Correspondence to: Thomas Vida, Department of Integrative Biology and Pharmacology, The University of Texas Medical School, 6431 Fannin, Houston, TX 77030., tvida{at}farmr1.med.uth.tmc.edu (E-mail), (713) 500-7445 (phone), (713) 500-7455 (fax)
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Abstract |
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We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). Yeast spheroplasts are lysed by extrusion through polycarbonate filters. After differential centrifugation, a 125,000-g pellet is enriched for radiolabeled proCPY and is used as "donor" membranes. A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as "acceptor" membranes. When these membranes are incubated together with ATP and cytosolic extracts, ~50% of the radiolabeled proCPY is processed to mature CPY. Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for the VPS33 gene was less efficient at driving transport. Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a VPS gene product.
Key Words: carboxypeptidase Y, lysosome, membrane fusion, Saccharomyces cerevisiae, vacuole
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Introduction |
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THE secretory and endocytic pathways in eukaryotic cells comprise a series of intercompartmental transport events. The directed movement of protein and lipid from the endoplasmic reticulum to the plasma membrane or from the plasma membrane to the lysosome necessarily engages transfer between multiple organelles. In most cases, carrier vesicles mediate the traffic of protein and lipid cargo from one subcellular compartment to another.
Two integral approaches, genetics and biochemistry, continue to contribute preeminently in elucidating the molecular details of vesicle-mediated transport in the secretory and endocytic pathways. Mutant isolation screens and selections in such diverse organisms as Drosophila melanogaster (
Despite the progress made in other vesicle-mediated events, a poorly understood intercompartmental step in eukaryotic cells continues to be transfer of proteins from prelysosomal compartments (PLC)1 to the lysosome. The PLC, or late endosome, plays a pivotal role in protein traffic since it is the organelle where the secretory and endocytic pathways converge. Resident lysosomal proteins pass through the PLC after being sorted away from secretory proteins in the trans-Golgi network (
Saccharomyces cerevisiae contains not only a lysosome-like vacuole, but also a PLC-like prevacuolar compartment (PVC) (
In this report, we describe an intercompartmental protein transport assay using partially purified organelles. This cell-free system measures proteolytic maturation of soluble vacuolar proenzymes such as carboxypeptidase Y and proteinase A after transfer from the PVC to the vacuole. The reaction is sensitive to membrane dilution, requires ATP, and cytosol. Importantly, cytosol made from a vps33 strain is deficient at stimulating transport in the new cell-free system. Furthermore, antibody raised against Vps33p can inhibit the assay >90% and cytosolic extracts made from strains overexpressing Vps33p can restore this inhibition. Thus, we have developed a transport-coupled assay for the function of a VPS gene product.
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Materials and Methods |
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Media
Yeast strains were maintained on YPD media (1% yeast extract, 2% peptone, 2% dextrose, and 2.5% bacto-agar). Liquid media for radiolabeling and plasmid maintenance was Wickerham's minimal proline (WIMP) (
Strains and Plasmids
The yeast strains used in this study include BGY3300 ( ura3-52 leu2-3,112 his3-
200 trp-
901 lys2-801 suc2-
9 vps33
::HIS3. SEY6210 (
ura3-52 leu2-3,112 his3-
200 trp 1-
901 lys2-801 suc2-
9 prc1
::HIS3 prb1
::hisG pep4
::LEU2; and TVY1 (
ura3-52 leu2-3,112 his3-
200 trp 1-
901 lys2-801 suc2-
9 pep4
::LEU2. Several constructs were made to put the VPS33 gene under control of the glyceraldehyde-3-phosphate dehydrogenase promoter (GPD1pr). First, site-directed mutagenesis (
Antibody Production
Two previously described trpe-VPS33 fusion constructs (
Preparation of Cytosol
Yeast strain TVY614 was grown at 30°C in YPD (with 5% glucose) to an OD600 of 46 (usually 8001,600 total OD600 units of cells were used). The cells were harvested with centrifugation at 5,000 rpm in a Beckman JA-14 rotor for 15 min. The cells were washed once with sterile distilled water (using 50% of the original volume of media) and harvested again as above. The washed cell pellet was resuspended in ~35 ml of ice-cold 0.25 M sorbitol, 20 mM Hepes-KOH, 150 mM potassium acetate, and 5 mM magnesium acetate pH 7.0 (standard transport buffer, TB). The resuspended cells were transferred to a 50-ml conical tube and harvested via centrifugation at the highest setting (~1,750 g) of a clinical centrifuge (International Equipment Co., Inc.) for 15 min at 4°C. The cells were resuspended in TB to 200 OD600/ml and transferred to 2-ml polypropylene tubes in 1-ml aliquots. Approximately 1 g of acid-washed glass beads (0.5 mm) was added and then agitated for three 30-s intervals in a Mini Bead-Beater (BioSpec Products, Inc.) at 4°C. All tubes were subjected to centrifugation at 1,500 g for 2 min. The supernatant was removed from each tube and the pellet was rinsed with 1 ml of TB, agitated briefly on a vortex mixer, and subjected to centrifugation at 1,500 g for 2 min. The second supernatant was pooled with the first supernatant and subjected to centrifugation in a Beckman TLA 100.3 rotor at 50,000 rpm (~103,000 g average) for 10 min. The supernatant was removed, dispensed into small aliquots, and snap-frozen in liquid nitrogen. The protein concentration of all cytosolic extracts ranged from 2550 mg/ml.
