From the Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033
Received for publication, February 28, 2000, and in revised form, December 28, 2000
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ABSTRACT |
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When Saccharomyces cerevisiae are
shifted from medium containing poor carbon sources to medium containing
fresh glucose, the key gluconeogenic enzyme fructose-1,6-bisphosphatase
(FBPase) is imported into Vid (vacuole import and degradation) vesicles and then to the vacuole for degradation. Here, we show that FBPase import is independent of vacuole functions and proteasome degradation. However, FBPase import required the ubiquitin-conjugating enzyme Ubc1p.
A strain containing a deletion of the UBC1 gene exhibited defective FBPase import. Furthermore, FBPase import was inhibited when
cells overexpressed the K48R/K63R ubiquitin mutant that fails to
form multiubiquitin chains. The defects in FBPase import seen for the
The vacuole of the yeast Saccharomyces cerevisiae is
homologous to the lysosome of higher eucaryotes and as such, plays an important role in protein degradation (1-4). The function of the
vacuole requires the targeting of a number of vacuole resident proteins
into this organelle. These proteins are sorted to this organelle by
several mechanisms and require the assistance of numerous genes. For
example, targeting of the vacuole lumenal protein carboxypeptidase Y
(CPY)1 from the late Golgi
requires more than 40 VPS genes (1-4).
Proteins and organelles can be delivered to the vacuole from the
cytoplasm by the microautophagy or macroautophagy pathways (5-11).
Regulation of the autophagic process can have important consequences on
cellular physiology. For example, the tumor suppresser gene
beclin-1 is homologous to APG6/VPS30 and induces
autophagy in yeast and mammalian cells. Therefore, a decrease in
autophagic protein degradation may contribute to the development or
progression of human malignancy (13).
A nonselective macroautophagy pathway is induced when S. cerevisiae are starved of nitrogen (5-11). This pathway requires a novel ubiquitin-like conjugating system (14). Furthermore, this
pathway also overlaps with the cytoplasm to vacuole targeting pathway for targeting aminopeptidase I from the cytoplasm (5-11). Aminopeptidase I trafficking to the vacuole occurs by two routes (11).
Under normal growth conditions, aminopeptidase I is targeted to the
vacuole by cytoplasm to vacuole targeting vesicles. When cells are
starved of nitrogen, however, aminopeptidase I is delivered to the
vacuole by the macroautophagy pathway (11). Recent evidence suggests
that the cytoplasm to vacuole targeting pathway also shares components
with the peroxisome microautophagy pathway (15-17).
Fructose-1,6-bisphosphatase (FBPase), the key regulatory enzyme in
gluconeogenesis in S. cerevisiae, is induced when yeast cells are grown in medium containing poor carbon sources (18). When
fresh glucose is added to the medium, however, FBPase is targeted to
the vacuole and degraded (19, 20). This redistribution of FBPase to the
vacuole has been observed by immunofluorescence microscopy, cell
fractionation, and electron microscopy (19, 20). More recently, FBPase
targeting to the vacuole has been reconstituted in vitro
using permeabilized yeast cells incubated with purified radiolabeled
FBPase in the presence of ATP, an ATP regenerating system and cytosolic
proteins (21).
FBPase is imported into a novel type of Vid (vacuole import and
degradation) vesicle prior to its uptake by the vacuole (22). These
vesicles have been purified to near homogeneity from wild-type cells
(22). The identification of Vid vesicles in the FBPase degradation
pathway suggests that this pathway can be divided into at least two
steps. The first step is the targeting and sequestration of FBPase into
Vid vesicles. The second step is the delivery of FBPase from Vid
vesicles to the vacuole for degradation.
Since Vid vesicles do not contain markers from known organelles, they
may represent a novel transport structure, although it is possible that
Vid vesicles are derived from existing structures. Thus far, the heat
shock protein Ssa2p is the only molecule that has been shown to play a
role in the import of FBPase into Vid vesicles (23). To identify more
molecules involved in this process, we analyzed the import of FBPase
into Vid vesicles using various inhibitors and mutants. We found that
FBPase import was not affected by inhibitors or mutants that block
vacuole acidification, vacuole proteolysis, or proteasome degradation.
However, FBPase import did require ubiquitin chain formation and the
ubiquitin conjugation enzyme Ubc1p. The In the absence of the UBC1 gene, the level of the Vid
vesicle-specific marker Vid24p was reduced in the Vid vesicle pellet fraction, suggesting that UBC1 is required for Vid vesicle
production. Alternatively, Vid24p binding to Vid vesicles may be
compromised in the absence of ubiquitination. However, overproduction
of the K48R/K63R mutant did not prevent Vid24p binding to Vid vesicles. Since ubiquitin chain formation is necessary for Vid vesicle function, but is dispensable for Vid24p binding to Vid vesicles, these results are consistent with the hypothesis that Vid vesicle formation is
regulated by ubiquitin conjugation and ubiquitin chain formation. Thus,
our work complements previous studies in which ubiquitin conjugation is
important for peroxisome biogenesis (24), mitochondrial inheritance
(25), mitochondrial targeting (26), and receptor-mediated endocytosis
(27-31).
Yeast Strains, Chemicals, and Antibodies--
S.
cerevisiae strains used in this study are listed in Table
I. For the in vitro
experiments, the endogenous FBP1 gene was deleted and a
known quantity of purified FBPase was added to the reaction. To produce
the fbp1 null strain, the FBP1 gene was cloned into pBR322 to yield the plasmid pJS31. The fbp1 deletion
construct was generated by removing 90% of the FBP1 gene
from pJS31 with StuI and religating with a LEU2
containing fragment which was produced by digestion of the YEP13
plasmid with BglII. The deletion construct was then digested
with BamHI and HindIII and transformed into yeast
strains using the standard lithium acetate method. The deletion of
FBP1 was confirmed by Western blotting with anti-FBPase antibodies.
