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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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Deletion of the v-SNARE Nyv1p has no effect on the localization of other SNAREs. (A) GFP-Nyv1p vacuoles were subject to in vitro docking assay and ratiometric fluorescence microscopy analysis, as described in Materials and methods. The GFP:FM4–64 ratio values were generated for outside edge membranes (red curves) and vertex membranes (black curves), and plotted against their percentile. (B) Trans-pairing of SNAREs. Trans-SNARE pairing was assayed directly using purified vacuoles from four strains: (1) VTI1-GFP, NYV1, pep4–3; (2) HA3-VAM3, NYV1, pep4::HIS3; (3) VTI1-GFP, pep4::HIS3, nyv1::URA3; and (4) HA3-VAM3, pep4::HIS3, nyv1::URA3. After a 60-min fusion reaction, membranes were dissolved in Triton X-100, and Vti1-GFP was immunoprecipitated by immobilized antibody to GFP (unpublished data). Trans-associated HA-Vam3p was detected by immunoblot and quantified by densitometry as a percentage of the total HA-Vam3p in the sample. Recombinant Gyp1–46p (0.4 μg/ml) was added where indicated (lane 3). To determine the level of background association, detergent extracts of reactions with ATP and single vacuole populations were prepared and mixed before immunoprecipitation (lane 4). (C) Vacuoles were isolated from nvy1Δ strains with GFP-tagged Vam3p or Vam7p and assayed for protein localization during docking as above (Fig. 4).
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fig6: Deletion of the v-SNARE Nyv1p has no effect on the localization of other SNAREs. (A) GFP-Nyv1p vacuoles were subject to in vitro docking assay and ratiometric fluorescence microscopy analysis, as described in Materials and methods. The GFP:FM4–64 ratio values were generated for outside edge membranes (red curves) and vertex membranes (black curves), and plotted against their percentile. (B) Trans-pairing of SNAREs. Trans-SNARE pairing was assayed directly using purified vacuoles from four strains: (1) VTI1-GFP, NYV1, pep4–3; (2) HA3-VAM3, NYV1, pep4::HIS3; (3) VTI1-GFP, pep4::HIS3, nyv1::URA3; and (4) HA3-VAM3, pep4::HIS3, nyv1::URA3. After a 60-min fusion reaction, membranes were dissolved in Triton X-100, and Vti1-GFP was immunoprecipitated by immobilized antibody to GFP (unpublished data). Trans-associated HA-Vam3p was detected by immunoblot and quantified by densitometry as a percentage of the total HA-Vam3p in the sample. Recombinant Gyp1–46p (0.4 μg/ml) was added where indicated (lane 3). To determine the level of background association, detergent extracts of reactions with ATP and single vacuole populations were prepared and mixed before immunoprecipitation (lane 4). (C) Vacuoles were isolated from nvy1Δ strains with GFP-tagged Vam3p or Vam7p and assayed for protein localization during docking as above (Fig. 4).

Mentions: Because v-SNAREs and t-SNAREs are localized to vertices via different pathways, SNARE localization may precede the formation of the trans-SNARE complex. The vacuolar v-SNARE Nyv1p enters trans-SNARE complexes (Ungermann and Wickner, 1998) and is needed for vacuole homotypic fusion (Nichols et al., 1997). Nyv1p was also enriched at vertices on docked vacuoles (Fig. 6 A). Vacuoles lacking Nyv1p cannot fuse, and antibody to Nyv1p blocks the fusion of wild-type vacuoles (Ungermann and Wickner, 1998). Using a recently developed assay of trans-pairing of SNAREs (unpublished data), we find that the absence of Nyv1p completely blocks trans-SNARE pairing between Vti1-GFP from one partner vacuole and HA-Vam3p from the other vacuole population (Fig. 6 B). Nevertheless, the absence of Nyv1p had no effect on the vertex enrichment of the t-SNAREs Vam3p or Vam7p (Fig. 6 C). Together with the effects of fusion inhibitors on the localization of SNAREs, our results indicate that SNAREs need not pair in trans in order to be enriched at vertices. The clustered localization of SNAREs at a vertex ring might be a prerequisite for efficient trans-SNARE pairing and downstream events leading to fusion.


