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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 cell-free assay reconstitutes intercompartmental transport between donor and acceptor membranes. (A) Strategy for using yeast strains deleted for processing protease genes to test for intercompartmental transport in vitro (see Results for details). (B) Processing of CPY in intact spheroplasts from wild-type strains and strains deleted for the proteinase A gene (pep4Δ). Yeast spheroplasts with the indicated genotypes were radiolabeled for 5 min with Tran35S-label (0 min) and chased with unlabeled methionine and cysteine for 60 min. After each time point, the cells were lysed, immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. (C) Cell-free assays with donor and acceptor membranes from wild-type strains and strains deleted for the proteinase A gene (pep4Δ). Donor membranes from radiolabeled, and acceptor membranes from nonradiolabeled yeast spheroplasts were prepared as previously described (Fig. 3) from strains with the indicated genotypes. Standard reaction conditions (Fig. 3) were used to incubate all combinations of donor and acceptor membranes (with and without ATP and cytosol), as indicated. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.
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Figure 5: The cell-free assay reconstitutes intercompartmental transport between donor and acceptor membranes. (A) Strategy for using yeast strains deleted for processing protease genes to test for intercompartmental transport in vitro (see Results for details). (B) Processing of CPY in intact spheroplasts from wild-type strains and strains deleted for the proteinase A gene (pep4Δ). Yeast spheroplasts with the indicated genotypes were radiolabeled for 5 min with Tran35S-label (0 min) and chased with unlabeled methionine and cysteine for 60 min. After each time point, the cells were lysed, immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. (C) Cell-free assays with donor and acceptor membranes from wild-type strains and strains deleted for the proteinase A gene (pep4Δ). Donor membranes from radiolabeled, and acceptor membranes from nonradiolabeled yeast spheroplasts were prepared as previously described (Fig. 3) from strains with the indicated genotypes. Standard reaction conditions (Fig. 3) were used to incubate all combinations of donor and acceptor membranes (with and without ATP and cytosol), as indicated. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.

Mentions: We took advantage of CPY processing characteristics in vivo (Fig. 5 A) using PEP4 and pep4Δ yeast strains as a source of both donor and acceptor membranes in vitro. To confirm the genotype of the strains, we performed pulse–chase analysis and compared CPY processing. Both PEP4 and pep4Δ strains showed no significant differences for p1 and p2CPY after a 5-min pulse (Fig. 5 B, lanes 1 and 3). However, after a 60-min chase the fate of the p1 and p2CPY precursors was different. The PEP4 strain produced mCPY and the pep4Δ strain did not produce any mCPY but the p2CPY precursor accumulated (Fig. 5 B, lanes 2 and 4). With these phenotypes established, we prepared radiolabeled P3 donor membranes and unlabeled P2 acceptor membranes from the wild-type PEP4 and the pep4Δ mutant strains. These membranes were then mixed and incubated for cell-free assays in all combinations. Importantly, the radiolabeled reaction product took on the processing phenotype of the unlabeled acceptor membranes, not the radiolabeled donor membranes. For instance, PEP4 acceptor membranes gave rise to mCPY even from pep4Δ donor membranes (Fig. 5 C; lanes 5 and 6). Acceptor membranes from the pep4Δ strain did not produce detectable p2CPY maturation (Fig. 5 C, lanes 7 and 8). The small amount of mCPY (∼9%) that occurred from mixing PEP4 donor membranes with pep4Δ acceptor membranes (Fig. 5 C, lane 7) was present in the reaction where no acceptor membranes were added back (Fig. 5 C, lane 3). This indicated that a trace amount of vacuoles contaminated the PEP4 donor membranes in this experiment. These reactions with donor and acceptor membranes from a strain deleted for the principal processing protease gene provided the strongest evidence that our new cell-free assay was indeed intercompartmental. This reconstitution was likely an intercompartmental transport process between the PVC and the vacuole.


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

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

The cell-free assay reconstitutes intercompartmental transport between donor and acceptor membranes. (A) Strategy for using yeast strains deleted for processing protease genes to test for intercompartmental transport in vitro (see Results for details). (B) Processing of CPY in intact spheroplasts from wild-type strains and strains deleted for the proteinase A gene (pep4Δ). Yeast spheroplasts with the indicated genotypes were radiolabeled for 5 min with Tran35S-label (0 min) and chased with unlabeled methionine and cysteine for 60 min. After each time point, the cells were lysed, immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. (C) Cell-free assays with donor and acceptor membranes from wild-type strains and strains deleted for the proteinase A gene (pep4Δ). Donor membranes from radiolabeled, and acceptor membranes from nonradiolabeled yeast spheroplasts were prepared as previously described (Fig. 3) from strains with the indicated genotypes. Standard reaction conditions (Fig. 3) were used to incubate all combinations of donor and acceptor membranes (with and without ATP and cytosol), as indicated. 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 5: The cell-free assay reconstitutes intercompartmental transport between donor and acceptor membranes. (A) Strategy for using yeast strains deleted for processing protease genes to test for intercompartmental transport in vitro (see Results for details). (B) Processing of CPY in intact spheroplasts from wild-type strains and strains deleted for the proteinase A gene (pep4Δ). Yeast spheroplasts with the indicated genotypes were radiolabeled for 5 min with Tran35S-label (0 min) and chased with unlabeled methionine and cysteine for 60 min. After each time point, the cells were lysed, immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. (C) Cell-free assays with donor and acceptor membranes from wild-type strains and strains deleted for the proteinase A gene (pep4Δ). Donor membranes from radiolabeled, and acceptor membranes from nonradiolabeled yeast spheroplasts were prepared as previously described (Fig. 3) from strains with the indicated genotypes. Standard reaction conditions (Fig. 3) were used to incubate all combinations of donor and acceptor membranes (with and without ATP and cytosol), as indicated. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.
Mentions: We took advantage of CPY processing characteristics in vivo (Fig. 5 A) using PEP4 and pep4Δ yeast strains as a source of both donor and acceptor membranes in vitro. To confirm the genotype of the strains, we performed pulse–chase analysis and compared CPY processing. Both PEP4 and pep4Δ strains showed no significant differences for p1 and p2CPY after a 5-min pulse (Fig. 5 B, lanes 1 and 3). However, after a 60-min chase the fate of the p1 and p2CPY precursors was different. The PEP4 strain produced mCPY and the pep4Δ strain did not produce any mCPY but the p2CPY precursor accumulated (Fig. 5 B, lanes 2 and 4). With these phenotypes established, we prepared radiolabeled P3 donor membranes and unlabeled P2 acceptor membranes from the wild-type PEP4 and the pep4Δ mutant strains. These membranes were then mixed and incubated for cell-free assays in all combinations. Importantly, the radiolabeled reaction product took on the processing phenotype of the unlabeled acceptor membranes, not the radiolabeled donor membranes. For instance, PEP4 acceptor membranes gave rise to mCPY even from pep4Δ donor membranes (Fig. 5 C; lanes 5 and 6). Acceptor membranes from the pep4Δ strain did not produce detectable p2CPY maturation (Fig. 5 C, lanes 7 and 8). The small amount of mCPY (∼9%) that occurred from mixing PEP4 donor membranes with pep4Δ acceptor membranes (Fig. 5 C, lane 7) was present in the reaction where no acceptor membranes were added back (Fig. 5 C, lane 3). This indicated that a trace amount of vacuoles contaminated the PEP4 donor membranes in this experiment. These reactions with donor and acceptor membranes from a strain deleted for the principal processing protease gene provided the strongest evidence that our new cell-free assay was indeed intercompartmental. This reconstitution was likely an intercompartmental transport process between the PVC and the 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