type cells expressing Vid22p-V5 were labeled for 20 minutes and chased
for 0, 30 and 60 minutes at 30°C. Cells were harvested and total lysates
were subjected to differential centrifugation as described above. Each
fraction was collected, solubilized in 2% Triton X-100 and immunoprecipitated
with anti-V5 antibodies. The immunoprecipitated materials were resolved by
SDS-PAGE and radiolabeled proteins were visualized using a phosphorimager.
Sucrose density gradients
Sucrose density gradients were constructed by sequential addition of 3 ml
50% sucrose, 2 ml 40% sucrose, 2 ml 35% sucrose, 2 ml 30% sucrose, and 2 ml
20% sucrose in FBPase lysis buffer. A 10 ml culture was grown for 2 days in
YPKG and then shifted to YPD for 30 minutes. Cell pellets were resuspended in
200 µl FBPase lysis buffer and broken as described above. The lysates were
loaded onto the sucrose gradient and centrifuged at 100,000 g
for 20 hours at 4°C. Following centrifugation, samples were aliquoted from
the top in 1 ml fractions and precipitated with 10% TCA. The precipitants were
resuspended in 50 µl of SDS sample buffer, resolved using SDS-PAGE, and
detected with western blotting and ECL reactions.
Immunofluorescence studies
For immunofluorescence studies, cells were grown to saturation in YPKG and
then shifted to YPD. One ml samples were collected at t=0, 30, and 60
minutes. Samples were fixed with 200 µl of a 37% formaldehyde solution for
30 minutes at 30°C with moderate shaking. Following fixation, cells were
washed with KP (0.1 M potassium phosphate, ph 6.5) and KPS buffer (0.1 M
potassium phosphate, pH 6.5, 1.2 M sorbitol). Cells were spheroplasted by
resuspension in 1 ml of KPS buffer containing 10 µl of
ß-mercaptoethanol and lyticase (20 U/OD). Following a 60 minute
incubation at 30°C, cells were washed, resuspended in KPS buffer and
adsorbed to multiwell slides pre-coated with 1% poly-lysine. Samples were
blocked with PBST (0.04 M potassium phosphate monobasic, 0.01 M potassium
phosphate dibasic, 0.15 M NaCl, 0.1% Tween-20, 10 mg/ml BSA and 0.1% sodium
azide) for 15 minutes at 25°C, and incubated with anti-V5 or anti-Pma1p
antibodies overnight at 4°C. The primary antibodies were removed and cells
were washed four times with PBST. They were then incubated with
FITC-conjugated goat anti-mouse or anti-rabbit antibodies for 1 hour at
25°C. The cells were washed four times with PBST and the slides were
sealed with mounting media. Cells were examined using a Zeiss Axiovert s100
(Carl Zeiss Inc., Thornwood, NY) fluorescence microscope equipped with a
digital camera (Hamamatsu Inc., Japan).
Miscellaneous assays
Degradation of FBPase was performed as described
(Hoffman and Chiang, 1996).
Briefly, wild-type cells, vid22-1 mutants,
vid22
mutants or mutant strains transformed with the VID22 gene were grown
in media containing low glucose to induce FBPase and then shifted to media
containing glucose for the indicated times. The degradation of peroxisomes was
examined using 3-oxoacyl Co A thiolase as a marker
(Chiang et al., 1996
). For
proteinase K experiments, total lysates from the
vid22 strain
were digested with proteinase K (1 mg/ml) in the absence or presence of 2%
Triton X-100 for 20 minutes. The reactions were terminated by TCA
precipitation and samples were boiled, resolved by SDS-PAGE and analyzed via
western blotting. Vid24p induction and distribution were examined as described
(Shieh et al., 2001
). Cells
were shifted to glucose for the indicated time and total lysates were
immunoblotted with anti-Vid24p antibodies. For Vid24p distribution
experiments, cells were shifted to glucose for 60 minutes and lysed. Samples
were first centrifuged at 13,000 g for 20 minutes and the
13,000 g supernatants (total) were centrifuged at 200,000
g for 2 hours. The 200,000 g supernatant
(high-speed supernatant) and 200,000 g pellet (high-speed
pellet) were immunoblotted with anti-Vid24p antibodies as described
(Shieh et al., 2001
).