Cell Preparation for Donor and Acceptor Membranes
All steps are reported for the preparation of donor membranes from 25 OD600 units of cells. Whenever preparing more than this amount, volumes were scaled up proportionately. Yeast cells were grown in Wickerham's minimal proline (
Preparation of Donor and Acceptor Membranes
If using frozen cells, they were thawed in a 25°C circulating water bath for 1 min and placed on ice. 600 µl of 0.6 M sorbitol with 5 mM Hepes-KOH, pH 7.0 (lysis buffer) was added and the cells were resuspended thoroughly. The cells were harvested by centrifugation for 1 min at 16,000 g and then resuspended to 8 OD600 units/ml in lysis buffer. The resuspended cells were pushed through a 13-mm polycarbonate filter (NucleoporeTM; Corning) with 3-µm pores using a 3-ml syringe. The filter effluent was subjected to centrifugation at 440 g for 5 min to generate a P1 pellet and S1 supernatant fraction. The S1 supernatant was subjected to centrifugation at 15,000 g for 10 min to generate a P2 pellet (acceptor membranes) and S2 supernatant fraction. The S2 supernatant was subjected to centrifugation at 125,000 g for 10 min to generate a P3 pellet (donor membranes) and S3 supernatant fraction.
Cell-free Assays
Radiolabeled donor membranes and nonradiolabeled acceptor membranes were resuspended in TB. Standard conditions for assays were 50 µl total volume containing donor membranes (equivalent to 5 OD600 units of cells), 100125 µg of acceptor membranes, 1 mM ATP, 40 mM creatine phosphate, 0.2 mg/ml creatine kinase, and 5 mg/ml cytosol. All reactions were assembled on ice and then incubated at 25°C in a circulating water bath for 60 min. To stop the reactions, 3 µl of 100 mM PMSF, 25 µl of 8.0 M urea, 5% SDS, and 5% NP-40 was added and they were boiled for 5 min. All samples were processed for immunoprecipitation, SDS-PAGE, and autoradiography as previously described (
Microscopy
All light microscopy images were obtained as previously described (
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Results |
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A New Method to Lyse Yeast Spheroplasts
The usefulness of cell-free assays cannot be overstated in their contribution to our understanding of mechanisms in protein transport, secretion, and endocytosis. Since the development of a permeabilized cell assay for transport to the yeast vacuole (
The technique of passing cells through a small orifice to generate a crude lysate from shear forces was first used for mammalian cells. For example, cell homogenates have been prepared from PC12 cells by passing cell suspensions 15 times through a narrow clearance (10 µm) stainless steel ball homogenizer (
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We performed centrifugation techniques on crude lysates after extrusion through polycarbonate filters and examined each supernatant and pellet fraction for marker proteins. Simple differential centrifugation allows separation of a variety of yeast organelles and membranes (
Various steps from the filter lysis procedure were also examined with microscopy. To follow the vacuole, we prestained yeast cells with FM4-64 and CDCFDA. As expected, the P1 pellet was devoid of unbroken cells and was enriched in cell wall remnants (Figure 2). As expected from the marker protein analysis, the P2 pellet was enriched in intact vacuoles, since many FM4-64stained membranes containing CDCFDA were observed (Figure 2). In contrast, the 125,000-g P3 pellet was devoid of vacuoles and instead was enriched for very small particulate structures. Importantly, if cells were stained with FM4-64 at 15°C, many of the small particulate structures in the 125,000-g pellet exhibited fluorescence (Figure 2, inset). Additionally, membrane fluorescence in the 15,000-g P2 pellet was markedly reduced at 15°C (Figure 2, inset). Since FM4-64 is kinetically trapped in prevacuolar compartments at 15°C (
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Reconstitution of p2CPY Maturation after Mixing the Donor and Acceptor Membrane Pellets
