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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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Characteristics of the cell-free system. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared and incubated (as described in Fig. 3) with modifications in reaction volume, time, or temperature. (A) Dilution sensitivity. All reactions were performed with a constant amount of donor and acceptor membranes while the reaction volume was increased (50–1,000 μl), as indicated. The concentration of ATP (plus regeneration components) and cytosol was equivalent in all reactions. (B) Maturation kinetics. A standard 50-μl reaction (see Fig. 3) was scaled up fivefold, incubated at 25°C, and aliquots were removed at the indicated times. (C) Temperature dependence. Standard 50-μl reactions were incubated at the indicated temperatures. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.
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Figure 4: Characteristics of the cell-free system. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared and incubated (as described in Fig. 3) with modifications in reaction volume, time, or temperature. (A) Dilution sensitivity. All reactions were performed with a constant amount of donor and acceptor membranes while the reaction volume was increased (50–1,000 μl), as indicated. The concentration of ATP (plus regeneration components) and cytosol was equivalent in all reactions. (B) Maturation kinetics. A standard 50-μl reaction (see Fig. 3) was scaled up fivefold, incubated at 25°C, and aliquots were removed at the indicated times. (C) Temperature dependence. Standard 50-μl reactions were incubated at the indicated temperatures. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.

Mentions: 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 (Fig. 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 15–20 min (Fig. 4 B). A linear phase followed for the next 20 min and reached a plateau between 40 and 60 min (Fig. 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 (Fig. 4 C). The optimal efficiency occurred between 20 and 30°C and sharply tapered off at temperatures above 30°C (Fig. 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.


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

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

Characteristics of the cell-free system. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared and incubated (as described in Fig. 3) with modifications in reaction volume, time, or temperature. (A) Dilution sensitivity. All reactions were performed with a constant amount of donor and acceptor membranes while the reaction volume was increased (50–1,000 μl), as indicated. The concentration of ATP (plus regeneration components) and cytosol was equivalent in all reactions. (B) Maturation kinetics. A standard 50-μl reaction (see Fig. 3) was scaled up fivefold, incubated at 25°C, and aliquots were removed at the indicated times. (C) Temperature dependence. Standard 50-μl reactions were incubated at the indicated temperatures. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199724&req=5

Figure 4: Characteristics of the cell-free system. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared and incubated (as described in Fig. 3) with modifications in reaction volume, time, or temperature. (A) Dilution sensitivity. All reactions were performed with a constant amount of donor and acceptor membranes while the reaction volume was increased (50–1,000 μl), as indicated. The concentration of ATP (plus regeneration components) and cytosol was equivalent in all reactions. (B) Maturation kinetics. A standard 50-μl reaction (see Fig. 3) was scaled up fivefold, incubated at 25°C, and aliquots were removed at the indicated times. (C) Temperature dependence. Standard 50-μl reactions were incubated at the indicated temperatures. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.
Mentions: 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 (Fig. 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 15–20 min (Fig. 4 B). A linear phase followed for the next 20 min and reached a plateau between 40 and 60 min (Fig. 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 (Fig. 4 C). The optimal efficiency occurred between 20 and 30°C and sharply tapered off at temperatures above 30°C (Fig. 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.

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