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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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Vacuolar precursor proteins undergo maturation after incubating donor and acceptor membranes in a cell-free system. Wild-type yeast spheroplasts were radiolabeled (as in Fig. 1 B). The cells were subjected to lysis through a polycarbonate filter with subsequent differential centrifugation to generate a 125,000 g, P3 donor membrane pellet. The same yeast strain was used to make a 15,000 g, P2 acceptor membrane pellet from nonradiolabeled spheroplasts. The radiolabeled donor membranes (from ∼5 × 107 spheroplasts per reaction) were incubated at 25°C for 60 min with various combinations of buffer, ATP (plus a regeneration system), cytosol (5 mg/ml), and acceptor membranes (∼100 μg) in a total volume of 50 μl, as indicated. All reactions were sequentially immunoprecipitated for CPY and PrA, subjected to SDS-PAGE, and autoradiography. For the reactions in lanes 10 and 11, both donor and acceptor membranes were washed once with lysis buffer and reharvested before incubation with ATP and cytosol.
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Figure 3: Vacuolar precursor proteins undergo maturation after incubating donor and acceptor membranes in a cell-free system. Wild-type yeast spheroplasts were radiolabeled (as in Fig. 1 B). The cells were subjected to lysis through a polycarbonate filter with subsequent differential centrifugation to generate a 125,000 g, P3 donor membrane pellet. The same yeast strain was used to make a 15,000 g, P2 acceptor membrane pellet from nonradiolabeled spheroplasts. The radiolabeled donor membranes (from ∼5 × 107 spheroplasts per reaction) were incubated at 25°C for 60 min with various combinations of buffer, ATP (plus a regeneration system), cytosol (5 mg/ml), and acceptor membranes (∼100 μg) in a total volume of 50 μl, as indicated. All reactions were sequentially immunoprecipitated for CPY and PrA, subjected to SDS-PAGE, and autoradiography. For the reactions in lanes 10 and 11, both donor and acceptor membranes were washed once with lysis buffer and reharvested before incubation with ATP and cytosol.

Mentions: 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 (Fig. 3, lanes 1–5). 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 (Fig. 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 (Fig. 3, lane 9). The cytosolic extract stimulated their maturation just over twofold compared with incubating the donor and acceptor membranes with ATP alone (Fig. 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. (Fig. 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.


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

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

Vacuolar precursor proteins undergo maturation after incubating donor and acceptor membranes in a cell-free system. Wild-type yeast spheroplasts were radiolabeled (as in Fig. 1 B). The cells were subjected to lysis through a polycarbonate filter with subsequent differential centrifugation to generate a 125,000 g, P3 donor membrane pellet. The same yeast strain was used to make a 15,000 g, P2 acceptor membrane pellet from nonradiolabeled spheroplasts. The radiolabeled donor membranes (from ∼5 × 107 spheroplasts per reaction) were incubated at 25°C for 60 min with various combinations of buffer, ATP (plus a regeneration system), cytosol (5 mg/ml), and acceptor membranes (∼100 μg) in a total volume of 50 μl, as indicated. All reactions were sequentially immunoprecipitated for CPY and PrA, subjected to SDS-PAGE, and autoradiography. For the reactions in lanes 10 and 11, both donor and acceptor membranes were washed once with lysis buffer and reharvested before incubation with ATP and cytosol.
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Related In: Results  -  Collection

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Figure 3: Vacuolar precursor proteins undergo maturation after incubating donor and acceptor membranes in a cell-free system. Wild-type yeast spheroplasts were radiolabeled (as in Fig. 1 B). The cells were subjected to lysis through a polycarbonate filter with subsequent differential centrifugation to generate a 125,000 g, P3 donor membrane pellet. The same yeast strain was used to make a 15,000 g, P2 acceptor membrane pellet from nonradiolabeled spheroplasts. The radiolabeled donor membranes (from ∼5 × 107 spheroplasts per reaction) were incubated at 25°C for 60 min with various combinations of buffer, ATP (plus a regeneration system), cytosol (5 mg/ml), and acceptor membranes (∼100 μg) in a total volume of 50 μl, as indicated. All reactions were sequentially immunoprecipitated for CPY and PrA, subjected to SDS-PAGE, and autoradiography. For the reactions in lanes 10 and 11, both donor and acceptor membranes were washed once with lysis buffer and reharvested before incubation with ATP and cytosol.
Mentions: 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 (Fig. 3, lanes 1–5). 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 (Fig. 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 (Fig. 3, lane 9). The cytosolic extract stimulated their maturation just over twofold compared with incubating the donor and acceptor membranes with ATP alone (Fig. 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. (Fig. 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.

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