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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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The function of Vps33p is required early during the cell-free reconstitution system and precedes the function of Vam3p. (A) Experimental strategy. (B) Transport kinetics. A standard 50-μl reaction (see Fig. 3) was scaled up, incubated at 25°C, and then aliquots were removed and stopped at the indicated times. (C) Antibody inhibition. Aliquots from the same reactions as in A were also removed and received either 128 μg of immune IgG against Vps33p or 5 μl of antiserum against Vam3p (as indicated), incubated 15 min on ice, and then shifted to 25°C for 60 min. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. Nonimmune serum was added to control for the effect of anti-Vam3p serum at each time point and did not inhibit the assay. A 2nd degree polynomial was used to fit the curve for the inhibition data points (C).
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Figure 8: The function of Vps33p is required early during the cell-free reconstitution system and precedes the function of Vam3p. (A) Experimental strategy. (B) Transport kinetics. A standard 50-μl reaction (see Fig. 3) was scaled up, incubated at 25°C, and then aliquots were removed and stopped at the indicated times. (C) Antibody inhibition. Aliquots from the same reactions as in A were also removed and received either 128 μg of immune IgG against Vps33p or 5 μl of antiserum against Vam3p (as indicated), incubated 15 min on ice, and then shifted to 25°C for 60 min. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. Nonimmune serum was added to control for the effect of anti-Vam3p serum at each time point and did not inhibit the assay. A 2nd degree polynomial was used to fit the curve for the inhibition data points (C).

Mentions: 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 Fig. 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 Fig. 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 (Fig. 8 C). Moreover, this inhibition was most effective during the first 10–15 min of the time course (Fig. 8 C). This interval of time in the cell-free assay was the latent period showing very little maturation of p2CPY (Fig. 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 (Fig. 8 C). For example, at 15 min only 10% of intercompartmental transport took place (Fig. 8 B) and the inhibition was only 40% (Fig. 8 C). This effect was more notable at the 30 min time point where ∼60% (Fig. 8 B) of intercompartmental transport occurred but the inhibition was only 10% (Fig. 8 C).


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

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

The function of Vps33p is required early during the cell-free reconstitution system and precedes the function of Vam3p. (A) Experimental strategy. (B) Transport kinetics. A standard 50-μl reaction (see Fig. 3) was scaled up, incubated at 25°C, and then aliquots were removed and stopped at the indicated times. (C) Antibody inhibition. Aliquots from the same reactions as in A were also removed and received either 128 μg of immune IgG against Vps33p or 5 μl of antiserum against Vam3p (as indicated), incubated 15 min on ice, and then shifted to 25°C for 60 min. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. Nonimmune serum was added to control for the effect of anti-Vam3p serum at each time point and did not inhibit the assay. A 2nd degree polynomial was used to fit the curve for the inhibition data points (C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199724&req=5

Figure 8: The function of Vps33p is required early during the cell-free reconstitution system and precedes the function of Vam3p. (A) Experimental strategy. (B) Transport kinetics. A standard 50-μl reaction (see Fig. 3) was scaled up, incubated at 25°C, and then aliquots were removed and stopped at the indicated times. (C) Antibody inhibition. Aliquots from the same reactions as in A were also removed and received either 128 μg of immune IgG against Vps33p or 5 μl of antiserum against Vam3p (as indicated), incubated 15 min on ice, and then shifted to 25°C for 60 min. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. Nonimmune serum was added to control for the effect of anti-Vam3p serum at each time point and did not inhibit the assay. A 2nd degree polynomial was used to fit the curve for the inhibition data points (C).
Mentions: 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 Fig. 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 Fig. 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 (Fig. 8 C). Moreover, this inhibition was most effective during the first 10–15 min of the time course (Fig. 8 C). This interval of time in the cell-free assay was the latent period showing very little maturation of p2CPY (Fig. 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 (Fig. 8 C). For example, at 15 min only 10% of intercompartmental transport took place (Fig. 8 B) and the inhibition was only 40% (Fig. 8 C). This effect was more notable at the 30 min time point where ∼60% (Fig. 8 B) of intercompartmental transport occurred but the inhibition was only 10% (Fig. 8 C).

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