Processing of CPY and API was performed as described previously
(Rothman and Stevens, 1986
;
George et al., 2000
).
Starvation-induced autophagy studies were conducted as described previously
(Shintani et al., 2001
).
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Results |
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Partial sequences of the Tn disrupted vid22-1 gene were obtained using a vectorette PCR protocol described in Materials and Methods. Sequence analysis indicated that the transposon was inserted at nucleotide residue 224 in the open reading frame of YLR373C as determined by a BLAST search of the Saccharomyces Genome Database. A role for VID22 in FBPase degradation was confirmed via complementation experiments. When the VID22 gene was cloned and transformed into the vid22-1 mutant, the FBPase degradation rate was restored to that seen in wild-type cells (Fig. 1A).
To further verify that VID22 participates in the FBPase
degradation process, we used a yeast strain in which open reading frame
YLR373C was completely deleted (vid22). Wild-type cells
rapidly degrade FBPase when they are shifted from low glucose to high
glucose-containing media. Accordingly, there was a significant decrease in
FBPase levels in the wild-type strain at t=60 minutes and little if
any detectable levels of FBPase at t=120 minutes
(Fig. 1B). Conversely, the
vid22 cells exhibited relatively little change in the level of
FBPase over the entire time period when shifted to glucose rich media. When
vid22 cells were transformed with the VID22 gene,
their ability to degrade FBPase was restored
(Fig. 1B). Thus, VID22
must play some important role in the FBPase degradation process.
Cells lacking VID22 accumulate FBPase in the cytosol
Various FBPase degradation deficient mutants accumulate FBPase either
within the cytosol or within Vid vesicles
(Hoffman and Chiang, 1996;
Chiang and Chiang, 1998
). This
suggests that mutants can be defective either in the import of FBPase into Vid
vesicles or in the trafficking of Vid vesicles to the vacuole. To determine
the point at which the FBPase degradation pathway is blocked in the
vid22 mutant, we examined FBPase distribution following
differential centrifugation and proteinase K treatment assays. The
vid22 mutant cells were glucose starved and then shifted to
glucose for t=0 or t=60 minutes. Cells were homogenized and
total lysates were subjected to proteinase K digestion in the absence or
presence of Triton X-100 (Fig.
2A). At t=0 minutes, FBPase was degraded by proteinase K
whether Triton X-100 was present or not, suggesting that FBPase was in a
proteinase K sensitive compartment, most likely the cytosol. At t=60
minutes, FBPase was also sensitive to proteinase K in the presence or absence
of Triton X-100 (Fig. 2A). Since FBPase remained in the proteinase K sensitive cytosolic compartment
following a glucose shift in the
vid22 mutant, these results
suggest that the targeting of FBPase from the cytosol to Vid vesicles is
blocked in this mutant.
|
The site of blockage in the FBPase degradation pathway in the
vid22 mutant was further examined by differential
centrifugation experiments. Glucose starved cells were shifted to glucose
containing media for t=0 or t=60 minutes and then harvested.
Cell lysates were subjected to differential centrifugation procedures and
FBPase distribution in these fractions was examined. In the
vid22 mutant, FBPase was detected in the supernatant
fractions, while only a minor amount of FBPase was found in the Vid vesicle
pellet fraction (Fig. 2B). Note
that the distribution of FBPase in the supernatant versus the pellet fractions
did not change from t=0 to t=60 minutes, suggesting that the
absence of Vid22p results in a defect of FBPase targeting to Vid vesicles. By
contrast, the
vid24 mutant strain did exhibit a change in
FBPase localization following a glucose shift. FBPase was found primarily in
the supernatant fraction at t=0 minute in this mutant. However,
FBPase accumulated within the Vid vesicle fraction after a shift of this
mutant to glucose for 60 minutes (Fig.
2B). This was as expected, since
vid24 has
previously been shown to be defective in the trafficking of Vid vesicles to
the vacuole, an occurrence that results in the accumulation of FBPase within
Vid vesicles (Chiang and Chiang,
1998
).
A defect in FBPase targeting to the Vid vesicle fraction could result from
either an absence or a reduction in the level of Vid vesicles. Therefore, we
examined whether Vid vesicle formation was affected in the
vid22 mutants using the Vid vesicle-specific marker Vid24p.