A pep4 null mutation was produced using the pTS15 plasmid
provided by Dr. T. Stevens (University of Oregon). This plasmid was
digested with EcoRI and XhoI to disrupt the
PEP4 locus (32). The defect in the pep4 null
strains was confirmed by the accumulation of the p2 form of CPY
intracellularly. A strain with a null mutation of the VID1
gene was also utilized. The VID1 gene is identical to the
ISE1 or ERG6
gene.2 The gene was amplified
by polymerase chain reaction and cloned into a TA cloning vector
(Invitrogen) using a 5' primer AGCGGCCGCGGGATGGGGAGTGAAACAGAATTGAGAAAA and a 3' primer TGAGGCGGCCGCCTTGAGTTGCTTCTTGGGAAGTTTGGG. A deletion construct was produced by removing 80% of the gene via KpnI
and PflMI digestion, and religating with a URA3
containing fragment produced by digesting the YIP352 plasmid with
SmaI and HpaI. The resultant construct was
linearized with NotI and transformed into a wild type
strain. The deletion was confirmed by polymerase chain reaction
analysis. The pUB141 plasmid containing the wild type Myc-tagged
ubiquitin, the pUB223 plasmid containing the Myc-tagged K48R/K63R
ubiquitin mutant and the Ub-Pro-
YPD is a complete medium (10 g/liter of Bacto-yeast extract, 20 g/liter
of Bacto-peptone, Difco Labs Inc.) supplemented with 20 g/liter
dextrose (Fisher Scientific). YPKG contained 10 g/liter Bacto-yeast
extract, 20 g/liter Bacto-peptone, 10 g/liter potassium acetate, and 5 g/liter dextrose. Synthetic minimal medium consisted of 6.7 g/liter
yeast nitrogen base without amino acids, supplemented with 5 g/liter
casamino acids, 40 mg/liter adenine, 60 mg/liter leucine, and 20 g/liter dextrose. Inhibitors used in this study included ATP The FBPase Import Assay--
The FBPase import assay was
performed according to Shieh and Chiang (21). In a typical experiment,
the reaction mixture (100 µl) contained 3 A600
nm units of semi-intact cells, 11 µg of
35S-FBPase, an ATP regenerating system (0.5 mM
ATP, 0.2 mg/ml creatine phosphokinase, 40 mM creatine
phosphate), and 0.5 mg/ml cytosolic proteins. The mixture was incubated
at 30 °C for the indicated times, after which 0.8 mg/ml proteinase K
was added to identify the fraction of FBPase that was sequestered in a
proteinase K-resistant compartment. Samples were processed and
resuspended in 200 µl of SDS-loading buffer. The proteins (15 µl)
were then resolved by SDS-PAGE and analyzed by a Fuji FUJIX BAS 1000 Bioimaging Analyzer (Fuji Medical Systems).
Miscellaneous Assays--
Isolation of Vid vesicles by
differential centrifugation was performed as described (23). Briefly,
total lysates were subjected to differential centrifugation at
13,000 × g for 20 min and the supernatant was further
centrifuged at 200,000 × g for 2 h. The distribution of Vid24p in the high speed pellet (200,000 × g pellet) and the high speed supernatant (200,000 × g supernatant) was determined by Western blotting with
anti-Vid24p antibodies. The biosynthesis of CPY was studied using the
protocol described by Graham et al. (35). The exponentially
grown FBPase Import in Vitro--
To biochemically analyze FBPase import
into Vid vesicles, we used an in vitro system that
reproduces the defects seen for mutants affecting the FBPase
degradation pathway. For example, both the
To examine FBPase import in the The FBPase Import Is Independent of Vacuole Proteolysis and Vacuole
Acidification--
Next, we utilized our in vitro assay to
investigate whether FBPase import into Vid vesicles was dependent on
other cellular processes such as vacuole proteolysis or vacuole
acidification. The PEP4 gene is required for the maturation
of several major vacuolar proteinases including CPY. Hence, the
deletion of the PEP4 gene renders cells defective in
vacuolar proteolysis (1, 3). In wild type cells, CPY is synthesized as
prepro-CPY and then translocated into the endoplasmic reticulum where
it is glycosylated to p1-CPY in the endoplasmic reticulum (1-4). CPY
is further modified in the Golgi to p2-CPY and finally processed to the
mature form in the vacuole (1-4). Therefore, the deletion of the
PEP4 gene resulted in the accumulation of p2-CPY in the
As is shown in Fig. 1, the addition of ATP and cytosol stimulates
FBPase import into Vid vesicles. This suggests that ATPases and/or ATP
hydrolysis (see below) may play some role in FBPase import. The
VMA3 gene, which encodes the 16-kDa proteolipid subunit of
the membrane sector of the V-ATPase (1, 39), has previously been shown
to play a role in autophagy (40). However, when FBPase import was
measured in the vma3 deletion mutant, there was no significant defect (Fig. 3B, lane 5). Therefore, V-ATPase is
not essential for FBPase import into Vid vesicles.
