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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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Vacuole tethering. Inhibitors, such as 300 ng/μl Gdi1p, 600 ng/μl Gyp1p, and 60 ng/μl anti-Vam3p Fab were added as indicated to an in vitro docking assay. For each condition, images were acquired from random fields and an average of 500 vacuoles were scored for cluster size.
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fig3: Vacuole tethering. Inhibitors, such as 300 ng/μl Gdi1p, 600 ng/μl Gyp1p, and 60 ng/μl anti-Vam3p Fab were added as indicated to an in vitro docking assay. For each condition, images were acquired from random fields and an average of 500 vacuoles were scored for cluster size.

Mentions: Vacuole docking has been studied using an in vitro tethering assay (Fig. 3; Mayer and Wickner, 1997; Wang et al., 2002) in which tethered vacuoles form large clusters (Fig. 3, black bars). The tethering reaction requires the GTPase Ypt7p. Gdi1p, which extracts Ypt7p from the membrane, inhibited cluster formation (Fig. 3, open bars). Ypt7p cycles between the GTP-bound state and the GDP-bound state, though it has not been known which state catalyzes tethering. Because Gyp proteins promote GTP hydrolysis by Ypt7p, driving it to the GDP-bound state (Vollmer et al., 1999; Rak et al., 2000; Du and Novick, 2001), we analyzed the effects of recombinant Gyp1–46p (an active fragment of Gyp1p) on Ypt7p-mediated vacuole tethering. Gyp1–46p did not reduce vacuole tethering (Fig. 3, gray bars), though fusion was completely inhibited (Fig. 2, lane 4; Eitzen et al., 2000). Cluster formation was still sensitive to Gdi1p in the presence of Gyp1p (hatched bars), indicating that these vacuoles were tethered through a Ypt7p-dependent pathway. Anti-Vam3p Fab, which blocked vacuole fusion (Fig. 2, lane 2), did not affect Ypt7p dependent vacuole tethering (Fig. 3, slanted line bars), in accord with other studies showing that tethering requires Rabs but not SNAREs (Cao et al., 1998). Thus, tethering requires Ypt7p, but does not require its nucleotide exchange to Ypt7:GTP or the Vam3p t-SNARE.


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)

Vacuole tethering. Inhibitors, such as 300 ng/μl Gdi1p, 600 ng/μl Gyp1p, and 60 ng/μl anti-Vam3p Fab were added as indicated to an in vitro docking assay. For each condition, images were acquired from random fields and an average of 500 vacuoles were scored for cluster size.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Vacuole tethering. Inhibitors, such as 300 ng/μl Gdi1p, 600 ng/μl Gyp1p, and 60 ng/μl anti-Vam3p Fab were added as indicated to an in vitro docking assay. For each condition, images were acquired from random fields and an average of 500 vacuoles were scored for cluster size.
Mentions: Vacuole docking has been studied using an in vitro tethering assay (Fig. 3; Mayer and Wickner, 1997; Wang et al., 2002) in which tethered vacuoles form large clusters (Fig. 3, black bars). The tethering reaction requires the GTPase Ypt7p. Gdi1p, which extracts Ypt7p from the membrane, inhibited cluster formation (Fig. 3, open bars). Ypt7p cycles between the GTP-bound state and the GDP-bound state, though it has not been known which state catalyzes tethering. Because Gyp proteins promote GTP hydrolysis by Ypt7p, driving it to the GDP-bound state (Vollmer et al., 1999; Rak et al., 2000; Du and Novick, 2001), we analyzed the effects of recombinant Gyp1–46p (an active fragment of Gyp1p) on Ypt7p-mediated vacuole tethering. Gyp1–46p did not reduce vacuole tethering (Fig. 3, gray bars), though fusion was completely inhibited (Fig. 2, lane 4; Eitzen et al., 2000). Cluster formation was still sensitive to Gdi1p in the presence of Gyp1p (hatched bars), indicating that these vacuoles were tethered through a Ypt7p-dependent pathway. Anti-Vam3p Fab, which blocked vacuole fusion (Fig. 2, lane 2), did not affect Ypt7p dependent vacuole tethering (Fig. 3, slanted line bars), in accord with other studies showing that tethering requires Rabs but not SNAREs (Cao et al., 1998). Thus, tethering requires Ypt7p, but does not require its nucleotide exchange to Ypt7:GTP or the Vam3p t-SNARE.

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