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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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Polyclonal IgG against Vps33p inhibits cell-free reconstitution of intercompartmental transport between donor and acceptor membranes. (A) Specificity of the anti-Vps33p serum. Whole yeast cells (SEY6210) containing a plasmid with the VPS33 gene under control of the GPD1 promoter (pGPD426-33) were radiolabeled for 10 min (Tran35S-label) and chased (methionine and cysteine) for 30 min. After cell lysis, the extracts were immunoprecipitated with preimmune (P, lane 1) or immune (I, lane 2) serum against Vps33p, subjected to SDS-PAGE, and autoradiography. The positions of molecular weight standards are given in kD. (B) Purified immune IgG inhibits cell-free transport of CPY. Protein A–Sepharose was used to purify IgG from both preimmune and immune rabbit serum. Different amounts (as indicated) of the purified IgG  were used in standard cell-free reactions with donor and acceptor membranes. All components of the reactions were added and incubated on ice with the indicated amounts of preimmune and immune IgG for 15 min before the standard 60-min incubation at 25°C. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.
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Figure 7: Polyclonal IgG against Vps33p inhibits cell-free reconstitution of intercompartmental transport between donor and acceptor membranes. (A) Specificity of the anti-Vps33p serum. Whole yeast cells (SEY6210) containing a plasmid with the VPS33 gene under control of the GPD1 promoter (pGPD426-33) were radiolabeled for 10 min (Tran35S-label) and chased (methionine and cysteine) for 30 min. After cell lysis, the extracts were immunoprecipitated with preimmune (P, lane 1) or immune (I, lane 2) serum against Vps33p, subjected to SDS-PAGE, and autoradiography. The positions of molecular weight standards are given in kD. (B) Purified immune IgG inhibits cell-free transport of CPY. Protein A–Sepharose was used to purify IgG from both preimmune and immune rabbit serum. Different amounts (as indicated) of the purified IgG were used in standard cell-free reactions with donor and acceptor membranes. All components of the reactions were added and incubated on ice with the indicated amounts of preimmune and immune IgG for 15 min before the standard 60-min incubation at 25°C. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.

Mentions: 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 (Fig. 7 A, lane 2). The preimmune serum did not immunoprecipitate any proteins in this cell lysate (Fig. 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 (Fig. 7 B). At 128 μg and above, the immune IgG was able to block >90% of intercompartmental transport in the assay (Fig. 7 B, lane 8). Importantly, preimmune IgG was without any measurable inhibitory effect (Fig. 7 B, lanes 3–8).


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

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

Polyclonal IgG against Vps33p inhibits cell-free reconstitution of intercompartmental transport between donor and acceptor membranes. (A) Specificity of the anti-Vps33p serum. Whole yeast cells (SEY6210) containing a plasmid with the VPS33 gene under control of the GPD1 promoter (pGPD426-33) were radiolabeled for 10 min (Tran35S-label) and chased (methionine and cysteine) for 30 min. After cell lysis, the extracts were immunoprecipitated with preimmune (P, lane 1) or immune (I, lane 2) serum against Vps33p, subjected to SDS-PAGE, and autoradiography. The positions of molecular weight standards are given in kD. (B) Purified immune IgG inhibits cell-free transport of CPY. Protein A–Sepharose was used to purify IgG from both preimmune and immune rabbit serum. Different amounts (as indicated) of the purified IgG  were used in standard cell-free reactions with donor and acceptor membranes. All components of the reactions were added and incubated on ice with the indicated amounts of preimmune and immune IgG for 15 min before the standard 60-min incubation at 25°C. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.
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Related In: Results  -  Collection

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Figure 7: Polyclonal IgG against Vps33p inhibits cell-free reconstitution of intercompartmental transport between donor and acceptor membranes. (A) Specificity of the anti-Vps33p serum. Whole yeast cells (SEY6210) containing a plasmid with the VPS33 gene under control of the GPD1 promoter (pGPD426-33) were radiolabeled for 10 min (Tran35S-label) and chased (methionine and cysteine) for 30 min. After cell lysis, the extracts were immunoprecipitated with preimmune (P, lane 1) or immune (I, lane 2) serum against Vps33p, subjected to SDS-PAGE, and autoradiography. The positions of molecular weight standards are given in kD. (B) Purified immune IgG inhibits cell-free transport of CPY. Protein A–Sepharose was used to purify IgG from both preimmune and immune rabbit serum. Different amounts (as indicated) of the purified IgG were used in standard cell-free reactions with donor and acceptor membranes. All components of the reactions were added and incubated on ice with the indicated amounts of preimmune and immune IgG for 15 min before the standard 60-min incubation at 25°C. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.
Mentions: 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 (Fig. 7 A, lane 2). The preimmune serum did not immunoprecipitate any proteins in this cell lysate (Fig. 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 (Fig. 7 B). At 128 μg and above, the immune IgG was able to block >90% of intercompartmental transport in the assay (Fig. 7 B, lane 8). Importantly, preimmune IgG was without any measurable inhibitory effect (Fig. 7 B, lanes 3–8).

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