FBPase Import Requires the UBC1 Gene--
Ubiquitination plays an
important role in distinct biological functions including DNA repair,
protein degradation, organelle biogenesis, and protein trafficking (41,
42). For example, the ubiquitin protein ligase Rsp5p is essential for
mitochondrial inheritance and mitochondrial import (25, 26). Rsp5p is
also involved in receptor-mediated internalization of Ste2p, Ste3p, and
other cell surface proteins (31). In addition, the
ubiquitin-conjugating enzyme Ubc10p plays a critical role in
peroxisomal biogenesis (24). Ubc10p is one of 13 ubiquitin-conjugating
enzymes found in yeast (41, 42). UBC1, UBC4, and
UBC5 are functionally overlapping and are involved in
degrading abnormal or short-lived proteins (43, 44). As expected, the
One of the major functions of ubiquitin conjugation is to target
proteins for degradation by the proteasome (41, 42). However, ubiquitin
conjugation can also have other important functions unrelated to
protein degradation (24-31, 41, 42). We investigated whether the
proteasome plays a role in FBPase import using the pre1-1pre2-1 proteasome mutant. PRE1 and
PRE2 encode subunits of the 20 S core particle of the
proteasome and an interaction between Pre1p and Pre2p is necessary for
formation of the chymotrypsin-like active site in the proteasome (47,
48). A decrease in the degradation rate of short-lived proteins was
observed for the pre1-1pre2-1 mutant strain (Fig.
4A). However, the import of FBPase in the
pre1-1pre2-1 mutant was not altered (Fig. 4B, lane
4). Thus, the proteasome is unlikely to be involved in the import process.
Inhibitor Studies--
We next investigated whether FBPase import
was dependent upon vacuole acidification or proteasome degradation
using inhibitors that block these processes (Fig.
5). For these experiments,
UBC1 Is Necessary for FBPase Import--
Since the
We next examined whether the defect in FBPase import observed for the
The impaired ability of the
If UBC1 is required for Vid vesicle formation, the number of
Vid vesicles should be reduced in the The K48R/K63R Ubiquitin Mutant Inhibits FBPase
Degradation--
Ubiquitin molecules are most often linked to one
another by isopeptide bonds between the carboxyl terminus of one
ubiquitin and the FBPase Import into Vid Vesicles Is Inhibited by the K48R/K63R
Ubiquitin Mutant--
We investigated whether ubiquitin chain
formation is necessary for FBPase import in vitro. FBPase
was imported when cytosol and semi-intact cells were prepared from the
wild type strain overexpressing wild type ubiquitin (Fig. 8B,
lane 1). By contrast, in vitro FBPase import was
significantly reduced when both cytosol and semi-intact cells were
prepared from the strain that overproduced the K48R/K63R mutant
(lane 2). When cytosol from the K48R/K63R strain was
incubated with semi-intact cells from the strain overexpressing wild
type ubiquitin, a high level of FBPase was imported (Fig. 8B,
lane 3). By contrast, FBPase import decreased when cytosol from
the strain overexpressing wild type ubiquitin was incubated with
semi-intact cells from the K48R/K63R strain (lane 4).
Therefore, the K48R/K63R mutant inhibits the function of Vid vesicles
to import FBPase, but does not affect the ability of cytosol to
stimulate FBPase import into competent Vid vesicles.
The K48R/K63R Mutant Does Not Prevent Vid24p Binding to Vid
Vesicles--
As mentioned above, the decreased level of Vid24p in the
We next examined whether Vid24p was ubiquitinated by transforming wild
type and
To determine whether Vid24p was ubiquitinated, this protein was
immunoprecipitated from total lysates of wild type cells and then
immunoblotted with anti-Myc antibodies. Vid24p was expressed in wild
type cells, but was absent in the
Although the site of FBPase degradation has been a matter of debate
(52, 53), a PEP4-dependent degradation of FBPase
was confirmed by an independent research group (54). To examine whether
FBPase was polyubiquitinated, wild type and In this study, we analyzed FBPase import into Vid vesicles to
identify molecules involved in early stages of the FBPase degradation pathway. Our results suggest that vacuole proteolysis, vacuole acidification, and proteasome degradation are unlikely to be involved in FBPase import. The Our results indicate that the cytosolic ubiquitin-conjugating enzyme
Ubc1p is an important regulator of the FBPase import process. FBPase
import into Vid vesicles is defective in the Our data show that UBC1 is required for the proper function
of Vid vesicles. The Based upon results from this study and from previous studies (23, 37),
we have proposed a model for the FBPase degradation pathway (Fig.
10). In the initial step, FBPase is
imported into Vid vesicles through a process that requires the presence
of the heat shock protein Ssa2p. Following FBPase sequestration inside these structures, the loaded vesicles then traffic to the vacuole via a
process controlled by Vid24p. At present, the site of origin for Vid
vesicles is unknown, although the formation of these organelles appears
to be regulated by the cytosolic ubiquitin-conjugating enzyme Ubc1p. In
the absence of this enzyme, levels of Vid vesicles are reduced and
FBPase degradation is compromised. Therefore, we propose that
ubiquitination plays an important role in the degradation of FBPase
through its effect on the machinery (Vid vesicles) that transports
FBPase to the vacuole. Identification of the factors that are
polyubiquitinated by Ubc1p may ultimately help identify their sites of
action as well as to elucidate how FBPase is imported into Vid
vesicles.
ubc1 and the K48R/K63R mutants were attributed to the Vid vesicle fraction. In the
ubc1 mutant, the level of
the Vid vesicle-specific marker Vid24p was reduced in the vesicle
fraction, suggesting that UBC1 is required for either Vid
vesicle production or Vid24p binding to Vid vesicles. However, the
K48R/K63R mutant did not prevent Vid24p binding to Vid vesicles,
indicating that ubiquitin chain formation is dispensable for Vid24p
binding to these structures. Our results support the findings that
ubiquitin conjugation and ubiquitin chain formation play important
roles in a number of cellular processes including organelle biogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ubc1 mutant
contained defective vesicles, but competent cytosol. Furthermore,
FBPase import was inhibited when cells overexpressed a ubiquitin mutant
(K48R/K63R) that prevents the formation of multiubiquitin chains. The
defect of the K48R/K63R mutant was associated with Vid vesicles,
indicating that ubiquitin chain formation is required to produce
competent Vid vesicles.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
gal plasmid (33, 34) were obtained
from Dr. D. Finley (Harvard Medical School).