The polycarbonate filter lysis technique and simple differential centrifugation cleanly separated membranes containing vacuolar precursor proteins, the P3 pellet, from membranes containing vacuoles, the P2 pellet. These conditions set up the ability to use the P3 pellet as a donor membrane fraction and the P2 pellet as an acceptor membrane fraction. We prepared a P3 pellet after radiolabeling yeast spheroplasts with Tran35S-label (5-min pulse, 2-min chase) and incubated the membranes under various conditions of ATP, cytosol, and acceptor membranes. Each reaction was sequentially immunoprecipitated for both CPY and proteinase A (PrA), subjected to SDS-PAGE, and autoradiography. The level of both p2CPY and p2PrA remained unchanged after incubating the donor membranes with buffer, ATP alone, cytosol alone, or with ATP plus cytosol (Figure 3, lanes 15). Even after adding back P2 acceptor membranes (made from unlabeled spheroplasts), alone and with cytosol, the amount of both p2CPY and p2PrA also remained constant (Figure 3, lanes 6 and 8). Importantly, when ATP, cytosol, and unlabeled acceptor membranes were added back to the P3 radiolabeled donor membranes, ~50% of the p2CPY and ~60% of the proPrA (e.g., p2PrA) were converted to their mature, active forms (Figure 3, lane 9). The cytosolic extract stimulated their maturation just over twofold compared with incubating the donor and acceptor membranes with ATP alone (Figure 3, compare lane 7 with 9). However, this cytosol stimulation increased to at least 40-fold after incubating the donor and acceptor membranes for 15 min on ice and reharvesting them with centrifugation before setting up the reactions. (Figure 3, lanes 10 and 11). This argued that some activity(ies) associated with either the donor membranes, acceptor membranes, or both was removed, rendering the p2CPY and p2PrA maturation completely dependent on exogenous cytosol.
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Characteristics of the Cell-free Assay
The characteristics of cell-free assays with P3 donor membranes and P2 acceptor membranes were examined to determine if they suggested that the reaction was intercompartmental. The first characteristic that we examined was dilution sensitivity. Normally, reactions were carried out in a 50-µl volume with the efficiency of p2CPY maturation ranging from 35 to 55%. To test the effect of dilution, the reaction volume was increased to dilute the concentration of donor/acceptor membranes while the concentration of ATP and cytosol was maintained at a constant level. An exponential decrease in p2CPY maturation efficiency was observed concomitant with an incremental increase in the reaction volume (Figure 4 A). For example, a sixfold decrease in efficiency (38% vs 6%) took place with a 10-fold increase in reaction volume (from 50 to 500 µl). This suggested that the concentration of donor and acceptor membranes had a critical threshold for optimal reconstitution of p2CPY maturation. The second characteristic that we examined of the cell-free assay was the reaction kinetics. A prominent lag period was observed in the first 1520 min (Figure 4 B). A linear phase followed for the next 20 min and reached a plateau between 40 and 60 min (Figure 4 B). Although not shown in this experiment, an increase in p2CPY maturation did not occur after a further 60 min incubation. This kinetic analysis suggested that a rate-limiting event(s) occurred early in the incubation, which might be the formation of a transport intermediate. The third characteristic that we examined of the cell-free assay was its temperature dependence. The maturation of p2CPY was undetectable when the incubation was carried out at 0 or 5°C (Figure 4 C). The optimal efficiency occurred between 20 and 30°C and sharply tapered off at temperatures above 30°C (Figure 4 C). Overall, the dilution sensitivity, kinetics, and temperature dependence of this new cell-free assay for p2CPY maturation indicated a complex event(s) was reconstituted after incubating P3 donor membranes and P2 acceptor membranes in the presence of ATP and cytosol.
