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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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In vitro vacuole tethering. (A) A schematic of tethered vacuoles. Membrane microdomains include outside edges (O), boundary membranes (B), and vertex membranes (V). (B) Vam7p is enriched at vertices. GFP-tagged Vam7p vacuoles were tethered in vitro (see Materials and methods) and labeled with FM4–64. Images were taken in GFP (left) and rhodamine (middle) channels; ratiometric images are on the right.
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fig1: In vitro vacuole tethering. (A) A schematic of tethered vacuoles. Membrane microdomains include outside edges (O), boundary membranes (B), and vertex membranes (V). (B) Vam7p is enriched at vertices. GFP-tagged Vam7p vacuoles were tethered in vitro (see Materials and methods) and labeled with FM4–64. Images were taken in GFP (left) and rhodamine (middle) channels; ratiometric images are on the right.

Mentions: Recently, we exploited the large (>1 μm) diameter of purified vacuoles to study their membrane morphology and protein spatial dynamics during fusion (Wang et al., 2002). Vacuoles accumulate in tethered clusters due to the relative rates of docking and fusion. Clustered vacuoles have three domains: (1) “outside” membrane that is not in contact with other vacuoles; (2) “boundary” membrane that is apposed to a neighboring docked vacuole; and (3) “vertex” membrane where two boundary domains, or boundary and outside membrane domains, meet (Fig. 1 A, right). In three dimensions, the vertices circumscribe the apposed boundary membranes (Fig. 1 A, left). Fusion occurs at the vertex ring domains on docked vacuole membranes, yielding internalized membranes after the completion of fusion (Wang et al., 2002). Docking and fusion factors, such as the Ypt7p GTPase, its effector complex for homotypic fusion and vacuole protein sorting (HOPS;* Price et al., 2000; Seals et al., 2000), or class C Vps (Wurmser et al., 2000), SNARE proteins, and vacuole-bound actin (Eitzen et al., 2002), assayed by ratiometric fluorescence microscopy (Fig. 1 B), accumulate at these vertex sites before catalyzing fusion. The vertex enrichment of these proteins may follow a specific causal pathway that regulates the fusion process.


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)

In vitro vacuole tethering. (A) A schematic of tethered vacuoles. Membrane microdomains include outside edges (O), boundary membranes (B), and vertex membranes (V). (B) Vam7p is enriched at vertices. GFP-tagged Vam7p vacuoles were tethered in vitro (see Materials and methods) and labeled with FM4–64. Images were taken in GFP (left) and rhodamine (middle) channels; ratiometric images are on the right.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: In vitro vacuole tethering. (A) A schematic of tethered vacuoles. Membrane microdomains include outside edges (O), boundary membranes (B), and vertex membranes (V). (B) Vam7p is enriched at vertices. GFP-tagged Vam7p vacuoles were tethered in vitro (see Materials and methods) and labeled with FM4–64. Images were taken in GFP (left) and rhodamine (middle) channels; ratiometric images are on the right.
Mentions: Recently, we exploited the large (>1 μm) diameter of purified vacuoles to study their membrane morphology and protein spatial dynamics during fusion (Wang et al., 2002). Vacuoles accumulate in tethered clusters due to the relative rates of docking and fusion. Clustered vacuoles have three domains: (1) “outside” membrane that is not in contact with other vacuoles; (2) “boundary” membrane that is apposed to a neighboring docked vacuole; and (3) “vertex” membrane where two boundary domains, or boundary and outside membrane domains, meet (Fig. 1 A, right). In three dimensions, the vertices circumscribe the apposed boundary membranes (Fig. 1 A, left). Fusion occurs at the vertex ring domains on docked vacuole membranes, yielding internalized membranes after the completion of fusion (Wang et al., 2002). Docking and fusion factors, such as the Ypt7p GTPase, its effector complex for homotypic fusion and vacuole protein sorting (HOPS;* Price et al., 2000; Seals et al., 2000), or class C Vps (Wurmser et al., 2000), SNARE proteins, and vacuole-bound actin (Eitzen et al., 2002), assayed by ratiometric fluorescence microscopy (Fig. 1 B), accumulate at these vertex sites before catalyzing fusion. The vertex enrichment of these proteins may follow a specific causal pathway that regulates the fusion process.

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