From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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Fructose-1,6-bisphosphatase (FBPase), the key enzyme in gluconeogenesis in the yeast Saccharomyces cerevisiae, is induced when cells are grown in medium containing poor carbon sources. FBPase is targeted from the cytosol to the vacuole for degradation when glucose-starved yeast cells are replenished with fresh glucose. In this study, we report the reconstitution of the glucose-induced import of FBPase into the vacuole in semi-intact yeast cells using radiolabeled FBPase, an ATP regenerating system and cytosol. The import of FBPase was defined as the fraction of the FBPase that was sequestered inside a membrane-sealed compartment. FBPase import requires ATP hydrolysis and is stimulated by cytosolic proteins. Furthermore, the import of FBPase is a saturable process. FBPase import is low in the glucose-starved cells and is stimulated in the glucose-replenished cells. FBPase accumulates to a higher level in the pep4 cell, suggesting that FBPase is targeted to the vacuole for degradation. Indirect immunofluorescence microscopy studies demonstrate that the imported FBPase is localized to the vacuole in the permeabilized cells. Thus, the glucose-induced targeting of FBPase into the vacuole can be reproduced in our in vitro system.
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INTRODUCTION |
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The vacuole of the yeast Saccharomyces cerevisiae is an acidic compartment surrounded by a lipid bilayer. Vacuoles contain a variety of soluble and membrane-bound proteolytic enzymes and are considered to be equivalent to the lysosome of higher eukaryotes (1). The proteolytic processes in the vacuole play an important role when the cells are under nutritional stress. The vacuole is also responsible for the degradation of overexpressed proteins, normal cellular proteins, and some abnormal proteins (2).
Fructose-1,6-bisphosphatase (FBPase),1 the key regulatory enzyme in gluconeogenesis in S. cerevisiae, is induced when yeast cells are grown in medium containing poor carbon sources. FBPase is rapidly degraded when fresh glucose is added to the glucose-starved yeast cells (3). FBPase is targeted from the cytosol to the vacuole for degradation in response to glucose (4, 5). The degradation of FBPase is dependent on the PEP4 gene encoding proteinase A, which is necessary for the activation of proteinase B and C (6). As a result, the deletion of the PEP4 gene reduces the vacuolar proteolytic activity to 30% that of the wild type level (6). In the pep4 deletion strain, FBPase is found in the vacuole when glucose-starved cells are transferred to fresh glucose (4, 7). In addition to FBPase, several other proteins and organelles are also delivered to the vacuole for degradation when cells are shifted to the glucose medium. They include formate dehydrogenase (8), plasma membrane proteins such as the maltose transporter (9) and the galactose transporter (7), and peroxisomes (7, 8, 10).
Most vacuole resident proteins pass through early parts of the
secretory pathway en route to the vacuole (2, 6, 11, 12). For instance,
carboxypeptidase Y (CPY) is synthesized and processed sequentially
in the endoplasmic reticulum and the Golgi. CPY is sorted in the late
Golgi by the CPY receptor and is delivered to the vacuole through the
prevacuolar/endosomal compartment (13, 14). In contrast, targeting of
vacuolar aminopeptidase I and -mannosidase to the vacuole is
independent of the secretory pathway (15-17). Plasma membrane proteins
can be internalized by endocytosis and transported through endosomes to
the vacuole for degradation (9, 18-22). Other organelles such as
peroxisomes or mitochondria can be engulfed by the vacuole by autophagy
(7, 8).
Biochemical reconstitution of protein sorting has been successfully applied to study the transport of proteins through the secretory pathway including translocation into the endoplasmic reticulum (23, 24), endoplasmic reticulum to Golgi transport (25-29), and intra-Golgi (30, 31) and late Golgi to vacuole transport (32, 33). Protein import into the nucleus (34), mitochondria (35), and peroxisomes (36) have also been reproduced in vitro. Homotypic fusion of vacuoles in vitro has been used to study processes required for vacuolar inheritance (37, 38). In addition, targeting of aminopeptidase I from the cytoplasm to the vacuole has also been reconstituted (39). Degradation of cytosolic proteins by isolated lysosomes has been documented in mammalian cells (40-42). However, targeting of cytosolic proteins into the yeast vacuole has not been reconstituted in vitro.