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)

Deletion of the v-SNARE Nyv1p has no effect on the localization of other SNAREs. (A) GFP-Nyv1p vacuoles were subject to in vitro docking assay and ratiometric fluorescence microscopy analysis, as described in Materials and methods. The GFP:FM4–64 ratio values were generated for outside edge membranes (red curves) and vertex membranes (black curves), and plotted against their percentile. (B) Trans-pairing of SNAREs. Trans-SNARE pairing was assayed directly using purified vacuoles from four strains: (1) VTI1-GFP, NYV1, pep4–3; (2) HA3-VAM3, NYV1, pep4::HIS3; (3) VTI1-GFP, pep4::HIS3, nyv1::URA3; and (4) HA3-VAM3, pep4::HIS3, nyv1::URA3. After a 60-min fusion reaction, membranes were dissolved in Triton X-100, and Vti1-GFP was immunoprecipitated by immobilized antibody to GFP (unpublished data). Trans-associated HA-Vam3p was detected by immunoblot and quantified by densitometry as a percentage of the total HA-Vam3p in the sample. Recombinant Gyp1–46p (0.4 μg/ml) was added where indicated (lane 3). To determine the level of background association, detergent extracts of reactions with ATP and single vacuole populations were prepared and mixed before immunoprecipitation (lane 4). (C) Vacuoles were isolated from nvy1Δ strains with GFP-tagged Vam3p or Vam7p and assayed for protein localization during docking as above (Fig. 4).
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fig6: Deletion of the v-SNARE Nyv1p has no effect on the localization of other SNAREs. (A) GFP-Nyv1p vacuoles were subject to in vitro docking assay and ratiometric fluorescence microscopy analysis, as described in Materials and methods. The GFP:FM4–64 ratio values were generated for outside edge membranes (red curves) and vertex membranes (black curves), and plotted against their percentile. (B) Trans-pairing of SNAREs. Trans-SNARE pairing was assayed directly using purified vacuoles from four strains: (1) VTI1-GFP, NYV1, pep4–3; (2) HA3-VAM3, NYV1, pep4::HIS3; (3) VTI1-GFP, pep4::HIS3, nyv1::URA3; and (4) HA3-VAM3, pep4::HIS3, nyv1::URA3. After a 60-min fusion reaction, membranes were dissolved in Triton X-100, and Vti1-GFP was immunoprecipitated by immobilized antibody to GFP (unpublished data). Trans-associated HA-Vam3p was detected by immunoblot and quantified by densitometry as a percentage of the total HA-Vam3p in the sample. Recombinant Gyp1–46p (0.4 μg/ml) was added where indicated (lane 3). To determine the level of background association, detergent extracts of reactions with ATP and single vacuole populations were prepared and mixed before immunoprecipitation (lane 4). (C) Vacuoles were isolated from nvy1Δ strains with GFP-tagged Vam3p or Vam7p and assayed for protein localization during docking as above (Fig. 4).
Mentions: Because v-SNAREs and t-SNAREs are localized to vertices via different pathways, SNARE localization may precede the formation of the trans-SNARE complex. The vacuolar v-SNARE Nyv1p enters trans-SNARE complexes (Ungermann and Wickner, 1998) and is needed for vacuole homotypic fusion (Nichols et al., 1997). Nyv1p was also enriched at vertices on docked vacuoles (Fig. 6 A). Vacuoles lacking Nyv1p cannot fuse, and antibody to Nyv1p blocks the fusion of wild-type vacuoles (Ungermann and Wickner, 1998). Using a recently developed assay of trans-pairing of SNAREs (unpublished data), we find that the absence of Nyv1p completely blocks trans-SNARE pairing between Vti1-GFP from one partner vacuole and HA-Vam3p from the other vacuole population (Fig. 6 B). Nevertheless, the absence of Nyv1p had no effect on the vertex enrichment of the t-SNAREs Vam3p or Vam7p (Fig. 6 C). Together with the effects of fusion inhibitors on the localization of SNAREs, our results indicate that SNAREs need not pair in trans in order to be enriched at vertices. The clustered localization of SNAREs at a vertex ring might be a prerequisite for efficient trans-SNARE pairing and downstream events leading to fusion.

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