S,
N-ethylmaleimide, brefeldin A, bafilomycin A, and
concanamycin A and were purchased from Sigma. MG132
(carbobenzoxyl-leucinyl-leucinyl-leucinal) and
-lactone were gifts
from Dr. A. Goldberg (Harvard Medical School).
Tran35S-label (10 mCi/mmol) was obtained from ICN. Rabbit
anti-FBPase and rabbit anti-CPY polyclonal antibodies were raised by
Berkeley Antibody Co. (Berkeley, CA) using purified FBPase and CPY
(Sigma). Mouse and rabbit anti-Myc antibodies were purchased from
Berkeley Antibody Co. Mouse anti-
-galactosidase antibodies were
purchased from Promega.
ise1,
ise1
pep4,
vid24, and
vid24
pep4 strains
were labeled with Tran35S-label for 10 min at 30 °C and
then chased for 40 min at 30 °C. To examine the effect of brefeldin
A on CPY processing, an ise1 strain was preincubated in the
presence or absence of brefeldin A (75 µg/ml) at 22 °C for 10 min.
Cells were pulsed for 10 min, chased for 40 min, and then harvested.
Total lysates were immunoprecipitated with CPY antiserum, subjected to
SDS-PAGE using 7.5% polyacrylamide gels, and analyzed with a Fuji
Bioimaging Analyzer. The degradation of short-lived and long-lived
proteins was examined using the protocols described by Lee and Goldberg
(36).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
vid1
(
ise1) and
vid24 mutants inhibit the
degradation of FBPase in vivo (Fig.
1A). However, these mutations
affect different steps in the FBPase degradation pathway. The
vid24 mutant strain imports FBPase into Vid vesicles
normally, but this mutation blocks the trafficking of Vid vesicles to
the vacuole. As such, this mutation results in the accumulation of
FBPase in Vid vesicles (37). On the other hand, a mutation of the
VID1 gene (a gene that is identical to the ISE1
or ERG6 gene)2 blocks FBPase import into Vid
vesicles (38) and serves as a negative control for in vitro
import.
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Fig. 1.
The kinetics of FBPase import into the
ise1 and
vid24 semi-intact cells.
A, wild type (HLY223),
ise1 (HLY001), and
vid24 (HLY227) were grown in YPKG to induce FBPase. Cells
were shifted to glucose for 0, 60, and 120 min. Total lysates from
these cells were solubilized in SDS buffer, separated by SDS-PAGE and
FBPase degradation was followed in these cells. B, both
ise1 (HLY208) and
vid24 (HLY232) mutants
were shifted to glucose for 20 min. Semi-intact cells and cytosol were
prepared as described (21). FBPase import was measured for 0, 10, 20, and 30 min in the absence or presence of ATP and cytosol. The % FBPase
import is indicated.
ise1 and
vid24 strains, the endogenous FBP1 gene was
deleted so that a known quantity of radiolabeled, purified FBPase could
be added and followed in the in vitro system. Each strain
was glucose starved and then shifted to glucose containing medium prior
to their conversion to semi-intact cells. Purified FBPase was incubated
with semi-intact cells in the absence or presence of ATP, an ATP
regenerating system and cytosol. At selected times, proteinase K was
added to digest the FBPase that was not protected in a membrane-sealed
compartment. In the absence of both ATP and cytosol, FBPase import into
the
vid24 semi-intact cells was minimal (Fig.
1B). In the presence of ATP and cytosol, however, FBPase
import increased in a time-dependent manner. When
quantitated, ~25-35% of the total added FBPase was proteinase K
protected after 30 min of import. In contrast, the
ise1
mutant had background levels of FBPase import either in the presence or
in the absence of ATP and cytosol (Fig. 1B).
ise1 Mutant Contains Defective Vesicles--
The defect of
FBPase import seen for the
ise1 mutant could result from
an inability of cytosol to stimulate FBPase import or an inability of
Vid vesicles to take up FBPase. To determine the site of this defect,
we performed an in vitro assay using various combinations of
semi-intact cells and cytosol from the
ise1 and
vid24 mutants. When the
ise1 semi-intact
cells were used, FBPase import was defective regardless of whether the
cytosol was isolated from the
ise1 (Fig.
2, lane 1) or the
vid24 mutants (lane 3). By contrast, FBPase
import into the
vid24 semi-intact cells was observed when
cytosol was prepared from either the
vid24 mutant
(lane 2) or the
ise1 mutant (lane
4). This experiment suggests that the
ise1 mutant
strain has competent cytosol that can stimulate FBPase import into
competent Vid vesicles. However, the
ise1 mutant contains
defective vesicles that cannot support FBPase import, even when
combined with import-competent cytosol.
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Fig. 2.
The ise1
mutant contains defective vesicles. The
ise1
(HLY208) and
vid24 (HLY 232) mutants were shifted to
glucose for 20 min. Semi-intact (SI) cells and cytosol were
prepared from the
ise1 and
vid24 mutants
and combined as indicated. Lane 1, FBPase import using
ise1 cytosol and
ise1 semi-intact cells.
