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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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Cytosolic extracts from a vps33  strain are deficient in stimulating intercompartmental transport in the cell-free system. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared from wild-type yeast spheroplasts (SEY6210). Standard reaction conditions (Fig. 3) were used to incubate the donor and acceptor membranes with ATP (plus regeneration components) and cytosol (5 mg/ml) from the VPS33 or vps33Δ strains, as indicated. The reactions in lanes 4–6 contained donor and acceptor membranes that had been washed once with lysis buffer (similar to Fig. 3, lanes 10 and 11) while the reactions in lanes 1–3 contained unwashed membranes. The bar graph depicts the average transport efficiency from three independent determinations and is normalized to the percent of maximal transport.
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Figure 6: Cytosolic extracts from a vps33 strain are deficient in stimulating intercompartmental transport in the cell-free system. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared from wild-type yeast spheroplasts (SEY6210). Standard reaction conditions (Fig. 3) were used to incubate the donor and acceptor membranes with ATP (plus regeneration components) and cytosol (5 mg/ml) from the VPS33 or vps33Δ strains, as indicated. The reactions in lanes 4–6 contained donor and acceptor membranes that had been washed once with lysis buffer (similar to Fig. 3, lanes 10 and 11) while the reactions in lanes 1–3 contained unwashed membranes. The bar graph depicts the average transport efficiency from three independent determinations and is normalized to the percent of maximal transport.

Mentions: One difficulty in reconstituting an intercompartmental transport event in our previous permeabilized cell assay was incomplete removal of many cytoplasmic VPS gene products such as Vps33p (Vida et al. 1990). For example, no transport defect has been observed when a cytosolic extract devoid of Vps33p from a vps33 strain (vps33Δ) was added back to wild-type permeabilized cells (data not shown). However, a significant defect was observed in vps33Δ cytosol when it was added back to the cell-free transport assay. The transport efficiency was decreased ∼2.5-fold compared with cytosol made from a wild-type VPS33 strain (Fig. 6, lanes 2 and 3, 5 and 6). Although the standard concentration of cytosol in our cell-free reactions was 5 mg/ml, these experiments also demonstrated that overall transport efficiency was remarkably consistent with the concentration of protein in crude, undiluted wild-type cytosol. For example, using extracts with a protein concentration of 50 mg/ml produced an average transport efficiency of 47.0% ± 1.3% . We observed an average transport efficiency of 32.6% ± 2.5% with an undiluted cytosolic protein concentration of 35 mg/ml. The 30% decrease in transport efficiency correlated well with the 30% decrease in protein concentration, which suggested that the level of a soluble protein factor(s) was critical for driving intercompartmental transport.


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

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

Cytosolic extracts from a vps33  strain are deficient in stimulating intercompartmental transport in the cell-free system. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared from wild-type yeast spheroplasts (SEY6210). Standard reaction conditions (Fig. 3) were used to incubate the donor and acceptor membranes with ATP (plus regeneration components) and cytosol (5 mg/ml) from the VPS33 or vps33Δ strains, as indicated. The reactions in lanes 4–6 contained donor and acceptor membranes that had been washed once with lysis buffer (similar to Fig. 3, lanes 10 and 11) while the reactions in lanes 1–3 contained unwashed membranes. The bar graph depicts the average transport efficiency from three independent determinations and is normalized to the percent of maximal transport.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199724&req=5

Figure 6: Cytosolic extracts from a vps33 strain are deficient in stimulating intercompartmental transport in the cell-free system. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were prepared from wild-type yeast spheroplasts (SEY6210). Standard reaction conditions (Fig. 3) were used to incubate the donor and acceptor membranes with ATP (plus regeneration components) and cytosol (5 mg/ml) from the VPS33 or vps33Δ strains, as indicated. The reactions in lanes 4–6 contained donor and acceptor membranes that had been washed once with lysis buffer (similar to Fig. 3, lanes 10 and 11) while the reactions in lanes 1–3 contained unwashed membranes. The bar graph depicts the average transport efficiency from three independent determinations and is normalized to the percent of maximal transport.
Mentions: One difficulty in reconstituting an intercompartmental transport event in our previous permeabilized cell assay was incomplete removal of many cytoplasmic VPS gene products such as Vps33p (Vida et al. 1990). For example, no transport defect has been observed when a cytosolic extract devoid of Vps33p from a vps33 strain (vps33Δ) was added back to wild-type permeabilized cells (data not shown). However, a significant defect was observed in vps33Δ cytosol when it was added back to the cell-free transport assay. The transport efficiency was decreased ∼2.5-fold compared with cytosol made from a wild-type VPS33 strain (Fig. 6, lanes 2 and 3, 5 and 6). Although the standard concentration of cytosol in our cell-free reactions was 5 mg/ml, these experiments also demonstrated that overall transport efficiency was remarkably consistent with the concentration of protein in crude, undiluted wild-type cytosol. For example, using extracts with a protein concentration of 50 mg/ml produced an average transport efficiency of 47.0% ± 1.3% . We observed an average transport efficiency of 32.6% ± 2.5% with an undiluted cytosolic protein concentration of 35 mg/ml. The 30% decrease in transport efficiency correlated well with the 30% decrease in protein concentration, which suggested that the level of a soluble protein factor(s) was critical for driving intercompartmental transport.

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