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A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.

Vida T, Gerhardt B - J. Cell Biol. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, Texas 77030, USA. tvida@farmr1.med.uth.tmc.edu

ABSTRACT
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, approximately 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.

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Biochemical reconstitution of Vps33p-dependent transport between donor and acceptor membranes. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared from wild-type yeast spheroplasts (Fig. 1 A). The donor and acceptor membranes were incubated for 15 min at 0°C with 128 μg of anti Vps33p IgG. Cytosol (5 mg/ml) was then added from a vps33Δ (lane 2), a VPS33 (lane 3), or a VPS33 strain containing pGPDHIS633-2 (lane 4), and incubated at 25°C for 60 min (as indicated). All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. The bar graph depicts average transport efficiency from three independent determinations and normalized to the percent of maximal transport.
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Figure 9: Biochemical reconstitution of Vps33p-dependent transport between donor and acceptor membranes. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared from wild-type yeast spheroplasts (Fig. 1 A). The donor and acceptor membranes were incubated for 15 min at 0°C with 128 μg of anti Vps33p IgG. Cytosol (5 mg/ml) was then added from a vps33Δ (lane 2), a VPS33 (lane 3), or a VPS33 strain containing pGPDHIS633-2 (lane 4), and incubated at 25°C for 60 min (as indicated). All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. The bar graph depicts average transport efficiency from three independent determinations and normalized to the percent of maximal transport.

Mentions: 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 (Mumberg et al. 1995). Indeed, a 100–200-fold increase in the amount of GPD1pr-Vps33p was observed compared with endogenous levels of the protein and the overexpressed Vps33p behaved like a soluble protein (data not shown). The high level overproduction of soluble Vps33p in yeast allowed us to determine if we could reverse the antibody inhibition of the cell-free reconstitution assay. After incubating the donor/acceptor assay with IgG against Vps33p, a transport efficiency of ∼50% maximum was observed with a cytosolic extract from a strain expressing GPD1pr-VPS33 (Fig. 9, lane 4). A significant level of transport was not observed when a cytosolic extract from the vps33Δ strain was added back to the IgG-inhibited reaction (Fig. 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.


A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.

Vida T, Gerhardt B - J. Cell Biol. (1999)

Biochemical reconstitution of Vps33p-dependent transport between donor and acceptor membranes. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared from wild-type yeast spheroplasts (Fig. 1 A). The donor and acceptor membranes were incubated for 15 min at 0°C with 128 μg of anti Vps33p IgG. Cytosol (5 mg/ml) was then added from a vps33Δ (lane 2), a VPS33 (lane 3), or a VPS33 strain containing pGPDHIS633-2 (lane 4), and incubated at 25°C for 60 min (as indicated). All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. The bar graph depicts average transport efficiency from three independent determinations and normalized to the percent of maximal transport.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199724&req=5

Figure 9: Biochemical reconstitution of Vps33p-dependent transport between donor and acceptor membranes. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared from wild-type yeast spheroplasts (Fig. 1 A). The donor and acceptor membranes were incubated for 15 min at 0°C with 128 μg of anti Vps33p IgG. Cytosol (5 mg/ml) was then added from a vps33Δ (lane 2), a VPS33 (lane 3), or a VPS33 strain containing pGPDHIS633-2 (lane 4), and incubated at 25°C for 60 min (as indicated). All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. The bar graph depicts average transport efficiency from three independent determinations and normalized to the percent of maximal transport.
Mentions: 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 (Mumberg et al. 1995). Indeed, a 100–200-fold increase in the amount of GPD1pr-Vps33p was observed compared with endogenous levels of the protein and the overexpressed Vps33p behaved like a soluble protein (data not shown). The high level overproduction of soluble Vps33p in yeast allowed us to determine if we could reverse the antibody inhibition of the cell-free reconstitution assay. After incubating the donor/acceptor assay with IgG against Vps33p, a transport efficiency of ∼50% maximum was observed with a cytosolic extract from a strain expressing GPD1pr-VPS33 (Fig. 9, lane 4). A significant level of transport was not observed when a cytosolic extract from the vps33Δ strain was added back to the IgG-inhibited reaction (Fig. 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, Texas 77030, USA. tvida@farmr1.med.uth.tmc.edu

ABSTRACT
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, approximately 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.

Show MeSH
Related in: MedlinePlus