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The Cell-free Assay Reconstitutes Intercompartmental Protein Transport
To truly determine if this new cell-free assay reconstituted intercompartmental protein transport, we performed reactions where the donor and acceptor fractions were prepared from yeast strains defective in vacuolar processing enzymes. A hallmark of most cell-free intercompartmental protein transport assays is using donor membranes deficient in the activity that marks the transport event. Two proteases are responsible for cleaving the propeptide from p2CPY, proteinase A (PEP4 gene) and proteinase B (PRB1 gene). In yeast strains mutant for the PEP4 gene (pep4-1, or pep4), p2CPY travels to the vacuole but is not processed to the mature form of the protein (
We took advantage of CPY processing characteristics in vivo (Figure 5 A) using PEP4 and pep4 yeast strains as a source of both donor and acceptor membranes in vitro. To confirm the genotype of the strains, we performed pulsechase analysis and compared CPY processing. Both PEP4 and pep4
strains showed no significant differences for p1 and p2CPY after a 5-min pulse (Figure 5 B, lanes 1 and 3). However, after a 60-min chase the fate of the p1 and p2CPY precursors was different. The PEP4 strain produced mCPY and the pep4
strain did not produce any mCPY but the p2CPY precursor accumulated (Figure 5 B, lanes 2 and 4). With these phenotypes established, we prepared radiolabeled P3 donor membranes and unlabeled P2 acceptor membranes from the wild-type PEP4 and the pep4
mutant strains. These membranes were then mixed and incubated for cell-free assays in all combinations. Importantly, the radiolabeled reaction product took on the processing phenotype of the unlabeled acceptor membranes, not the radiolabeled donor membranes. For instance, PEP4 acceptor membranes gave rise to mCPY even from pep4
donor membranes (Figure 5 C; lanes 5 and 6). Acceptor membranes from the pep4
strain did not produce detectable p2CPY maturation (Figure 5 C, lanes 7 and 8). The small amount of mCPY (~9%) that occurred from mixing PEP4 donor membranes with pep4
acceptor membranes (Figure 5 C, lane 7) was present in the reaction where no acceptor membranes were added back (Figure 5 C, lane 3). This indicated that a trace amount of vacuoles contaminated the PEP4 donor membranes in this experiment. These reactions with donor and acceptor membranes from a strain deleted for the principal processing protease gene provided the strongest evidence that our new cell-free assay was indeed intercompartmental. This reconstitution was likely an intercompartmental transport process between the PVC and the vacuole.
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A Role for Vps33p in the Cell-free Reconstitution Assay
One difficulty in reconstituting an intercompartmental transport event in our previous permeabilized cell assay was incomplete removal of many cytoplasmic VPS gene products such as Vps33p () was added back to wild-type permeabilized cells (data not shown). However, a significant defect was observed in vps33
cytosol when it was added back to the cell-free transport assay. The transport efficiency was decreased ~2.5-fold compared with cytosol made from a wild-type VPS33 strain (Figure 6, lanes 2 and 3, 5 and 6). Although the standard concentration of cytosol in our cell-free reactions was 5 mg/ml, these experiments also demonstrated that overall transport efficiency was remarkably consistent with the concentration of protein in crude, undiluted wild-type cytosol. For example, using extracts with a protein concentration of 50 mg/ml produced an average transport efficiency of 47.0% ± 1.3% (n = 10). We observed an average transport efficiency of 32.6% ± 2.5% (n = 10) with an undiluted cytosolic protein concentration of 35 mg/ml. The 30% decrease in transport efficiency correlated well with the 30% decrease in protein concentration, which suggested that the level of a soluble protein factor(s) was critical for driving intercompartmental transport.
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Polyclonal antiserum (raised against Vps33p-trpe fusion proteins) also directly implicated Vps33p in playing a specific role during the cell-free assay. We prepared a new antiserum against Vps33p and it proved to be monospecific, recognizing a single polypeptide of ~72 kD after immunoprecipitation of a total yeast cell lysate (Figure 7 A, lane 2). The preimmune serum did not immunoprecipitate any proteins in this cell lysate (Figure 7 A, lane 1). In pilot experiments, the Vps33p immune serum inhibited the cell-free assay while the preimmune had no effect. To avoid potential inhibitory problems from whole serum, we purified total IgG from both the preimmune and immune sera and measured the inhibition in titration experiments with the cell-free assay. As more of the immune IgG against Vps33p was added to the cell-free assay, we observed a proportional decrease in p2CPY transport (Figure 7 B). At 128 µg and above, the immune IgG was able to block >90% of intercompartmental transport in the assay (Figure 7 B, lane 8). Importantly, preimmune IgG was without any measurable inhibitory effect (Figure 7 B, lanes 38).
