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Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion.

Wang L, Merz AJ, Collins KM, Wickner W - J. Cell Biol. (2003)

Bottom Line: The v-SNARE Vti1p is enriched at vertices by a distinct pathway that is independent of the t-SNAREs, whereas both t-SNAREs will localize to vertices when trans-pairing of SNAREs is blocked.Thus, trans-SNARE pairing is not required for SNARE vertex enrichment; and (d) The t-SNAREs regulate the vertex enrichment of both G-actin and the Ypt7p effector complex for homotypic fusion and vacuole protein sorting (HOPS).In accord with this hierarchy concept, the HOPS complex, at the end of the vertex assembly hierarchy, is most enriched at those vertices with abundant Ypt7p, which is at the start of the hierarchy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

ABSTRACT
Vacuole tethering, docking, and fusion proteins assemble into a "vertex ring" around the apposed membranes of tethered vacuoles before catalyzing fusion. Inhibitors of the fusion reaction selectively interrupt protein assembly into the vertex ring, establishing a causal assembly hierarchy: (a) The Rab GTPase Ypt7p mediates vacuole tethering and forms the initial vertex ring, independent of t-SNAREs or actin; (b) F-actin disassembly and GTP-bound Ypt7p direct the localization of other fusion factors; (c) The t-SNAREs Vam3p and Vam7p regulate each other's vertex enrichment, but do not affect Ypt7p localization. The v-SNARE Vti1p is enriched at vertices by a distinct pathway that is independent of the t-SNAREs, whereas both t-SNAREs will localize to vertices when trans-pairing of SNAREs is blocked. Thus, trans-SNARE pairing is not required for SNARE vertex enrichment; and (d) The t-SNAREs regulate the vertex enrichment of both G-actin and the Ypt7p effector complex for homotypic fusion and vacuole protein sorting (HOPS). In accord with this hierarchy concept, the HOPS complex, at the end of the vertex assembly hierarchy, is most enriched at those vertices with abundant Ypt7p, which is at the start of the hierarchy. Our findings provide a unique view of the functional relationships between GTPases, SNAREs, and actin in membrane fusion.

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Protein enrichment at vertices of tethered vacuoles. Reaction inhibitors, including 50 ng/μl excess Sec17p, 60 ng/μl anti-Vam3 Fab, 600 ng/μl recombinant Gyp1p, 500 μM jasplakinolide, 15 μM PX domain, 500 μM latrunculin B, and 5 μM full-length Vam7p were added to docking assays (see Materials and methods) with GFP-tagged proteins as indicated. After incubation, vacuoles were observed by fluorescence microscopy and images were acquired in GFP and rhodamine channels. Vacuole clusters from random fields were analyzed and ratio values of GFP:FM4–64 were generated from outside edge membranes (black) and vertex membranes (red). An average of 200 normalized ratio values for each treatment were plotted against their percentile (cumulative distribution plot).
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fig4: Protein enrichment at vertices of tethered vacuoles. Reaction inhibitors, including 50 ng/μl excess Sec17p, 60 ng/μl anti-Vam3 Fab, 600 ng/μl recombinant Gyp1p, 500 μM jasplakinolide, 15 μM PX domain, 500 μM latrunculin B, and 5 μM full-length Vam7p were added to docking assays (see Materials and methods) with GFP-tagged proteins as indicated. After incubation, vacuoles were observed by fluorescence microscopy and images were acquired in GFP and rhodamine channels. Vacuole clusters from random fields were analyzed and ratio values of GFP:FM4–64 were generated from outside edge membranes (black) and vertex membranes (red). An average of 200 normalized ratio values for each treatment were plotted against their percentile (cumulative distribution plot).

Mentions: Using ratiometric imaging, we analyzed the spatial enrichment of GFP-tagged proteins on vacuoles that were labeled with the fluorescent lipophilic dye FM4–64 (Wang et al., 2002). For example, the ratio of GFP-tagged Vam7p to FM4–64 (Fig. 1 B) showed enrichment of this protein at vertices relative to outside edges. After measuring clusters from random fields, we generated ∼200 ratio values at outside edges (black) and vertices (red), normalized them to outside edge values, and plotted them against their percentile to form a cumulative distribution plot, or CD plot (Fig. 4). Statistical analysis (Fig. 5) shows the extent and significance of spatial enrichment and its modulation by these ligands. The protein enrichments observed in this work and in our recent papers (Eitzen et al., 2002; Wang et al., 2002) are similar in magnitude to values previously obtained in other works. For example, Roberts et al. (1999) studied the localization of GFP-labeled, overexpressed Rab5, and Lang et al. (2001) examined the localization of syntaxin using GFP-fusions. In these works, the mean or median enrichments were approximately twofold. In each case, the structures studied are likely of submicron size, i.e., near or below the optical resolution limits imposed by diffraction. For this reason, the measured enrichments are expected to substantially underestimate the actual values. Like the other fusions used in this work (Wang et al., 2002), the GFP-tagged versions of Vam3p, Vti1p, Nyv1p, and Vam7p were expressed as gene replacements under their native promoters and were functional in vivo, as assayed by vacuole morphology phenotype, and in vitro, as shown by the homotypic vacuole fusion assay. Tagging the NH2 or COOH terminus of Ykt6p was not compatible with viability in our strain.


Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion.