To investigate the pathway of FBPase degradation, we reconstituted in vitro FBPase targeting into the vacuole using permeabilized yeast cells incubated with radiolabeled FBPase, an ATP regenerating system, and cytosolic proteins. The import is a saturable process and is stimulated by glucose. Import of radiolabeled FBPase into semi-intact cells is competed by excess unlabeled FBPase but not a control protein. The imported FBPase accumulates to a higher level in the pep4 cell, suggesting that FBPase is targeted to the vacuole for degradation. Immunofluorescence microscopic studies show that the imported FBPase is localized to the vacuole. Thus, this in vitro system reconstitutes glucose-induced targeting of FBPase into the vacuole and provides a functional assay to identify molecules required for the FBPase targeting and degradation pathway.
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EXPERIMENTAL PROCEDURES |
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Media and Yeast Strains--
S. cerevisiae strains
used in this study were HLY193 (Mat his3-
200 ura3-52 leu2,
3-112 trp1 FBP1::LEU2) and HLY205 (Mat
his3-
200 ura3-52 leu2, 3-112 trp1 FBP1::LEU2
PEP4::TRP1). All the chemicals, unless otherwise noted,
were obtained from Sigma or ICN Biochemicals Inc. YPD was a complete
medium (10 g/liter Bacto yeast extract, 20 g/liter Bacto peptone;
Difco) supplemented with 20 g/liter dextrose (Fisher) and was used to
induce FBPase degradation. Synthetic minimal medium contained 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. YPKG was the FBPase-inducing medium and contained 10 g/liter Bacto yeast extract, 20 g/liter Bacto peptone, 10 g/liter potassium acetate, and 5 g/liter dextrose. 35S-Protein
labeling mix ([35S]methionine, 1,175 Ci/mmol) was
obtained from NEN Life Science Products. The FBPase expression plasmid
(AU125) was a gift from Dr. David T. Rogers (43). Rabbit anti-FBPase
and rabbit anti-CPY polyclonal antibodies were raised by Berkeley
Antibody Company using purified FBPase and CPY (Sigma).
Purification of Fructose-1,6-bisphosphatase--
The FBPase
expression plasmid AU125 (43) was introduced into S. cerevisiae strains by lithium acetate transformation (44). The
transformed cells overexpressing the FBPase gene were grown overnight
at 30 °C in the uracil- and methionine-free synthetic medium
(without ammonium sulfate) containing 20 g/liter dextrose and 10 mCi/liter of 35S-methionine. The radiolabeled FBPase was
purified according to the procedure described by Rittenhouse et
al. (45), except that P11 column was omitted. FBPase was the major
protein band (~80% that of the total proteins) on the Coomassie
Blue-stained gels. The protein concentrations from these preparations
were 5-10 mg/ml as determined by Bio-Rad Dc protein assay
kit (Bio-Rad). The specific activity was 4-7 × 104
cpm/µg of the protein. The enzyme was stored at 70 °C in small aliquots. For antibody production, the FBPase-overexpressing cells were
grown in synthetic minimal medium overnight, and FBPase was purified as
described (45).
Preparation of Semi-intact (Permeabilized) Cells--
S.
cerevisiae cells (2,000 A600 nm/liter
culture) were grown in YPKG for 2 days at 30 °C. Cells were
harvested and resuspended in YPD and incubated at 30 °C for
different periods of time. At the end of the incubation, cells were
chilled by adding ice water, collected by centrifugation (1,500 × g, 5 min) at 4 °C, and washed once with water.
Spheroplasts were prepared as described previously (4) and collected by
centrifugation (15,000 × g, 2 min). Spheroplasts were
washed with ice-cold sorbitol buffer (1 M sorbitol, 150 mM potassium acetate, 5 mM magnesium acetate,
20 mM HEPES-KOH, pH 6.6) once, resuspended in the same
buffer to 50-100 A600 nm/ml, and stored at
70 °C in small aliquots.
Preparation of Cytosol--
The same yeast cells as those
described above for the preparation of semi-intact cells were grown,
cultured, and harvested under the same conditions described above.
Cells were resuspended in 1 cell volume of import buffer supplemented
with 200 µg/ml phenylmethylsulfonyl fluoride, and 2 volumes of
ice-chilled glass beads (0.45-0.50 mm) were added. The mixture was
vortexed at the highest speed for 1 min and chilled on ice for 5 min.
The vortexing procedure was repeated 5 times. Usually, more than 90%
of the cells were disrupted as examined by light microscopy. The cell debris was removed by centrifugation (500 × g, 5 min).