When glucose starved cells are replenished with fresh glucose, Vid24p is
induced and localizes to Vid vesicles
(Chiang and Chiang, 1998
). As
is shown in Fig. 2C, Vid24p was
undetectable at t=0 minute in wild-type and
vid22
total cell lysates. However, this protein was induced to similar levels in
both strains following a glucose shift of t=20 and t=60
minutes. To determine whether Vid24p distribution was altered in the mutant
strain, wild-type and
vid22 cells were shifted to glucose for
30 minutes. Cells were homogenized and total lysates were subjected to
differential centrifugation. Under these conditions, Vid vesicles are enriched
in the high-speed pellet fraction, but not in the high-speed supernatant
fraction, which is enriched in cytosol
(Brown et al., 2000
). In
wild-type cells, most of the Vid24p was found in the Vid vesicle containing
pellet fraction (Fig. 2D).
Likewise, in the
vid22 mutants, the majority of Vid24p was
also found in the Vid vesicle containing fraction
(Fig. 2D), suggesting that the
formation of Vid vesicles is not impaired in
vid22 mutants.
Therefore, the VID22 gene does not appear to play a role in Vid
vesicle biogenesis.
VID22 is not required for other vacuolar trafficking
pathways
We next examined whether VID22 is involved in other vacuolar
targeting pathways. API is synthesized as a precursor form in the cytoplasm
and then targeted to the vacuole by the Cvt pathway. API is then processed to
the mature form in the vacuole via a process that is dependent on the
PEP4 gene (Klionsky et al.,
1992; Harding et al.,
1995
; Klionsky and Ohsumi,
1999
; Kim and Klionsky,
2000
). As is shown in Fig.
3A, the control wild-type cells processed API to the mature form
(lane 1). However, a
pep4 mutant failed to process API and
accumulated the precursor form of API within cells (lane 2). Likewise, the
apg1 mutant, which is defective in the Cvt pathway,
accumulated the precursor form of API within cells (lane 3). By contrast, the
majority of API was processed to the mature form in the
vid22
mutant (lane 4). Therefore, API processing is not impaired in cells lacking
the VID22 gene.
|
CPY is sorted to the vacuole from the secretory pathway by the Vps
targeting pathway (Rothman and Stevens,
1986; Johnson et al.,
1987
; Valls et al.,
1987
; Robinson et al.,
1988
; Raymond et al.,
1992
; Marcusson et al.,
1994
; Cooper and Stevens,
1996
). In wild-type cells, CPY is synthesized and processed to the
p1 form in the ER. CPY is further modified to the p2 form in the Golgi and
then processed to the mature form in the vacuole in a process that requires
the PEP4 gene (Klionsky et al.,
1990
; Jones, 1991
;
Bryant and Stevens, 1996). As is shown in
Fig. 3B, most of the CPY was in
the mature form in wild-type cells (lane 1), while the p2 form accumulated in
pep4 mutants (lane 2). Note that the
apg1
mutant did not affect the maturation of CPY, as indicated by the presence of
the mature form of this protein (lane 3). In a similar manner, the
vid22 mutant strain processed CPY to the mature form (lane 4).
Taken together, these results suggest that the VID22 gene is not
involved in the Vps or Cvt targeting pathways.
Peroxisomes are also targeted to the vacuole for degradation in response to
glucose (Chiang et al., 1996).
As an indicator of peroxisomal trafficking to the vacuole, we examined the
levels of the peroxisomal marker protein 3-oxoacyl Co A thiolase
(Fig. 3C). Thiolase expression
was induced when wild-type or
vid22 cells were grown in media
containing oleic acid. However, the levels of thiolase decreased to
approximately 35% of the original levels when these same cells were shifted to
media containing fresh glucose. This is consistent with our previous results
in which a similar percentage of peroxisomes remained after a prolonged shift
in glucose (Chiang et al.,
1996
). Thus, this indicates that VID22 does not play a
role in the trafficking of peroxisomes to the vacuole.