Lane 2, FBPase import into
vid24 semi-intact
cells with
vid24 cytosol. Lane 3, FBPase
import into
ise1 semi-intact cells with cytosol from
vid24. Lane 4, FBPase import into
vid24 semi-intact cells with cytosol from the
ise1 mutant.
ise1
pep4 and
vid24
pep4 strains (Fig.
3A). When FBPase import was
measured, the level was low in the
ise1 single mutant
(Fig. 3B, lane 1) and there was no significant increase in
the FBPase import in the
ise1
pep4 double
mutant (lane 2). Likewise, there was no significant change
in FBPase import in the
vid24
pep4 double mutant (lane 4) as compared with the
vid24
single mutant (lane 3). Since uptake of FBPase by Vid
vesicles is independent of the PEP4 gene, this supports our
model that FBPase import into Vid vesicles occurs prior to trafficking
to the vacuole.
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Fig. 3.
FBPase import into Vid vesicles is not
affected by PEP4 or VMA3
mutants. A, the biosynthesis of CPY was examined
by pulse-chase experiments in the ise1,
ise1
pep4,
vid24, and
vid24
pep4 strains. B, the
strains
ise1 (HLY208),
ise1
pep4 (HLY247),
vid24
(HLY232),
vid24
pep4 (HLY233), and
vma3 (HLY217) were shifted to glucose for 20 min. The
cytosol and semi-intact cells were prepared and FBPase import was
measured in the presence of ATP and cytosol. The percentage of FBPase
import in each strain is indicated.
ubc1 strain displayed a reduced rate of degradation of
short-lived proteins as compared with the wild type control (Fig.
4A). In contrast,
UBC6 and UBC7 are involved in the ubiquitination
of misfolded or unassembled proteins in the endoplasmic
reticulum degradation pathway (45, 46). Therefore,
ubc6 and
ubc7 strains did not
inhibit the degradation of short-lived proteins (Fig. 4A).
When the
ubc1,
ubc6, and
ubc7
strains were tested for FBPase import, a reduced level of import was
observed for
ubc1 (Fig. 4B, lane 1), but not
for the
ubc6 and
ubc7 strains (lanes
2 and 3), suggesting a specific role for
UBC1 in the import process.
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Fig. 4.
FBPase import into Vid vesicles is defective
in the ubc1 mutant.
A, the degradation of short-lived proteins was examined in
wild type (WT), pre1-1pre2-1,
ubc1,
ubc6, and
ubc7 cells. The
pre1-1pre2-1 was pulsed at 22 °C and chased at 37 °C,
while
ubc1,
ubc6, and
ubc7
were pulsed and chased at 30 °C. B, the strains
ubc1 (HLY212),
ubc6 (HLY213), and
ubc7 (HLY214) were shifted to glucose for 20 min. The
pre1-1pre2-1 (HLY 215) was shifted to glucose at 37 °C.
FBPase import into Vid vesicles was conducted as described under
"Experimental Procedures."
vid24 semi-intact cells and cytosol were preincubated
with various concentrations of inhibitors. These concentrations were
chosen based upon previous studies demonstrating maximal inhibition in
the yeast system (35, 36, 49-51). FBPase, ATP, and an ATP regenerating
system were then added to the reaction mixture to commence the import
process. The in vitro import of FBPase was inhibited by
nonhydrolyzable ATP
S (Fig. 5A, lane 3). However,
N-ethylmaleimide, which inhibits V-ATPase (1) did not affect
FBPase import in vitro (lane 4). Likewise,
brefeldin A had no effect on in vitro FBPase import (lane 5), even though this inhibitor caused accumulation of
p1-CPY in the ise1 (brefeldin A permeable) strain (Fig.
5B, lane 2). FBPase import was also unaffected by the
proteasome inhibitors MG132 or
-lactone (Fig. 5A, lanes 6 and 7), although these inhibitors did reduce the degradation
of short-lived proteins in vivo (Fig. 5C).
Inhibitors that perturb vacuole acidification such as bafilomycin A and
concanamycin A (1, 50, 51) also had no effect on FBPase import (Fig.
5A, lanes 8 and 9), but they did reduce the degradation of long-lived proteins in vivo (Fig.
5D). Taken together, the mutant analyses and the inhibitor
studies suggest that FBPase import into Vid vesicles is independent of
vacuole proteolysis, vacuole acidification, and proteasome degradation.
However, this import does require ATP hydrolysis and the
UBC1 gene.
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Fig. 5.
The effects of inhibitors on FBPase
import. A, FBPase import into semi-intact
vid24 cells (HLY232) was carried out in the absence
(lane 1) or presence (lane 2) of ATP and cytosol
or with preincubation of various inhibitors (lanes 3-12).
ATP
S (50 µM), N-ethylmaleimide (10 mM), brefeldin A (75 µg/ml), MG132 (100 µM),
-lactone (50 µM), bafilomycin A (20 µM), and concanamycin A (0.3 µM) were added
to semi-intact cells and cytosol for 20 min before the addition of
FBPase, ATP, and an ATP regenerating system. FBPase import was measured
as described. The percentage of FBPase import in semi-intact cells
treated with various inhibitors is indicated. B, the
addition of brefeldin A caused p1-CPY to accumulate in the
ise1 strain (lane 2). C, the
degradation of short-lived proteins was inhibited by MG132 and
-lactone. D, inhibitors that perturb the acidification of
the vacuole reduced the degradation of long-lived proteins.
ubc1 strain displayed defective FBPase import in
vitro, we next determined whether this strain was also defective in FBPase degradation in vivo. As is shown in Fig.
6A, wild type cells degraded
FBPase after a shift to glucose for 180 min. In contrast, FBPase
degradation was significantly retarded in the
ubc1
mutant, but was normal in the
ubc6 mutant. Therefore,
UBC1 is required for FBPase degradation, whereas
UBC6 is not.