The supernatant was clarified by centrifugation (150,000 × g, 2 h) and stored at 70 °C in small aliquots. The
protein concentrations in these preparations were 10-20 mg/ml. This
high speed supernatant is referred to as cytosol.
The Standard FBPase Import Assay -- The semi-intact cells and cytosol were prepared from the same cells. In a typical experiment, the reaction mixture (100 µl) contained 3 A600 nm units of semi-intact cells, 0.5 mg/ml cytosolic proteins, an ATP regenerating system (0.5 mM ATP, 0.2 mg/ml creatine phosphokinase, and 40 mM creatine phosphate), and 11 µg of 35S-FBPase in the import buffer. The mixture was incubated at 30 °C for 20 min. At the end of incubation, 0.8 mg/ml proteinase K (final concentration) with or without 2% Triton X-100 was added to the reactions (50 µl of proteinase K solution containing 2.4 mg/ml proteinase K, 100 mM KCl, 50 mM Tris-HCl, pH 7.5, 0.3 M sorbitol, 1 mM EDTA with or without 6% Triton X-100). The mixture was incubated at room temperature for 15 min, and the reaction was terminated by adding 1 ml of 15% w/v trichloroacetic acid solution. Samples were centrifuged at 13,000 × g for 10 min at 4 °C. The precipitate was washed once with ice-cold acetone and resuspended in 200 µl of SDS loading buffer. The proteins (15 µl) were then resolved on SDS-PAGE. Gels were stained with Coomassie Blue, dried, and analyzed by a Fuji FUJIX BAS 1000 Bioimaging analyzer (Fuji Medical Systems). Alternatively, proteins were resolved on SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell) for immunoblotting. FBPase and CPY were detected by anti-FBPase and anti-CPY antibodies using the enhanced chemiluminescence (ECL) immunoblotting procedure (46). The import of FBPase was defined as the fraction of FBPase that was protected from proteinase K digestion in the absence of Triton X-100 but was sensitive to proteinase K in the presence of the detergent. In general, 20-40% of import could be achieved in the in vitro system.
Indirect Immunofluorescence-- The semi-intact cells were treated with proteinase K at the end of import reactions. Semi-intact cells were collected by centrifugation (13,000 × g, 20-30 s) and resuspended in 1 ml of buffer C (100 mM KCl, 50 mM Tris-HCl, pH 7.5, 0.3 M sorbitol, 1 mM EDTA). Cells were fixed and stained with anti-FBPase antibody as described previously (4) with the following modifications. Cells were fixed with 7% formaldehyde for 40 min at room temperature, washed twice with 0.5 ml of 1.2 M sorbitol and incubated with pre-absorbed rabbit anti-FBPase antibodies (1:20 dilution) overnight and with fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies for 1 h. Cells were visualized by Nomarski optics, and FBPase was detected by immunofluorescence using Zeiss Axiophot microscopy. Images were analyzed with the Northern Exposure and Adobe Photoshop softwares.
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RESULTS |
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The Import System--
To reconstitute FBPase import into the
vacuole, we prepared semi-intact cells from the pep4 and
fbp1 deletion strain. The deletion of the endogenous FBPase
gene (FBP1) was necessary to introduce radiolabeled FBPase
exogenously. The pep4 deletion strain reduces the vacuolar
proteolytic activity to 30% that of the wild type level and allowed
FBPase to accumulate to a higher level and hence facilitated the
detection of the import process. The overall strategy to reconstitute
in vitro import of FBPase is illustrated in Fig.
1. The semi-intact cells, unless
otherwise noted, were prepared from the pep4
fbp1(HLY205) yeast cells that were grown to induce FBPase
and then transferred to medium containing fresh glucose for 20 min.
Semi-intact cells were prepared from the double deletion strain by
removing cell walls to produce spheroplasts, followed by slow freeze
and thaw and treating with hypotonic buffer. This releases soluble
proteins and small molecules into the medium and still preserves the
integrity of most organelles in a functional state (25). Using this
approach, we introduced purified, radiolabeled FBPase into semi-intact
cells and followed the fate of radiolabeled FBPase in the permeabilized
cells.