We next examined whether VID22 might play some essential role in
nitrogen starvation induced autophagy. Cells were subjected to nitrogen
starvation conditions, in order to induce the formation and trafficking of
autophagosomes to the vacuole. To allow for the accumulation of autophagic
bodies in the vacuole, cells were treated with PMSF, a compound that inhibits
the action of proteinase B. A number of autophagic bodies were observed in the
vacuole of wild-type cells following nitrogen starvation
(Fig. 3D). The
vid22 strain also contained a number of these structures
within their vacuoles. By contrast, autophagic bodies were not observed in the
vacuole of a strain in which the APG1 gene was deleted. Therefore,
this indicates that VID22 is not a required component of the
autophagy pathway. Taken together, these results suggest that VID22
plays a specific role in the targeting of FBPase to the vacuole. Accordingly,
this gene does not appear to be required for a variety of other vacuolar
trafficking processes.
Vid22p is a glycosylated integral membrane protein
VID22 resides on chromosome XII and encodes a protein with the
amino acid sequence shown in Fig.
4A. To date, the VID22 gene product has not been
analyzed, although a number of the characteristics of Vid22p can be inferred
based upon sequence analysis. Vid22p is a protein of 901 amino acids with a
predicted molecular weight of 102 kDa and a pI of 5.15. Based upon
Kyle-Doolittle hydropathy and TMpred analysis, Vid22p contains a hydrophobic
region that may serve as a transmembrane domain. Furthermore, there are 12
potential N-linked glycosylation sites found in the Vid22p amino acid
sequence, suggesting that Vid22p may be a glycoprotein
(Fig. 4A). Note that Vid22p
does not contain a N-terminal hydrophobic region that might be used as a
signal sequence for protein translocation into the ER.
|
A construct was generated in which a V5 epitope was fused in frame with Vid22p at the C terminus. The VID22V5 fusion gene was subcloned into the integration vector YIP352, linearized with SalI, and integrated into the yeast genome. The addition of the V5 tag to Vid22p did not affect the kinetics of FBPase degradation (Fig. 4D). Therefore, this procedure allowed for the detection of Vid22p with antibodies directed against the V5 epitope without compromising the function of this protein. Vid22p was observed as a doublet (Fig. 4B, lane 1). As stated above, Vid22p contains 12 potential glycosylation sites. Therefore, the faster migrating band might represent the unglycosylated form of Vid22p, while the slower migrating band could represent the glycosylated form. To test this, cellular lysates were treated with endo H glycosidase and then examined by western blot. The high molecular weight band appeared to significantly decrease in intensity following endo H glycosidase treatment (Fig. 4B, lane 2). This most likely represents the conversion of glycosylated Vid22p to unglycosylated Vid22p. Similar results were obtained when Vid22p was immunoprecipitated and then treated with endo H (not shown). These results suggest that Vid22p is a glycosylated protein.
To further verify that Vid22p is glycosylated, we tested whether this protein would bind to beads containing covalently crosslinked Con A. Cellular lysates were incubated with Con A beads and the Con A bound and unbound materials were examined via western blot analysis. As controls, we tested for the localization of the glycosylated protein CPY and the unglycosylated protein FBPase. As expected, CPY bound to Con A beads, while FBPase did not. Note that glycosylated Vid22p (the slower migrating band) bound to Con A, while the unglycosylated band (the faster migrating band) did not bind to Con A (Fig. 4C). Therefore, the endo H and Con A binding experiments indicate that the slower migrating band represents the glycoslyated form of Vid22p, while the faster migrating band represents the unglycoslylated form.
We next determined whether Vid22p expression or degradation was affected by
the presence of glucose. Wild-type cells expressing Vid22p-V5 were glucose
starved and then shifted to glucose-containing media. Cells were harvested and
lysates were immunoblotted with anti-V5 antibody to examine the steady state
levels of Vid22p. As is shown in Fig.
4D, Vid22p levels did not change in response to glucose, while the
levels of FBPase decreased significantly over the same period of time. As an
additional control, we examined Pma1p and observed that the levels were not
changed. By contrast, Vid24p was induced following a glucose shift, in
agreement with previous results (Chiang and
Chiang, 1998). To determine whether Vid22p degradation was
affected by the presence of glucose, cells expressing Vid22p-V5 were labeled
under starvation conditions (ethanol) and then chased for 0, 1 and 2 hours in
the presence or absence of glucose. Cells were harvested and Vid22p-V5 was
immunoprecipitated using anti-V5 antibodies. There was no significant
difference in the levels of radiolabeled Vid22p when cells were chased either
in ethanol or in glucose (Fig.
