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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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Lysis of yeast spheroplasts via extrusion through a polycarbonate filter. (A) Schematic diagram for polycarbonate filter lysis and subsequent differential centrifugation. After pushing a yeast spheroplast suspension in a syringe through a polycarbonate filter with 3-μm pores, the crude cell lysate was subjected to differential centrifugation using the indicated g forces and times. The P1, P2, and P3 pellets were enriched in the indicated organelles. (B) Fractionation of vacuolar marker proteins. Wild-type yeast spheroplasts (SEY6210) were radiolabeled with Tran35S-label for 5 min and chased with methionine and cysteine for 2 min at 30°C. The cells were subjected to lysis through a polycarbonate filter with subsequent differential centrifugation. Each supernatant and pellet from the 440 (S1 and P1), 15,000 (S2 and P2), and 125,000 g (S3 and P3) centrifugation steps was sequentially immunoprecipitated with antisera against carboxypeptidase Y (CPY), proteinase A (PrA), and alkaline phosphatase (ALP). Each immunoprecipitate was subjected to SDS-PAGE and autoradiography. The mid and late Golgi complex–modified precursor zymogen is designated p2, the endoplasmic reticulum and early Golgi complex precursor is designated p1 (CPY only), and the mature hydrolase is designated m for each protein.
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Figure 1: Lysis of yeast spheroplasts via extrusion through a polycarbonate filter. (A) Schematic diagram for polycarbonate filter lysis and subsequent differential centrifugation. After pushing a yeast spheroplast suspension in a syringe through a polycarbonate filter with 3-μm pores, the crude cell lysate was subjected to differential centrifugation using the indicated g forces and times. The P1, P2, and P3 pellets were enriched in the indicated organelles. (B) Fractionation of vacuolar marker proteins. Wild-type yeast spheroplasts (SEY6210) were radiolabeled with Tran35S-label for 5 min and chased with methionine and cysteine for 2 min at 30°C. The cells were subjected to lysis through a polycarbonate filter with subsequent differential centrifugation. Each supernatant and pellet from the 440 (S1 and P1), 15,000 (S2 and P2), and 125,000 g (S3 and P3) centrifugation steps was sequentially immunoprecipitated with antisera against carboxypeptidase Y (CPY), proteinase A (PrA), and alkaline phosphatase (ALP). Each immunoprecipitate was subjected to SDS-PAGE and autoradiography. The mid and late Golgi complex–modified precursor zymogen is designated p2, the endoplasmic reticulum and early Golgi complex precursor is designated p1 (CPY only), and the mature hydrolase is designated m for each protein.

Mentions: The technique of passing cells through a small orifice to generate a crude lysate from shear forces was first used for mammalian cells. For example, cell homogenates have been prepared from PC12 cells by passing cell suspensions 15 times through a narrow clearance (10 μm) stainless steel ball homogenizer (Martin and Kowalchyk 1997). Stainless steel ball homogenizers have been instrumental at reconstituting several steps in the secretory pathway such as fusion of secretory vesicles with the plasma membrane and ER to Golgi transport (Balch and Rothman 1985). Rather than use a steel ball homogenizer, we used polycarbonate filters to shear yeast spheroplasts. Intact spheroplasts were suspended with an osmotic support of 0.6 M sorbitol giving them a diameter in the range of 5–8 μm. The cells were then forced through a 3-μm polycarbonate filter from a syringe (Fig. 1 A). Typically, in a single pass through the filter, >98% of the cells lysed to generate a crude homogenate.


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

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

Lysis of yeast spheroplasts via extrusion through a polycarbonate filter. (A) Schematic diagram for polycarbonate filter lysis and subsequent differential centrifugation. After pushing a yeast spheroplast suspension in a syringe through a polycarbonate filter with 3-μm pores, the crude cell lysate was subjected to differential centrifugation using the indicated g forces and times. The P1, P2, and P3 pellets were enriched in the indicated organelles. (B) Fractionation of vacuolar marker proteins. Wild-type yeast spheroplasts (SEY6210) were radiolabeled with Tran35S-label for 5 min and chased with methionine and cysteine for 2 min at 30°C. The cells were subjected to lysis through a polycarbonate filter with subsequent differential centrifugation. Each supernatant and pellet from the 440 (S1 and P1), 15,000 (S2 and P2), and 125,000 g (S3 and P3) centrifugation steps was sequentially immunoprecipitated with antisera against carboxypeptidase Y (CPY), proteinase A (PrA), and alkaline phosphatase (ALP). Each immunoprecipitate was subjected to SDS-PAGE and autoradiography. The mid and late Golgi complex–modified precursor zymogen is designated p2, the endoplasmic reticulum and early Golgi complex precursor is designated p1 (CPY only), and the mature hydrolase is designated m for each protein.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199724&req=5

Figure 1: Lysis of yeast spheroplasts via extrusion through a polycarbonate filter. (A) Schematic diagram for polycarbonate filter lysis and subsequent differential centrifugation. After pushing a yeast spheroplast suspension in a syringe through a polycarbonate filter with 3-μm pores, the crude cell lysate was subjected to differential centrifugation using the indicated g forces and times. The P1, P2, and P3 pellets were enriched in the indicated organelles. (B) Fractionation of vacuolar marker proteins. Wild-type yeast spheroplasts (SEY6210) were radiolabeled with Tran35S-label for 5 min and chased with methionine and cysteine for 2 min at 30°C. The cells were subjected to lysis through a polycarbonate filter with subsequent differential centrifugation. Each supernatant and pellet from the 440 (S1 and P1), 15,000 (S2 and P2), and 125,000 g (S3 and P3) centrifugation steps was sequentially immunoprecipitated with antisera against carboxypeptidase Y (CPY), proteinase A (PrA), and alkaline phosphatase (ALP). Each immunoprecipitate was subjected to SDS-PAGE and autoradiography. The mid and late Golgi complex–modified precursor zymogen is designated p2, the endoplasmic reticulum and early Golgi complex precursor is designated p1 (CPY only), and the mature hydrolase is designated m for each protein.
Mentions: The technique of passing cells through a small orifice to generate a crude lysate from shear forces was first used for mammalian cells. For example, cell homogenates have been prepared from PC12 cells by passing cell suspensions 15 times through a narrow clearance (10 μm) stainless steel ball homogenizer (Martin and Kowalchyk 1997). Stainless steel ball homogenizers have been instrumental at reconstituting several steps in the secretory pathway such as fusion of secretory vesicles with the plasma membrane and ER to Golgi transport (Balch and Rothman 1985). Rather than use a steel ball homogenizer, we used polycarbonate filters to shear yeast spheroplasts. Intact spheroplasts were suspended with an osmotic support of 0.6 M sorbitol giving them a diameter in the range of 5–8 μm. The cells were then forced through a 3-μm polycarbonate filter from a syringe (Fig. 1 A). Typically, in a single pass through the filter, >98% of the cells lysed to generate a crude homogenate.

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