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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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Light microscopy of cell-free membrane pellets after polycarbonate filter lysis. Wild-type yeast cells (as in Fig. 1) were first stained with FM4-64 (15-min pulse, 45-min chase) followed with dichlorocarboxyfluorescein diacetate (15 min at pH 4.0) to mark the vacuole membrane and lumen, respectively. The double-stained cells were then enzymatically converted to spheroplasts at 30 or 15°C (as indicated). The spheroplasts were extruded through a polycarbonate filter with 3-μm pores (as described in Fig. 1 A). The lysate, P1, P2, and P3 pellets (as indicated) were then examined under a light microscope with differential interference contrast (DIC), phase contrast, and epifluorescence optics using a FITC and Texas red filter set (as indicated). The fluorescence images were digitally overlaid for a composite. The cells at 15°C (inset) were stained with just FM4-64 for a 30-min pulse. Bar, 5 μm.
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Figure 2: Light microscopy of cell-free membrane pellets after polycarbonate filter lysis. Wild-type yeast cells (as in Fig. 1) were first stained with FM4-64 (15-min pulse, 45-min chase) followed with dichlorocarboxyfluorescein diacetate (15 min at pH 4.0) to mark the vacuole membrane and lumen, respectively. The double-stained cells were then enzymatically converted to spheroplasts at 30 or 15°C (as indicated). The spheroplasts were extruded through a polycarbonate filter with 3-μm pores (as described in Fig. 1 A). The lysate, P1, P2, and P3 pellets (as indicated) were then examined under a light microscope with differential interference contrast (DIC), phase contrast, and epifluorescence optics using a FITC and Texas red filter set (as indicated). The fluorescence images were digitally overlaid for a composite. The cells at 15°C (inset) were stained with just FM4-64 for a 30-min pulse. Bar, 5 μm.

Mentions: Various steps from the filter lysis procedure were also examined with microscopy. To follow the vacuole, we prestained yeast cells with FM4-64 and CDCFDA. As expected, the P1 pellet was devoid of unbroken cells and was enriched in cell wall remnants (Fig. 2). As expected from the marker protein analysis, the P2 pellet was enriched in intact vacuoles, since many FM4-64–stained membranes containing CDCFDA were observed (Fig. 2). In contrast, the 125,000-g P3 pellet was devoid of vacuoles and instead was enriched for very small particulate structures. Importantly, if cells were stained with FM4-64 at 15°C, many of the small particulate structures in the 125,000-g pellet exhibited fluorescence (Fig. 2, inset). Additionally, membrane fluorescence in the 15,000-g P2 pellet was markedly reduced at 15°C (Fig. 2, inset). Since FM4-64 is kinetically trapped in prevacuolar compartments at 15°C (Vida et al. 1993), the membrane fluorescence in the 125,000-g pellet suggests that these differential centrifugation conditions separated vacuoles from prevacuolar compartments. Although not shown in this experiment, the P1 pellet also was enriched with intact nuclei after first staining yeast cells with DNA dyes (i.e., 4′,6-diamidino-2-phenylindole, dihydrochloride, DAPI). The P2 pellet was enriched for mitochondria after first staining cells with the mitochondrial vital dye 2,4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (DASPMI). We also examined the P2 and P3 pellets with electron microscopy. The P2 pellet comprised numerous electron-dense 1,000–1,500-nm membrane-delineated structures, which was consistent with the size expected for vacuoles (data not shown). In contrast, the P3 pellet was devoid of the relatively large, electron-dense membranes and instead was composed of 50–100 nm and 250–400 nm membrane-delineated structures (data not shown).


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

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

Light microscopy of cell-free membrane pellets after polycarbonate filter lysis. Wild-type yeast cells (as in Fig. 1) were first stained with FM4-64 (15-min pulse, 45-min chase) followed with dichlorocarboxyfluorescein diacetate (15 min at pH 4.0) to mark the vacuole membrane and lumen, respectively. The double-stained cells were then enzymatically converted to spheroplasts at 30 or 15°C (as indicated). The spheroplasts were extruded through a polycarbonate filter with 3-μm pores (as described in Fig. 1 A). The lysate, P1, P2, and P3 pellets (as indicated) were then examined under a light microscope with differential interference contrast (DIC), phase contrast, and epifluorescence optics using a FITC and Texas red filter set (as indicated). The fluorescence images were digitally overlaid for a composite. The cells at 15°C (inset) were stained with just FM4-64 for a 30-min pulse. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 2: Light microscopy of cell-free membrane pellets after polycarbonate filter lysis. Wild-type yeast cells (as in Fig. 1) were first stained with FM4-64 (15-min pulse, 45-min chase) followed with dichlorocarboxyfluorescein diacetate (15 min at pH 4.0) to mark the vacuole membrane and lumen, respectively. The double-stained cells were then enzymatically converted to spheroplasts at 30 or 15°C (as indicated). The spheroplasts were extruded through a polycarbonate filter with 3-μm pores (as described in Fig. 1 A). The lysate, P1, P2, and P3 pellets (as indicated) were then examined under a light microscope with differential interference contrast (DIC), phase contrast, and epifluorescence optics using a FITC and Texas red filter set (as indicated). The fluorescence images were digitally overlaid for a composite. The cells at 15°C (inset) were stained with just FM4-64 for a 30-min pulse. Bar, 5 μm.
Mentions: Various steps from the filter lysis procedure were also examined with microscopy. To follow the vacuole, we prestained yeast cells with FM4-64 and CDCFDA. As expected, the P1 pellet was devoid of unbroken cells and was enriched in cell wall remnants (Fig. 2). As expected from the marker protein analysis, the P2 pellet was enriched in intact vacuoles, since many FM4-64–stained membranes containing CDCFDA were observed (Fig. 2). In contrast, the 125,000-g P3 pellet was devoid of vacuoles and instead was enriched for very small particulate structures. Importantly, if cells were stained with FM4-64 at 15°C, many of the small particulate structures in the 125,000-g pellet exhibited fluorescence (Fig. 2, inset). Additionally, membrane fluorescence in the 15,000-g P2 pellet was markedly reduced at 15°C (Fig. 2, inset). Since FM4-64 is kinetically trapped in prevacuolar compartments at 15°C (Vida et al. 1993), the membrane fluorescence in the 125,000-g pellet suggests that these differential centrifugation conditions separated vacuoles from prevacuolar compartments. Although not shown in this experiment, the P1 pellet also was enriched with intact nuclei after first staining yeast cells with DNA dyes (i.e., 4′,6-diamidino-2-phenylindole, dihydrochloride, DAPI). The P2 pellet was enriched for mitochondria after first staining cells with the mitochondrial vital dye 2,4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (DASPMI). We also examined the P2 and P3 pellets with electron microscopy. The P2 pellet comprised numerous electron-dense 1,000–1,500-nm membrane-delineated structures, which was consistent with the size expected for vacuoles (data not shown). In contrast, the P3 pellet was devoid of the relatively large, electron-dense membranes and instead was composed of 50–100 nm and 250–400 nm membrane-delineated structures (data not shown).

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