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Using immune IgG against Vps33p as a specific inhibitor of its function, we determined where Vps33p was most active during the cell-free assay. To test this, we added back IgG against Vps33p at different points during a time course (similar to the kinetics in Figure 4 B). After allowing antibody/antigen binding for 15 min on ice at each time point, we then continued the incubation for 60 min at 25°C (see Figure 8 A). The results from this analysis suggested that the role of Vps33p in intercompartmental transport to the vacuole was executed at an early stage in the cell-free assay. For example, when the antibody was added back before incubation (at 0 min), >90% inhibition was observed (Figure 8 C). Moreover, this inhibition was most effective during the first 1015 min of the time course (Figure 8 C). This interval of time in the cell-free assay was the latent period showing very little maturation of p2CPY (Figure 8 B and 4 B). The inhibition from adding immune IgG against Vps33p during the cell-free assay time course was significantly less at the 15 min time point and beyond (Figure 8 C). For example, at 15 min only 10% of intercompartmental transport took place (Figure 8 B) and the inhibition was only 40% (Figure 8 C). This effect was more notable at the 30 min time point where ~60% (Figure 8 B) of intercompartmental transport occurred but the inhibition was only 10% (Figure 8 C).
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We also determined the possible involvement of another protein in the cell-free assay, Vam3p. Vam3p is a Q-SNARE protein (60% throughout the time course (Figure 8 C). These results suggested that the function of both Vps33p and Vam3p was required for efficient transport in the cell-free assay. However, an early event(s) was more dependent on the function of Vps33p, particularly during the first 15 min, than later events and Vam3p appeared to be required both early and late in the assay. The ability to inhibit the assay with Vps33p-specific antibodies decreased nearly threefold faster than with antibodies against Vam3p during the first 15 min of the cell-free assay.
Biochemical Complementation of VPS33 Function
The cell-free assay has allowed us to directly implicate the function of a VPS gene product in a reconstituted intercompartmental transport event. The inefficient transport from vps33 cytosolic extracts and the inhibition by IgG against Vps33p demonstrate that the function of this protein is required during incubation of donor and acceptor membranes. We wanted to positively implicate the role of Vps33p in the cell-free assay, which would establish a transport-coupled biochemical assay for a VPS gene product.
To this end, we expressed Vps33p in bacteria and it was produced at high levels (data not shown). However, over a variety of induction conditions with changes in temperature, time, or inducer concentration, Vps33p repeatedly was insoluble in bacterial lysates (data not shown). To avoid the insolubility problems from overexpression in bacteria, we overexpressed the VPS33 gene in yeast. We placed Vps33p under control of the promoter for glyceraldehyde 3-phosophate dehydrogenase (GPD1pr) because it is one of the strongest promoters in S. cerevisiae ( strain was added back to the IgG-inhibited reaction (Figure 9, lane 2). This suggested that restoration of p2CPY transport to the vacuole may be specific to Vps33p. A wild-type cytosol (i.e., VPS33pr-VPS33) leads to a transport efficiency just under 20% maximum, further suggesting that reversal of inhibition reflected the level of Vps33p added back to the assay. The results of this experiment provide evidence for biochemical complementation of a Vps protein-dependent defect to the yeast vacuole.
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Discussion |
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Gaining access to the cell cytoplasm is essential for detailed understanding of intracellular transport between organelles. Genetics and molecular biological approaches are able to obtain entrance into cells with the manipulation of genes and gene products. Although this control can often be very thorough, it is also often limited without augmentation using biochemical approaches in parallel. The biochemistry of transport between organelles requires working in a cell-free system. Frequently, severe limitations to cell-free analyses are not only maintaining organelle structure, but also (and more importantly) organelle function. These are the two most important criteria in successful cell-free reconstitution of intercompartmental transport.