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Fig. 6.
The ubc1
mutant contains defective vesicles, but normal cytosol.
A, FBPase degradation was followed in wild type,
ubc1 and
ubc6 cells for 0, 45, 90, 120, and
180 min. B, both
vid24 (HLY232) and
ubc1 (HLY212) were shifted to glucose for 20 min.
Semi-intact cells and cytosol were prepared from the glucose-shifted
vid24 and
ubc1 strains. Lane 1, FBPase import into
ubc1 semi-intact cells with cytosol
from the
ubc1 strain. Lane 2, FBPase import
into
vid24 semi-intact cells with
vid24
cytosol. Lane 3, FBPase import into
ubc1
semi-intact cells with
vid24 cytosol. Lane 4, FBPase import into
vid24 semi-intact cells with
ubc1 cytosol.
ubc1 mutant resulted from an inability of cytosol to support FBPase import or an inability of Vid vesicles to take up
FBPase. As is shown in Fig. 6B, when cytosol and semi-intact cells from the
ubc1 strain were used, FBPase import was
impaired (lane 1). By contrast, when cytosol and semi-intact
cells from the
vid24 strain were combined, a high level
of FBPase import was observed (lane 2). FBPase import
decreased when
ubc1 semi-intact cells were incubated with
cytosol from the
vid24 strain (lane 3). Since
the
vid24 strain contained import competent cytosol, this
result indicates that the
ubc1 mutant had defective
vesicles. In contrast, the
ubc1 strain appears to contain
competent cytosol, because cytosol from the
ubc1 strain
supported FBPase import into import competent Vid vesicles in
vid24 semi-intact cells (lane 4).
ubc1 semi-intact cells to
import FBPase could be due to a decrease in Vid vesicle production. Alternatively, the reduced import could result from a defect in the
import machinery. In initial experiments, we examined the levels of the
Vid vesicle specific marker, Vid24p. Vid24p is induced in response to
glucose and a significant portion of this protein is associated with
Vid vesicles as a peripheral protein (37). When cells were maintained
in low glucose medium (t = 0 min), Vid24p was
undetectable in total lysates. However, this protein was induced to a
similar level after wild type,
ubc1, and
ubc6 strains were shifted to glucose for 20 min (Fig.
7A). Therefore, Vid24p
production is not altered in the
ubc1 mutant.
View larger version (20K):
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Fig. 7.
Vid vesicle function is impaired in the
ubc1 mutant. A, wild
type (HLY223),
ubc1, and
ubc6 strains were
glucose starved (t = 0), or glucose starved and then
shifted to glucose for 20 min (t = 20). Total lysates
from t = 0 and t = 20 were separated by
SDS-PAGE and Vid24p was detected by Western blotting with Vid24p
antibodies. B, wild type,
ubc1, and
ubc6 strains were shifted to glucose for 20 min. Cells
were homogenized and subjected to differential centrifugation. Proteins
from the high speed supernatant (S) and high speed pellet
(P) were solubilized in SDS buffer and resolved by SDS-PAGE.
The distribution of Vid24p in the S and P fractions was detected by
anti-Vid24p antibodies. The lower panel indicates the % recovery of Vid24p in each fraction from these strains.
ubc1 mutant. This
would be reflected as a decreased level of Vid24p within fractions that contain Vid vesicles. Conversely, if UBC1 is required for
the function of the import machinery, the level of Vid24p would not be
altered in the Vid vesicle containing fractions. To test these possibilities, the wild type,
ubc1, and
ubc6 strains were shifted to glucose and cell extracts
were subjected to differential centrifugation using the protocol
described previously (23). In wild type and
ubc6 mutant
cells, most of the Vid24p was in the Vid vesicle containing pellet
fraction (Fig. 7B). By contrast, the
ubc1
mutant exhibited a significantly decreased level of Vid24p in the
pellet fraction, but a greater concentration of Vid24p in the soluble fraction (Fig. 7B). The decreased level of Vid24p in the
pellet fraction most likely represents a reduced production of Vid
vesicles, since Vid24p induction is not altered in the
ubc1 strain. However, a decreased binding of Vid24p to
Vid vesicles in the
ubc1 strain could also account for
this observation.
-amino group of lysine 48 of the next ubiquitin
(41, 42). However, ubiquitin chains can also be formed at lysine 63 (41, 42). Therefore, when both lysine 48 and lysine 63 are replaced
with arginine (K48R/K63R), the formation of multiubiquitin chains is
inhibited. To study the effect of ubiquitin chain formation on FBPase
degradation, a strain overexpressing the K48R/K63R mutation was used.
When wild type ubiquitin was overproduced, FBPase was degraded in
response to glucose in vivo (Fig.
8A). However, when the
K48R/K63R ubiquitin mutant was overexpressed, FBPase degradation was
impaired (Fig. 8A). Therefore, the degradation of FBPase
requires the formation of multiubiquitin chains.
View larger version (32K):
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Fig. 8.
FBPase import is impaired when ubiquitin
chain formation is inhibited. A, wild type cells were
transformed with multicopy plasmids containing either the wild type
ubiquitin (HLY824) or the K48R/K63R ubiquitin mutant (HLY823) under an
inducible copper promoter. The transformants were grown in synthetic
medium and ubiquitin was induced by 100 mM
CuSO4 using the protocol described by Schork et
al. (52). These cells were then shifted to glucose for the
indicated times and FBPase degradation was examined. B, wild
type cells overexpressing either wild type ubiquitin (HLY820) or the
K48R/K63R ubiquitin mutant (HLY819) were shifted to glucose for 20 min.
