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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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Catalytic assay of vacuole fusion. Vacuole fusion was assayed in vitro as described in Materials and methods. Fusion 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 present where indicated.
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fig2: Catalytic assay of vacuole fusion. Vacuole fusion was assayed in vitro as described in Materials and methods. Fusion 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 present where indicated.

Mentions: Vacuole fusion has been studied biochemically, using inhibitors with known targets (Wickner, 2002). For example, fusion was inhibited by antibodies against SNARE proteins (Fig. 2, lane 2; Ungermann et al., 1999) by the F-actin–stabilizing reagent jasplakinolide (Fig. 2, lane 3; Eitzen et al., 2002) and by Gyp proteins (Fig. 2, lane 4; Eitzen et al., 2000), which accelerate GTP hydrolysis by GTPases such as Ypt7p (Albert et al., 1999). Sec18p and Sec17p are cochaperones for the cis-SNARE complex and are required for priming, but the addition of excess Sec17p recaptures SNAREs into a cis-complex and thereby inhibits fusion (Fig. 2, lane 5; Wang et al., 2000). Vam7p, a homologue of neuronal SNAP-25, is a soluble SNARE protein. It is released from vacuoles during priming, but rebinds through association of its PX domain with PtdIns(3)P during docking. Exogenous recombinant PX domain inhibited fusion by competing with Vam7p for PtdIns(3)P (Fig. 2, lane 6; Boeddinghaus et al., 2002), whereas the addition of recombinant Vam7p rescued fusion from PX inhibition (Fig. 2, lane 7; unpublished data). Latrunculin B, which prevents the repolymerization of G-actin, inhibited fusion (Fig. 2, lane 8; Eitzen et al., 2002). These fusion inhibitors provide reliable tools for studying protein localization during docking.


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)

Catalytic assay of vacuole fusion. Vacuole fusion was assayed in vitro as described in Materials and methods. Fusion 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 present where indicated.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172665&req=5

fig2: Catalytic assay of vacuole fusion. Vacuole fusion was assayed in vitro as described in Materials and methods. Fusion 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 present where indicated.
Mentions: Vacuole fusion has been studied biochemically, using inhibitors with known targets (Wickner, 2002). For example, fusion was inhibited by antibodies against SNARE proteins (Fig. 2, lane 2; Ungermann et al., 1999) by the F-actin–stabilizing reagent jasplakinolide (Fig. 2, lane 3; Eitzen et al., 2002) and by Gyp proteins (Fig. 2, lane 4; Eitzen et al., 2000), which accelerate GTP hydrolysis by GTPases such as Ypt7p (Albert et al., 1999). Sec18p and Sec17p are cochaperones for the cis-SNARE complex and are required for priming, but the addition of excess Sec17p recaptures SNAREs into a cis-complex and thereby inhibits fusion (Fig. 2, lane 5; Wang et al., 2000). Vam7p, a homologue of neuronal SNAP-25, is a soluble SNARE protein. It is released from vacuoles during priming, but rebinds through association of its PX domain with PtdIns(3)P during docking. Exogenous recombinant PX domain inhibited fusion by competing with Vam7p for PtdIns(3)P (Fig. 2, lane 6; Boeddinghaus et al., 2002), whereas the addition of recombinant Vam7p rescued fusion from PX inhibition (Fig. 2, lane 7; unpublished data). Latrunculin B, which prevents the repolymerization of G-actin, inhibited fusion (Fig. 2, lane 8; Eitzen et al., 2002). These fusion inhibitors provide reliable tools for studying protein localization during docking.

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