Lysing yeast spheroplasts by extrusion through polycarbonate filters maintains function of organelles in the yeast vacuolar system. Polycarbonate filters are hydrophilic and contain uniform cylindrical pores. The diameter of these pores can be carefully controlled via ion etching, which allows for an even distribution across one plane over the entire exposed membrane surface. The ability to change the overall diameter of a yeast spheroplast with osmotic forces permits swelling of the cells to just greater than the diameter of the polycarbonate filter pores. Thus, in one simple step, the plasma membrane can be gently sheared away from cells and most organelles are free to pass through with little damage. In fact, we have used this method of lysis on mammalian cells (Chinese hamster ovary), which required a simple increase in pore size (from 3 to 8 µm). The yeast vacuole does undergo some loss of luminal content during extrusion through polycarbonate filters as expected from its labile structure. This loss is most likely from leakage rather than lysis and is inconsequential because the amount of soluble proteases is sufficient for processing of propeptides from vacuolar zymogens.
Proteolytic maturation within the donor compartment, presumably the PVC, does not appear to be an efficient process in vitro. Another explanation for our cell-free assay that measures maturation of p2CPY could be intracompartmental activation of processing proteases. To a first approximation, proPrA is contained in the same compartments as p2CPY. Unlike proCPY, the proPrA precursor has the ability to autoactivate (
This cell-free system is easily manipulated to show a near absolute requirement for exogenous cytosol. With this cytosol requirement, we can tentatively assign the location of Vps33p function to cycling from the cytosol to either the donor or acceptor membrane fractions. Although Vps33p is predominately localized to the cytosol, a fraction of the protein sediments with membranes (
One prospect for functional interaction of Vps33p with an insoluble component(s) is a SNARE complex (
The Sec1p family has many members, suggesting that these proteins function at every vesicle-mediated step in eukaryotic cells (
The advent of our cell-free assay will help uncover biochemical activities of VPS gene products. The majority of these proteins do not show sequence similarity to proteins of known biochemical properties, although several VPS gene products have activities in vitro. Without exception, the ability to design assays for detection of these catalytic activities arose from sequence similarities to proteins that had been subjected to previous biochemical characterization. This underscores the importance that biochemistry plays in elucidating gene function and discovering new activities should progress rapidly with our intercompartmental assay. This assay will allow us to define biochemical functions of cytosolic and membrane-associated factors necessary to execute transport between the PVC and vacuole. Our results with the cell-free assay imply that a vesicle intermediate may truly shuttle cargo between the PVC and the vacuole in yeast. Preincubation of donor membranes (in the absence of acceptor membranes) with ATP and cytosol gives rise to a fraction that contains p2CPY and separates from donor membranes. This membrane-enclosed compartment can be used as a functional intermediate in a second incubation with acceptor membranes (Gerhardt and Vida, manuscript in preparation). Many of the factors involved in this process are likely VPS gene products and may play a role in vesicle formation, transport, targeting, and fusion with the yeast vacuole/lysosome.
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Acknowledgements |
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We thank Etienne-Pascal Journet (Laboratoire de Biologie Moleculaire des Relations Plantes-Microorganismes Castanet-Tolosan Cedex France) for showing one of us (T. Vida) the polycarbonate filter lysis technique before publication. We acknowledge the excellent expertise of Kenneth Dunner, Jr. for electron microscopy. We thank William Wickner (Dartmouth Medical School) for his generous gift of antiserum against Vam3p. We thank Jean Whitesell and Cocalico Biologicals, Inc. for expert polyclonal antiserum production against Vps33p. We are grateful to Anita Seibold for telling us about propanol-jacketed cryo freezers. We also thank Victoria Knutson, Andrew Bean, and Neal Waxham for valuable discussions, and Andrew Bean for critically reading the manuscript. B. Gerhardt thanks Dave Matthews for inspirational music.
These studies were supported by a grant to T. Vida from the National Institutes of Health-National Institute of General Medical Sciences (GM52092). We also acknowledge an Institutional Core Grant (no. CA16672) that maintains the High Resolution Transmission Electron Microscopy Facility (University of Texas M.D. Anderson Cancer Center).
Submitted: April 26, 1999; Revised: May 28, 1999; Accepted: June 8, 1999.
1.used in this paper: CPY, carboxypeptidase Y; CDCFDA, dichlorocarboxyfluorescein diacetate; PLC, prelysosomal compartment; PrA, proteinase A; PVC, prevacuolar compartment
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