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FBPase Is Imported into a Membrane-sealed Compartment-- We first determined whether FBPase was imported into a proteinase K-resistant compartment in the permeabilized cells in vitro. In the standard import conditions, the total FBPase, which consisted of both imported and nonimported FBPase, migrated at 38 kDa on the SDS-PAGE gels (Fig. 2A, lane 1). To distinguish the FBPase that was imported in a membrane-sealed compartment from the FBPase that was not, we added proteinase K to digest the free FBPase after the import had occurred. This treatment produced two species of FBPase; the full-length 38-kDa FBPase and the smaller sized 30-kDa FBPase (Fig. 2A, lane 3). The 38-kDa FBPase represented the FBPase that was protected in a membrane-sealed compartment, since it was sensitive to proteinase K digestion when Triton X-100 was added (Fig. 2A, lane 4). By contrast, the 30-kDa FBPase was produced only after proteinase K was added to the reactions in the absence of ATP and cytosol (Fig. 2A, lane 2). Quantitative analysis indicated that more than 98% of the full-length FBPase was converted to 30 kDa by proteinase K treatment under this condition (Fig. 2A, lane 2). Since the 30-kDa FBPase was resistant to proteinase K digestion, even in the presence of Triton X-100 (Fig. 2A, lane 4), it represented the FBPase that was not protected in a membrane-sealed compartment and served as an internal control.
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FBPase Import Is a Saturable Process-- In the wild type cell harboring a chromosomal copy of the FBP1 gene, FBPase is degraded with a half-life of 30 min in response to glucose. The degradation of FBPase is retarded if the cells overexpress the FBP1 gene; the half-life of FBPase degradation increases to 60-90 min in such cells (4). The dose-dependent increase in the FBPase degradation half-lives suggests that FBPase targeting to the vacuole is a saturable process. We tested whether the import of FBPase was a saturable process in the reconstituted system by incubating semi-intact cells with increasing amounts of radiolabeled FBPase in the absence or presence of an ATP regenerating system and cytosol. As shown in Fig. 3A, FBPase import was minimal in the absence of ATP, whether or not cytosol was added to the medium. FBPase import increased when ATP was added in the absence of cytosol. FBPase import was the highest when both ATP and cytosol were present in the incubation medium. Quantitation of the results obtained in the presence of ATP and cytosol showed that the import of FBPase increased when the amounts of FBPase were raised from 2.2 to 11 µg (Fig. 3B). However, when the amount of the exogenous FBPase was increased to 22 µg, the amount of FBPase that was imported within semi-intact cells remained unchanged (Fig. 3B). Thus, FBPase import is a saturable process.
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FBPase Import Requires Cytosol and ATP-- We titrated the concentrations of cytosolic proteins and determined the effects of cytosolic proteins in FBPase import in the in vitro system. As shown in Fig. 4A, the import was very low in the absence of both cytosol and an ATP regenerating system. The addition of 0.5 mM ATP in the absence of cytosol increased FBPase import. The maximal import was found when 0.5 mM ATP was combined with cytosol at the concentration of 0.5 mg/ml. No further increase was seen when the concentration of cytosol was raised to 1 mg/ml. Therefore, cytosol has a synergistic effect on FBPase import and stimulates the import 2-3-folds when combined with ATP in the import reactions.
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Glucose Stimulates FBPase Import-- FBPase is subjected to glucose-stimulated proteolytic degradation in the vacuole. Glucose may regulate the production of important factor(s) that are required for importing FBPase into the vacuole. We determined whether the import would be regulated by glucose. Cells were transferred to glucose for 0-60 min and were permeabilized. FBPase import was assayed in semi-intact cells prepared from glucose-starved (t = 0 min) or glucose-replenished (t = 10 to t = 60 min) cells. As shown in Fig. 5, FBPase import was low in the glucose-starved cells (Fig. 5A, t = 0 min). The import was higher in cells that were shifted to glucose for 20-30 min (Fig. 5, C and D). FBPase import was reduced in cells that had been shifted to glucose for 45 or 60 min (Fig. 5, E and F). These results suggest that the effect of glucose on FBPase import is at its maximum in cells that have been transferred to the glucose-rich medium for 20-30 min.
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FBPase Accumulates at a Higher Level in the pep4 Cell-- To examine whether FBPase was imported and degraded in the vacuole, we compared FBPase import in a wild type strain to that in the pep4 deletion strain. Both strains had been transferred to glucose for 20 min, since the highest import was observed under this condition in the pep4 cell (Fig. 5). Accumulation of FBPase in the pep4 cell would indicate that FBPase was targeted into the vacuole for degradation. Fig. 6A shows that, in the wild type cell, the amounts of FBPase were low. When the semi-intact cells and cytosol were prepared from the pep4 cell, FBPase accumulated at higher levels (Fig. 6B). These results suggest that FBPase was targeted into the vacuole for degradation in the wild type cell; the accumulation of FBPase in the pep4 strain was a consequence of reduced proteolytic activity in the pep4 cell.
