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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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Working model of vertex ring assembly. See text for details. Solid black arrows indicate a strict hierarchical requirement for vertex enrichment. Dotted arrows indicate partial influences on vertex enrichment.
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fig8: Working model of vertex ring assembly. See text for details. Solid black arrows indicate a strict hierarchical requirement for vertex enrichment. Dotted arrows indicate partial influences on vertex enrichment.

Mentions: Protein localization to membrane domains is critical for the functions of many biological systems. For large protein complexes, such as cell adhesion complexes (Martin et al., 2002; Schwartz and Ginsberg, 2002) and the immunological synapse (Bromley et al., 2001; Dustin et al., 2001), protein enrichment at specific sites on the plasma membrane relies on protein and lipid-mediated signaling networks and follows temporal and spatial hierarchies of molecular interactions. Membrane fusion is also catalyzed by a complex protein and lipid machinery that assembles at specific membrane domains. Prominent examples include Rab domains on endosomal membranes (Roberts et al., 1999; Sonnichsen et al., 2000), SNARE domains, and a RIM1a-mediated protein scaffold at neuronal synaptic junctions (Bennett et al., 1992; Lang et al., 2001; Schoch et al., 2002), and the yeast exocyst complex at the bud tip or septation ring (Finger et al., 1998; Guo et al., 2001). A vertex ring of proteins and lipids assembles on tethered vacuoles before catalyzing fusion (Wang et al., 2002). We have now identified a hierarchy in the spatial localization of these docking and fusion factors, revealing new features of the interactions between organelle-bound actin, Ypt/Rab GTPase, and SNAREs (Fig. 8). The strong correlation between enrichment of Ypt7p and HOPS at individual vertex sites, and selective loss of Vps33p enrichment by PX domain treatment provide independent support to this hierarchy. Though we have only used a select few inhibitors and GFP-tagged a subset of the proteins that catalyze vacuole fusion (Wickner, 2002), their interplay has revealed the first outlines of a vertex ring assembly hierarchy.


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)

Working model of vertex ring assembly. See text for details. Solid black arrows indicate a strict hierarchical requirement for vertex enrichment. Dotted arrows indicate partial influences on vertex enrichment.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Working model of vertex ring assembly. See text for details. Solid black arrows indicate a strict hierarchical requirement for vertex enrichment. Dotted arrows indicate partial influences on vertex enrichment.
Mentions: Protein localization to membrane domains is critical for the functions of many biological systems. For large protein complexes, such as cell adhesion complexes (Martin et al., 2002; Schwartz and Ginsberg, 2002) and the immunological synapse (Bromley et al., 2001; Dustin et al., 2001), protein enrichment at specific sites on the plasma membrane relies on protein and lipid-mediated signaling networks and follows temporal and spatial hierarchies of molecular interactions. Membrane fusion is also catalyzed by a complex protein and lipid machinery that assembles at specific membrane domains. Prominent examples include Rab domains on endosomal membranes (Roberts et al., 1999; Sonnichsen et al., 2000), SNARE domains, and a RIM1a-mediated protein scaffold at neuronal synaptic junctions (Bennett et al., 1992; Lang et al., 2001; Schoch et al., 2002), and the yeast exocyst complex at the bud tip or septation ring (Finger et al., 1998; Guo et al., 2001). A vertex ring of proteins and lipids assembles on tethered vacuoles before catalyzing fusion (Wang et al., 2002). We have now identified a hierarchy in the spatial localization of these docking and fusion factors, revealing new features of the interactions between organelle-bound actin, Ypt/Rab GTPase, and SNAREs (Fig. 8). The strong correlation between enrichment of Ypt7p and HOPS at individual vertex sites, and selective loss of Vps33p enrichment by PX domain treatment provide independent support to this hierarchy. Though we have only used a select few inhibitors and GFP-tagged a subset of the proteins that catalyze vacuole fusion (Wickner, 2002), their interplay has revealed the first outlines of a vertex ring assembly hierarchy.

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