Wang L, Merz AJ, Collins KM, Wickner W - J. Cell Biol. (2003)

Protein enrichment at vertices of tethered vacuoles. Reaction inhibitors, including 50 ng/μl excess Sec17p, 60 ng/μl anti-Vam3 Fab, 600 ng/μl recombinant Gyp1p, 500 μM jasplakinolide, 15 μM PX domain, 500 μM latrunculin B, and 5 μM full-length Vam7p were added to docking assays (see Materials and methods) with GFP-tagged proteins as indicated. After incubation, vacuoles were observed by fluorescence microscopy and images were acquired in GFP and rhodamine channels. Vacuole clusters from random fields were analyzed and ratio values of GFP:FM4–64 were generated from outside edge membranes (black) and vertex membranes (red). An average of 200 normalized ratio values for each treatment were plotted against their percentile (cumulative distribution plot).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172665&req=5

fig4: Protein enrichment at vertices of tethered vacuoles. Reaction inhibitors, including 50 ng/μl excess Sec17p, 60 ng/μl anti-Vam3 Fab, 600 ng/μl recombinant Gyp1p, 500 μM jasplakinolide, 15 μM PX domain, 500 μM latrunculin B, and 5 μM full-length Vam7p were added to docking assays (see Materials and methods) with GFP-tagged proteins as indicated. After incubation, vacuoles were observed by fluorescence microscopy and images were acquired in GFP and rhodamine channels. Vacuole clusters from random fields were analyzed and ratio values of GFP:FM4–64 were generated from outside edge membranes (black) and vertex membranes (red). An average of 200 normalized ratio values for each treatment were plotted against their percentile (cumulative distribution plot).
Mentions: Using ratiometric imaging, we analyzed the spatial enrichment of GFP-tagged proteins on vacuoles that were labeled with the fluorescent lipophilic dye FM4–64 (Wang et al., 2002). For example, the ratio of GFP-tagged Vam7p to FM4–64 (Fig. 1 B) showed enrichment of this protein at vertices relative to outside edges. After measuring clusters from random fields, we generated ∼200 ratio values at outside edges (black) and vertices (red), normalized them to outside edge values, and plotted them against their percentile to form a cumulative distribution plot, or CD plot (Fig. 4). Statistical analysis (Fig. 5) shows the extent and significance of spatial enrichment and its modulation by these ligands. The protein enrichments observed in this work and in our recent papers (Eitzen et al., 2002; Wang et al., 2002) are similar in magnitude to values previously obtained in other works. For example, Roberts et al. (1999) studied the localization of GFP-labeled, overexpressed Rab5, and Lang et al. (2001) examined the localization of syntaxin using GFP-fusions. In these works, the mean or median enrichments were approximately twofold. In each case, the structures studied are likely of submicron size, i.e., near or below the optical resolution limits imposed by diffraction. For this reason, the measured enrichments are expected to substantially underestimate the actual values. Like the other fusions used in this work (Wang et al., 2002), the GFP-tagged versions of Vam3p, Vti1p, Nyv1p, and Vam7p were expressed as gene replacements under their native promoters and were functional in vivo, as assayed by vacuole morphology phenotype, and in vitro, as shown by the homotypic vacuole fusion assay. Tagging the NH2 or COOH terminus of Ykt6p was not compatible with viability in our strain.

Bottom Line: The v-SNARE Vti1p is enriched at vertices by a distinct pathway that is independent of the t-SNAREs, whereas both t-SNAREs will localize to vertices when trans-pairing of SNAREs is blocked.Thus, trans-SNARE pairing is not required for SNARE vertex enrichment; and (d) The t-SNAREs regulate the vertex enrichment of both G-actin and the Ypt7p effector complex for homotypic fusion and vacuole protein sorting (HOPS).In accord with this hierarchy concept, the HOPS complex, at the end of the vertex assembly hierarchy, is most enriched at those vertices with abundant Ypt7p, which is at the start of the hierarchy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

ABSTRACT
Vacuole tethering, docking, and fusion proteins assemble into a "vertex ring" around the apposed membranes of tethered vacuoles before catalyzing fusion. Inhibitors of the fusion reaction selectively interrupt protein assembly into the vertex ring, establishing a causal assembly hierarchy: (a) The Rab GTPase Ypt7p mediates vacuole tethering and forms the initial vertex ring, independent of t-SNAREs or actin; (b) F-actin disassembly and GTP-bound Ypt7p direct the localization of other fusion factors; (c) The t-SNAREs Vam3p and Vam7p regulate each other's vertex enrichment, but do not affect Ypt7p localization. The v-SNARE Vti1p is enriched at vertices by a distinct pathway that is independent of the t-SNAREs, whereas both t-SNAREs will localize to vertices when trans-pairing of SNAREs is blocked. Thus, trans-SNARE pairing is not required for SNARE vertex enrichment; and (d) The t-SNAREs regulate the vertex enrichment of both G-actin and the Ypt7p effector complex for homotypic fusion and vacuole protein sorting (HOPS). In accord with this hierarchy concept, the HOPS complex, at the end of the vertex assembly hierarchy, is most enriched at those vertices with abundant Ypt7p, which is at the start of the hierarchy. Our findings provide a unique view of the functional relationships between GTPases, SNAREs, and actin in membrane fusion.

Show MeSH
Related in: MedlinePlus