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Localization of the Imported FBPase-- We examined whether the imported FBPase was localized in the vacuole. Cells were proteinase K-treated and homogenized by gentle lysis. The vacuole was separated by Ficoll gradient separation. The distribution of CPY was determined by Western blotting with CPY antibodies. Using this method, the recovery of intact vacuole was less than 10%, suggesting that significant lysis of the vacuole occurred during the separation process (data not shown). A similar problem was encountered when the vacuole was isolated by sucrose density gradient. Because the recovery of intact vacuole was too low, no conclusion could be drawn as to whether FBPase was indeed targeted to the vacuole in the reconstituted system.
As an alternative, we used indirect immunofluorescence microscopy to examine the localization of the imported FBPase. At the import time of 0, 20, and 40 min, proteinase K was added to the semi-intact cells. Cells were then fixed with formaldehyde. Localization of FBPase was detected by probing with anti-FBPase antibodies followed by fluorescein isothiocyanate-conjugated secondary antibodies. Consistent with the reports by several investigators (25, 34), the overall morphology of the semi-intact cells was significantly compromised. Fig. 7B, D, and F show representative images of the cells that contained recognizable vacuole as seen by Nomarski microscopy. When the Nomarski images were compared with fluorescence FBPase staining in these cells, localization of FBPase in the vacuole was observed at the import time of 20 and 40 min (Fig. 7, C and E). At the import time of 0 min, there was no detectable FBPase immunofluorescence (Fig. 7 A). After import for 20 min, most of the FBPase staining was in small dots inside the region that corresponded to the vacuole as seen by Nomarski images (Fig. 7C). A similar pattern of FBPase staining in small dots inside the vacuole has been reported in vivo (4, 7). At the import time of 40 min, most of the FBPase staining was also observed in the vacuole. At this time point, the immunofluorescence intensity was reduced, suggesting that some of the imported FBPase had been degraded by vacuole proteinases (Fig. 7E).
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DISCUSSION |
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The in vitro reconstitution system is a powerful tool
to dissect complicated biological events in cells. We developed an
in vitro assay to study the targeting of a cytosolic protein
FBPase to the yeast vacuole. We measured in vitro import of
FBPase by incubating semi-intact cells with 35S-FBPase in
the presence of an ATP regenerating system and cytosol. FBPase import
is a saturable process (Fig. 3). Cells that have been transferred to
glucose for 20 or 30 min and then permeabilized stimulate FBPase import
to higher levels than the glucose-starved cells (Fig. 5). The import of
FBPase requires ATP and cytosolic proteins (Fig. 4). It is inhibited by
ATPS, suggesting that ATP hydrolysis is important for FBPase import
to occur. ATP may be required for the action of molecular chaperonins,
targeting, or translocation of FBPase into organelle membranes or
maintaining of the vacuole acidity. ATP
S may have much higher
affinity than ATP for the ATP binding factors. Once ATP
S is bound to
such factors, it cannot be hydrolyzed and locks the ATP-binding
proteins in a configuration that prevents further binding of ATP to the
factors. This may explain why lower concentrations of ATP
S inhibit
FBPase import in this in vitro system.
The degradation of FBPase requires the synthesis of new proteins, as FBPase degradation is inhibited by cycloheximide, an inhibitor of protein synthesis. The inhibition can only be observed if cycloheximide is added at the same time with glucose (4). Cycloheximide has no effect if this agent is added 20-30 min after glucose incubation, suggesting that important proteins for FBPase degradation are synthesized in the first 20-30 min of glucose readdition. Our in vitro system demonstrates that cells that have been shifted to glucose for 20-30 min and then permeabilized stimulate FBPase import to higher levels. These cells may contain a higher level of the factors induced by glucose. Cells that have been transferred to fresh glucose medium for 45 min or longer may have consumed these important factors and therefore import FBPase at lower levels.
We have shown in our previous study that the degradation of FBPase is dependent on the function of the PEP4 gene product in vivo (4). In this in vitro system, FBPase level is low in the wild type cell and accumulates at a higher level in the pep4 cell (Fig. 6). Since the pep4 strain contains reduced proteolytic activity, the higher level of FBPase in the pep4 cell is expected if FBPase is targeted to the vacuole and is degraded slowly by residual proteinases.