Cytosol and semi-intact cells from these strains were combined as
indicated and in vitro import of FBPase was performed as
described under "Experimental Procedures." C, total
lysates from wild type cells over-expressing either wild type ubiquitin
(HLY824) or the K48R/K63R ubiquitin mutant (HLY823) were fractionated
by differential centrifugation. The distribution of Vid24p in total
(T), high speed pellet (P), and high speed
supernatant (S) fractions was examined by Western blotting
with anti-Vid24p antibodies.
ubc1 high speed pellet may result from a reduced number
of Vid vesicles, or it may be due to a decreased binding of this
protein to Vid vesicles. Accordingly, if ubiquitin chain formation is
necessary for Vid24p binding to Vid vesicles, the distribution of
Vid24p might be altered when the K48R/K63R mutant was overproduced.
When Vid24p was induced in cells overexpressing wild type ubiquitin, most of the Vid24p was in the pellet fraction and very little was in
the supernatant fraction (Fig. 8C). However, in cells
overproducing the K48R/K63R mutant, the level of Vid24p decreased to
one-third of that observed in cells overexpressing wild type ubiquitin
(Fig. 8C). It is unknown why the K48R/K63R mutant reduced
total amounts of Vid24p. However, this was not due to an overall
decrease in protein concentration, because both wild type and K48R/K63R
strains had similar protein concentrations in total lysates as well as in individual supernatant (9.92 versus 8.88 mg/ml) and
pellet (4.16 versus 4.61 mg/ml) fractions. When Vid24p
distribution was quantitated in the K48R/K63R mutant, more than 90% of
the Vid24p was in the pellet fraction and less than 10% was in the
soluble fraction. Thus, the ratio of bound versus unbound
Vid24p was not altered when the K48R/K63R mutant was overproduced.
Given that the association of Vid24p with Vid vesicles was not
prevented by the K48R/K63R mutant, polyubiquitination is not required
for Vid24p binding to the Vid vesicles. Therefore, these data are consistent with the hypothesis that the
ubc1 and
K48R/K63R mutations result in a decreased production of Vid vesicles.
vid24 strain with or without the Myc-tagged wild
type ubiquitin plasmid. These strains were incubated in glucose poor
medium containing copper to induce Myc ubiquitin and FBPase. Cells were
then shifted to glucose for 20 min to induce Vid24p. Ub-Pro-
-galactosidase was used as a positive control since
Ub-Pro-
-galactosidase is known to be polyubiquitinated
constitutively (34). Immunoblotting experiments indicate that high
levels of Myc ubiquitin were expressed in cells transformed with the
Myc ubiquitin plasmid, but not in cells that did not harbor the Myc ubiquitin plasmid (Fig. 9A, lanes
1-4). As shown by immunoblotting and immunoprecipitation
experiments, Ub-Pro-
-galactosidase was present as multiple bands in
cells transformed with the Ub-Pro-
-galactosidase plasmid
(lanes 5, 6, 9, and 10). However, these bands
were not observed in control cells that did not contain the
Ub-Pro-
-galactosidase plasmid (lanes 7, 8, 11, and
12). In cells transformed with both Ub-Pro-
-galactosidase
and Myc ubiquitin plasmids, multiple Ub-Pro-
-galactosidase bands
were detected by anti-Myc antibodies, suggesting that these bands were
polyubiqutinated forms of Ub-Pro-
-galactosidase (lane 14). By contrast, no Myc signal could be found in cells that did not harbor the Myc ubiquitin plasmid (lane 13) or in cells
that did not contain the Ub-Pro-
-galactosidase plasmid (lanes
15 and 16).
View larger version (55K):
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Fig. 9.
Vid24p is not ubiquitinated.
A, wild type cells were transformed with or without a
multicopy Ub-Pro- -galactosidase plasmid. These cells were then
transformed with or without a multicopy Myc ubiquitin plasmid. Cells
were grown in synthetic medium and then shifted to synthetic medium
containing 2% ethanol and 100 mM CuSO4 for
5 h to induce FBPase and ubiquitin. Cells were transferred to
synthetic medium containing fresh 2% glucose for 20 min. Total lysates
from these cells were aliquoted into two parts. One-half of the lysates
were immunoblotted with anti-Myc antibodies (lanes 1-4), or
anti-
-galactosidase antibodies (lanes 5-8). Another half
of the total lysates were immunoprecipitated first with
anti-
-galactosidase antibodies and then immunoblotted with either
anti-
-galactosidase antibodies (lanes 9-12) or with
anti-Myc antibodies (lanes 13-16). B, wild type
or
vid24 strains were transformed with or without a
multicopy Myc ubiquitin plasmid under a copper inducible promoter.
Total lysates were aliquoted into two portions. Half of the lysates
were subjected to immunoblotting with anti-Myc antibodies (lanes
1-4) or anti-Vid24p antibodies (lanes 5-8). The other
half of the total lysates were subjected to immunoprecipitation with
anti-Vid24p antibodies and then immunoblotted with either Vid24p
antibodies (lanes 9-12) or anti-Myc antibodies (lanes
13-16). C, wild type and
fbp1 strains
were transformed with or without a multicopy Myc ubiquitin plasmid.
Half of the total lysates were immunoblotted with anti-Myc antibodies
(lanes 1-4) or FBPase antibodies (lanes 5-8).
Another half of the lysates were immunoprecipitated with FBPase
antibodies and then immunoblotted with anti-FBPase antibodies
(lanes 9-12) or anti-Myc antibodies (lanes
13-16).
vid24 strain, as
indicated by immunoblotting (Fig. 9B, lanes 5-8) and
immunoprecipitation experiments (lanes 9-12). When the
precipitated Vid24p was immunoblotted with anti-Myc antibodies, there
was no detectable Myc signal (lane 14). Likewise, no
ubiquitination of Vid24p could be found in cells that did not contain
the Myc ubiquitin plasmid (lane 13), or in the
vid24 strain (lanes 15 and 16).