4E). Thus, Vid22p appears to be a stable protein and its rate of
degradation is not accelerated by glucose.
Vid22p contains putative transmembrane domains as determined by Kyte-Doolittle hydropathy plot and TMpred analysis. Therefore, Vid22p may be an integral membrane protein. In order to test this, cells were homogenized and total lysates were subjected to extraction procedures. Samples were resuspended in TE buffer, TE with Na2CO3, or TE with 2% Triton X-100. Samples were incubated at 4°C for 30 minutes and then centrifuged. The resultant supernatants and pellets were examined using SDS-PAGE and western blot analysis. As is shown in Fig. 4F, in the presence of TE buffer, Vid22p was associated with the pellet fraction (lane 2) and not the supernatant (lane 1). Likewise, Vid22p remained associated with the pellet fraction (lane 4) when samples were extracted with Na2CO3. Following extraction with Triton X-100, however, Vid22p was found in the supernatant fraction (lane 5) but not in the pellet fraction (lane 6). As a control, we examined the effects of these extraction procedures on the distribution of the integral membrane protein Pma1p. Pma1p was extracted into the supernatant fraction by Triton X-100 treatment, but remained in the pellet fraction when samples were treated with TE buffer alone or with Na2CO3 (Fig. 4F). Therefore, Vid22p exhibits the same characteristics as an integral membrane protein.
Vid22p is found in plasma membrane-containing fractions
To address whether Vid22p associates with subcellular organelles, we
performed a series of differential centrifugation experiments. The majority of
Vid22p was found in the P1 low-speed pellet following differential
centrifugation (Fig. 5a).
Potentially, this fraction could contain cell membranes, nuclei and unbroken
cells. To distinguish between these possibilities, we examined the
distribution of a number of marker proteins. The cytosolic marker protein
enolase was found primarily in the S200 fraction
(Fig. 5b), indicating that the
majority of cells had been lysed. The vacuolar marker dipeptidyl
aminopeptidase B (DPAP B) and the endosomal marker Pep12p
(Becherer et al., 1996) were
found primarily within the P13 fraction
(Fig. 5c,d), while the plasma
membrane protein Pma1p was located mainly within the P1 fraction
(Fig. 5e). Although a small
amount of Vid22p was detected within the P13 fraction, Vid22p was not detected
in the P100, P200 and S200 fractions. Taken together, these results indicate
that Vid22p is associated with fast sedimenting structures that colocalize
with the plasma membrane marker Pma1p.
|
To further characterize the localization of Vid22p within cells, we
subjected the total lysates to centrifugation through a sucrose density
gradient. Fractions were collected following centrifugation and the resultant
material was examined by western blot analysis using a V5-specific antibody.
The majority of Vid22p was found in fractions 7-10
(Fig. 6a). Likewise, most of
the Pma1p was localized to these fractions, although a small amount was found
in fraction 2 (Fig. 6b). By
contrast, the distribution of Vid22p did not overlap with other organelle
marker proteins that were examined. For example, the ER-derived COP I vesicle
protein Sec21p (Bednarek et al.,
1995) was found in fractions 3-4
(Fig. 6c), while the Golgi
protein Mnn1p (Graham et al.,
1994
) and the vacuolar protein CPY were found localized to
fractions 1-5 (Fig. 6d,e).
There was some overlap between Vid22p and the Vid vesicle marker protein
Vid24p (Fig. 6f). However, the
Vid22p and Vid24p peaks did not coincide. These results support the contention
that Vid22p is a plasma membrane protein, and is not associated with other
cellular organelles.
|
Vid22p is a plasma membrane protein
The localization of Vid22p was further examined via the use of indirect
immunofluorescence analysis. Wild-type cells expressing Vid22p-V5 were glucose
starved and then incubated in fresh glucose as described above. The cells were
fixed and processed for indirect immunofluorescence analysis using the
V5-specific antibody. Vid22p appeared to localize to the surface of the cells
in a pattern indicative of plasma membrane staining
(Fig. 7a-c). This plasma
membrane localization was confirmed via staining of cells with an antibody
specific for Pma1p. FITC-fluorescence labeling of Pma1p showed similar
staining as Vid22p (Fig. 7d-f).