We have attempted to isolate the vacuole using different procedures. However, we could only recover less than 10% of intact vacuole after homogenization and gradient separation. This was observed whether cells were homogenized by osmotic lysis or with a Dounce homogenizer. The low recovery of intact vacuole occurred whether the vacuole was isolated by Ficoll flotation gradients or by sucrose density gradient. The semi-intact cells had been subjected to cycles of freeze and thaw and then treatments with hypotonic solution repeatedly before the import reaction. Since 20-40% FBPase was protected in the proteinase K-resistant compartment, the organelle containing the sequestered FBPase, presumably the vacuole, was likely to be intact during the import reaction. However, when cells were homogenized and further fractionated, the yield of intact vacuole was significantly decreased. Because of this problem, no conclusion could be drawn as to whether the imported FBPase was indeed localized to the vacuole. Another factor that might contribute to the technical difficulties was the addition of excessive amounts of proteinase K at the end of the import reactions. Proteinase K was not a problem when samples were solubilized in SDS buffer, boiled immediately, and separated by SDS-PAGE electrophoresis. It might become a problem when cells were homogenized and fractionated. The large amounts of proteinase K might remain active and degrade cellular components nonselectively. As a result, the recovery of intact vacuole was significantly reduced.
This problem was partially circumvented by the immunofluorescence techniques that required fixing of the semi-intact cells with formaldehyde, which also quenched the activity of proteinase K. When the FBPase staining was compared with Nomarski images in these cells, most of the FBPase staining was localized to the vacuole at the import time of 20 min (Fig. 7). In this in vitro system, the cells had been shifted to glucose for 20 min at 30 °C and then permeabilized for the import reactions. The sequestered FBPase was detected in the vacuole after an import time of 20 min at 30 °C. If we add the glucose shift time of 20 min with the import time of 20 min, a total of 40 min is required to detect FBPase in the vacuole at 30 °C. This time course is in close agreement with our in vivo studies that FBPase is found in the vacuole after cells are shifted to glucose for 45 min at 30 °C (4, 7). In this reconstituted system, the fluorescence signal is significantly reduced at the import time of 40 min compared with that seen at t = 20 min. This suggests that FBPase is targeted to the vacuole and then degraded by vacuolar proteinases in the reconstituted system.
Recent evidence suggests that FBPase is targeted to intermediate vesicles before uptake by the vacuole (5). In this in vitro system, FBPase staining was in the cytoplasm and also in the vacuole at earlier time points. It is possible that FBPase staining is in the intermediate vesicles at earlier time points. Since we have experienced a high background staining of FBPase in certain yeast strains, we cannot rule out the possibility that FBPase staining in the cytoplasm is nonselective. Given that we only recovered less than 10% of intact vacuole using cell fractionation techniques, a better separation scheme will be necessary to resolve this issue.
The yeast vacuole is the final destination for sorting of vacuolar resident proteins, endocytosis of plasma membrane proteins, and degradation of cytosolic proteins such as FBPase. The in vitro reconstituted system has been used to study homotypic fusion between vacuoles (37), sorting of CPY from the late Golgi to the vacuole (33), and targeting of aminopeptidase I from the cytosol to the vacuole in S. cerevisiae (39). In vitro targeting and processing of aminopeptidase I presents a post-membrane binding transport event, because the precursor aminopeptidase I is labeled in vivo and is associated with some membranous structures before cells are permeabilized. Furthermore, targeting of the precursor aminopeptidase I to the vacuole does not require cytosol (39). In our in vitro reactions, purified FBPase is introduced to the semi-intact cells. FBPase is imported into the vacuole in a process that is stimulated by cytosol. The development of this in vitro FBPase import system provides a functional assay to understand the molecular mechanisms for targeting FBPase from the cytosol to vacuole for degradation.
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ACKNOWLEDGEMENTS |
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We thank Dr. David T. Rogers for FBPase expression plasmid (AU125) and co-workers in the Chiang lab for helpful suggestions and insightful discussions throughout the investigation. We thank Dr. Hans Hansen for comments and Dr. Kathleen J. Barrett for editing this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1GM49267 (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 Physiology,
Tufts University School of Medicine, 136 Harrison Ave., Boston, MA
02111. Tel: 617-636-6707; Fax: 617-636-0445.
1
The abbreviations used are: FBPase,
fructose-1,6-bisphosphatase; CPY, carboxypeptidase Y; PAGE,
polyacrylamide gel electrophoresis; ATPS, adenosine
5
-O-(thiotriphosphate).
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REFERENCES |
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