Furthermore, no ubiquitination of Vid24p could be detected using
pulse-chase experiments followed by immunoprecipitation with
anti-Vid24p antibodies (data not shown). Therefore, Vid24p is unlikely
to be ubiquitinated. This supports our contention that ubiquitination
is not required for the function of Vid24p.
fbp1 strains were transformed with or without the Myc-tagged ubiquitin plasmid using
the protocol described by the Wolf group (52, 53). FBPase was reported
to be polyubiquitinated under these conditions (52, 53). As shown by
both immunoblotting (Fig. 9C, lanes 5-8) and immunoprecipitation (lanes 9-12) experiments, FBPase was
detected in wild type cells, but not in the
fbp1 strain.
When the precipitated FBPase was immunoblotted with anti-Myc
antibodies, some faint bands migrating below the IgG band were detected
in wild type cells transformed with the Myc ubiquitin plasmid
(lane 14). However, these bands were also seen in cells that
did not harbor the Myc ubiquitin plasmid (lane 13) as well
as in the
fbp1 strain that did not have the
FBP1 gene (lanes 15 and 16). Thus,
these bands were unlikely to represent polyubiquitinated FBPase.
Similarly, no polyubiquitination of FBPase was detected when wild type
cells were pulsed-chased and then immunoprecipitated with anti-FBPase antibodies (data not shown). Since purified FBPase without
ubiquitination was imported into Vid vesicles in vitro,
polyubiquitination of FBPase is unlikely to be required for the import process.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ise1
pep4 or
vid24
pep4 double mutants did not alter
FBPase import as compared with the
ise1 or
vid24 single mutants, suggesting that FBPase import is
independent of the PEP4 gene. Furthermore, the
vma3 deletion mutant and compounds such as bafilomycin A or
concanamycin A that block acidification of the vacuole did not inhibit
FBPase import. Therefore, FBPase import is independent of the two major
vacuole functions, vacuole proteolysis and vacuole acidification. This
further supports our model that FBPase import into Vid vesicles occurs
prior to the trafficking to the vacuole.
ubc1 mutant,
but not in the
ubc6 or
ubc7 mutants,
suggesting a specific role for UBC1 in the import process.
However, this requirement is not linked to proteasome degradation. The
pre1-1pre2-1 proteasome mutant showed normal FBPase import
and proteasome inhibitors such as MG132 and
-lactone had no effect
on FBPase degradation.
ubc1 mutant contained defective
vesicles, but normal cytosol. In the absence of the UBC1
gene, cells may decrease the production of Vid vesicles or reduce the
efficiency of the import machinery. In the control wild type and
ubc6 strains, most of the Vid vesicle marker Vid24p was
found in fractions containing Vid vesicles. When quantitated, ~90%
of the Vid24p was recovered in the Vid vesicle containing pellet
fraction in wild type cells. However, in the
ubc1 strain,
about 25% of the Vid24p was in the pellet fraction, while most of the
Vid24p was in the soluble fraction. The reduced levels of Vid24p in the
pellet fraction could result from decreased Vid vesicle production or a
decreased binding of Vid24p to Vid vesicles. However, the K48R/K63R
mutant did not prevent Vid24p binding to Vid vesicles, even though it
inhibited vesicle import. Therefore, polyubiquitination is necessary
for FBPase import into Vid vesicles, but does not play an important role in Vid24p binding to Vid vesicles.
View larger version (11K):
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Fig. 10.
The FBPase degradation pathway. When
glucose-starved cells are shifted to medium containing fresh glucose,
FBPase is imported into Vid vesicles and then to the vacuole for
degradation. The cytosolic heat shock protein Ssa2p is required for
FBPase import into Vid vesicles. After FBPase is sequestered inside the
vesicles, Vid vesicles then carry FBPase to the vacuole in a process
that is dependent upon Vid24p. Although the origin of Vid vesicles is
not known, the formation of Vid vesicles is regulated by the cytosolic
ubiquitin conjugating enzyme Ubc1p through unidentified factors that
are likely to be polyubiquinated.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Graham Hung and Ly Brown for
critically reading this manuscript. We also thank Dr. Stefan Jentsch
(University of Heidelberg, Germany) for the ubc1,
ubc6, and
ubc7 strains and Dr. Alfred
Goldberg (Harvard Medical School) for the pre1-1pre2-1, ise1 strains, and proteasome inhibitors. We also thank Dr.
Dan Finley (Harvard Medical School) for the wild type ubiquitin
plasmid, the K48R/K63R ubiquitin plasmid and the
Ub-Pro-
-galactosidase plasmid.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant RO1GM59480 (to H-L. C.).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: Dept. of Cellular and
Molecular Physiology, Penn State College of Medicine, 500 University
Dr., Hershey, PA 17033. Tel.: 717-531-0860; Fax: 717-531-0859; E-mail:
hlchiang@psu.edu.
Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.M001767200
2 H-L. Shieh, Y. Chen, C. R. Brown, and H-L. Chiang, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CPY, carboxypeptidase Y;
FBPase, fructose-1,6-bisphosphatase;
VPS, vacuole
protein sorting;
VID, vacuole import and degradation;
PAGE, polyacrylamide gel electrophoresis;
MG132, carbobenzoxyl-leucinyl-leucinyl-leucinal;
ATPS, adenosine
5'-O-(thiotriphosphate).
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