Therefore, these immunofluorescence results are consistent with the conclusion
that Vid22p is localized within the plasma membrane. Interestingly, the
localization of the Vid22p to the plasma membrane remained unchanged following
the addition of glucose (Fig.
7a-c).
|
To test whether Vid22p is endocytosed and delivered to the vacuole in
response to glucose, Vid22p distribution was examined in a
pep4 mutant strain expressing Vid22p-V5. Cells were shifted to
glucose for the indicated times and examined for Vid22p distribution by
indirect immunofluorescence microscopy. Most of the Vid22p staining was found
on the plasma membrane, whether
pep4 cells were glucose
starved or shifted to glucose (Fig.
7g-i). Therefore, the majority of Vid22p appears to remain on the
plasma membrane and is not endocytosed in response to glucose.
Vid22p is targeted to the plasma membrane independent of the
classical secretory pathway
Most plasma membrane proteins are initially synthesized and translocated
into the ER. From there, they then travel through the secretory pathway and to
the plasma membrane. If Vid22p is targeted to the plasma membrane via the
secretory pathway, then the processing of Vid22p should be inhibited when the
secretory pathway is blocked. To test this idea, pulse chase experiments were
conducted using a temperature-sensitive sec18-1 mutant strain that
blocks the secretory pathway when incubated at the nonpermissive temperature
(Kaiser and Schekman, 1990;
Graham and Emr, 1991
). A
sec18-1 mutant strain expressing Vid22p-V5 was pulsed at the
nonpermissive temperature and chased at either the permissive or nonpermissive
temperature for the indicated time points. Cells were harvested and half of
the aliquots were immunoprecipitated with anti-V5 antibodies and the other
half of the aliquots were immunoprecipitated with anti-CPY antibodies. As
expected, CPY was processed to the p1 form, the p2 form and finally the mature
form following a chase of the sec18-1 mutant at the permissive
temperature (Fig. 8A). However,
CPY remained as the p1 form when the sec18-1 mutant strains were
chased at the nonpermissive temperature. By contrast, Vid22p processing was
not inhibited when the sec18-1 mutant was chased either at the
permissive or nonpermissive temperatures
(Fig. 8A). Since Vid22p
processing was not inhibited when ER-Golgi transport was blocked, Vid22p may
not travel through the classical secretory pathway. However, these results do
not rule out the possibility that Vid22p glycosylation occurs in the ER.
|
To determine whether glycosylation of Vid22p occurs in the ER, we examined the distribution of newly synthesized Vid22p using pulse-chase experiments combined with subcellular fractionation (Fig. 8B). Wild-type cells expressing Vid22p-V5 were labeled for 20 minutes and chased at t=0, 30 and 60 minutes. Cells were harvested and total lysates from each time point were subjected to differential centrifugation. To determine the distribution of Vid22p, fractions collected from differential centrifugation were detergent solubilized and immunoprecipitated with an anti-V5 antibody. As is shown in Fig. 8B, newly synthesized Vid22p was in the S200 fraction at t=0 minute. Vid22p appeared as a doublet, but still remained in the S200 fraction following a chase for 30 minutes. However, at t=60 minutes, Vid22p was detected in the plasma membrane fraction (P1). Note that Vid22p was not found in the P13 or P100 fractions that contain vacuole, ER or Golgi at any of the time points. Likewise, Vid22p was detected in the S200 fraction at t=30 minutes and P1 fraction at t=60 minutes, when the sec18-1 mutant was shifted to the nonpermissive temperature, but not in the ER fraction at any time point in this strain (not shown). This is consistent with the observation that Vid22p does not contain an N-terminal hydrophobic signal sequence for protein translocation into the ER. Therefore, these results suggest that Vid22p is targeted to the plasma membrane independent of the classical secretory pathway.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FBPase degradation-deficient mutants can be defective in one of several
steps in the FBPase degradation pathways. Mutations can prevent targeting of
FBPase from the cytosol into the Vid vesicle. Additionally, mutations can
inhibit trafficking of Vid vesicles to the vacuole. We have shown previously
that the VID24 gene is involved in the second step of the targeting
pathway (Chiang and Chiang,
1998). In the present study, we have begun to characterize the
VID22 gene, a gene that encodes a glycosylated integral membrane
protein. Subcellular fractionation by differential centrifugation and a
sucrose density gradient revealed that Vid22p was distributed in the same
fraction as Pma1p, a known plasma membrane marker. Finally, the localization
of Vid22p to the plasma membrane was further confirmed via the use of indirect
immunofluorescence analysis. Vid22p was localized to the plasma membrane and
exhibited a similar staining pattern to cells that were stained with
antibodies against Pma1p.
Most plasma membrane proteins and secretory proteins are translocated into
the ER and travel through the Golgi prior to their delivery to their final
destinations. However, protein export through the non-classical secretory
pathway has been demonstrated (Kuchler and
Thorner, 1992; Michaelis,
1993
; Cleves et al.,
1996
; Florkiewicz et al.,
1998
; Boulianne et al,
2000
; Dahl et al.,
2000
). For example, the yeast a-factor is secreted by the
non-classical secretory pathway
(Michaelis, 1993
;
Kuchler et al., 1989
;
Kuchler and Thorner, 1992
).
Human FGF-2 is also secreted by a mechanism independent of the ER/Golgi
pathway (Florkiewicz et al.,
1998
; Dahl et al.,
2000
). Mammalian galectins play important roles in diverse
biological events and are secreted by the non-classical ER/Golgi pathway
(Dodd and Drickamer, 2001
).
Yeast cells expressing galectin-1 export this protein at a normal rate when
the secretory pathway is blocked by the sec18 mutant at the
nonpermissive temperature, suggesting that galectin-1 export uses a new
pathway that is distinct from the classical secretory pathway
(Cleves et al., 1996
). In a
similar manner, Vid22p processing was not inhibited by the sec18-1
mutant, which blocks the secretory pathway at the nonpermissive temperature.
Furthermore, Vid22p first appears as a low molecular weight band in the S200
fraction following pulse labeling. The high molecular weight band also
appeared in the S200 fraction before Vid22p trafficked to the plasma membrane
fraction. Note that Vid22p was not found in the ER or Golgi-containing
fractions, suggesting that Vid22p does not travel through the ER/Golgi
pathway. Furthermore, there is no hydrophobic N-terminal region that might act
as a signal sequence for Vid22p to translocate into the ER. Therefore, these
results suggest that Vid22p is delivered to the plasma membrane independent of
the ER/Golgi secretory pathway. To the best of our knowledge, Vid22p is the
first example of a plasma membrane protein that is targeted from the cytoplasm
independent of the classical secretory pathway.
Vid22p was found to play an important role in the degradation of FBPase,
since the deletion of VID22 resulted in a significant decrease in the
rate of FBPase degradation. In the vid22 mutant, FBPase
accumulated primarily in the cytosolic fraction. This implies that the FBPase
degradation defect is at the trafficking step from the cytosol to Vid
vesicles. However, the reduced FBPase targeting to Vid vesicles in the
vid22 mutants was not due to a decrease in Vid vesicle
formation. Instead, the cytosolic component was found to be defective in this
strain. We have recently identified cyclophilin A as the cytosolic protein
that mediates the function of Vid22p in FBPase import process
(Brown et al., 2001
). The
levels of Cpr1p were significantly decreased in the
vid22
mutant strain compared with wild-type controls. Furthermore, the addition of
purified Cpr1p to
vid22 in vitro components stimulated the
import of FBPase. Therefore, Vid22p regulates the levels of Cpr1p, which in
turn stimulates FBPase import into Vid vesicles.
A number of proteins are required to carry out the degradation of FBPase. While we have identified some of these proteins, a clear picture of the individual roles that these proteins play in the process awaits further investigation. The characterization of FBPase degradation mutants will enhance our knowledge of the FBPase degradation pathway. Accordingly, our goals are to identify more genes involved in the FBPase degradation pathway and to determine the mechanisms by which these proteins induce the proper trafficking and degradation of FBPase.
![]() |
Acknowledgments |
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![]() |
Footnotes |
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