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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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Ypt7p and Vps33p colocalization at vertex sites. GFP-Ypt7p and Vps33-mRFP were quantified at vertex sites under docking conditions in the absence (open circles) or presence (filled circles) of PX domain. Lines show linear fits to log-transformed intensity data and 99% confidence intervals for the best-fit lines. The upper fit is to the control samples (open circles) and the lower fit is to the PX domain-treated samples (filled circles). The data are pooled from three independent experiments; interexperimental variation did not significantly influence the results.
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fig7: Ypt7p and Vps33p colocalization at vertex sites. GFP-Ypt7p and Vps33-mRFP were quantified at vertex sites under docking conditions in the absence (open circles) or presence (filled circles) of PX domain. Lines show linear fits to log-transformed intensity data and 99% confidence intervals for the best-fit lines. The upper fit is to the control samples (open circles) and the lower fit is to the PX domain-treated samples (filled circles). The data are pooled from three independent experiments; interexperimental variation did not significantly influence the results.

Mentions: Vertex sites are heterogeneous for their enrichment of each protein (Fig. 4; Wang et al., 2002). If this represents varying degrees of activation of the assembly hierarchy we describe herein, then the HOPS complex (at the end of the hierarchy) should be most enriched at those vertices which show the greatest enrichment for Ypt7p (at the start of the hierarchy). The recent availability of a fast-maturing monomeric red fluorescent protein variant (Campbell et al., 2002) allowed us to ask whether various vertex markers colocalize. We generated a yeast strain carrying both the HOPS subunit Vps33p fused to mRFP and Ypt7p fused to GFP. The blue fluorescent lipid probe TMA-DPH was used instead of the red dye FM4–64 to monitor lipid distribution on the vacuole. In a standard docking reaction, these three fluorophores were monitored on the same vacuole clusters (Fig. 7 A). GFP-Ypt7p and Vps33-mRFP significantly colocalized (P < 0.0001) at vertex sites (Fig. 7 B). As shown above using single GFP fusions, addition of PX domain did not significantly change GFP-Ypt7p localization at vertices. However, added PX domain (Fig. 7 B) significantly decreased the amount of Vps33-mRFP at the same sites (P < 0.0001). Interestingly, Vps33p and Ypt7p intensity were similarly correlated in both the presence and absence of PX domain (r2 = 0.58 and 0.62, respectively). Although less Vps33p accumulates at vertex sites in the presence of PX domain, this result suggests that the Vps33p that does accumulate is recruited through a Ypt7p-dependent mechanism. Regression models incorporating lipid intensity effects indicate that colocalization of GFP-Ypt7p and Vps33-mRFP did not depend on TMA-DPH intensity (unpublished data). A simpler regression model that incorporates effects due to GFP-Ypt7p localization and treatment (absence or presence of PX domain) accounts for almost 70% of the variation in Vps33-mRFP localization at vertex sites (r2 = 0.68; F[3,605] = 436.0; P < 0.0001). Thus, only ∼30% of the variability in the vertex localization of Vps33p localization is explained by experimental noise and other factors. These experiments corroborate and extend our findings that HOPS localization depends on both Ypt7p function and Vam7p function, and support the interpretation that Ypt7p acts upstream of HOPS and other factors (such as Vam7p) to specify the assembly of vertex sites.


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)

Ypt7p and Vps33p colocalization at vertex sites. GFP-Ypt7p and Vps33-mRFP were quantified at vertex sites under docking conditions in the absence (open circles) or presence (filled circles) of PX domain. Lines show linear fits to log-transformed intensity data and 99% confidence intervals for the best-fit lines. The upper fit is to the control samples (open circles) and the lower fit is to the PX domain-treated samples (filled circles). The data are pooled from three independent experiments; interexperimental variation did not significantly influence the results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172665&req=5

fig7: Ypt7p and Vps33p colocalization at vertex sites. GFP-Ypt7p and Vps33-mRFP were quantified at vertex sites under docking conditions in the absence (open circles) or presence (filled circles) of PX domain. Lines show linear fits to log-transformed intensity data and 99% confidence intervals for the best-fit lines. The upper fit is to the control samples (open circles) and the lower fit is to the PX domain-treated samples (filled circles). The data are pooled from three independent experiments; interexperimental variation did not significantly influence the results.
Mentions: Vertex sites are heterogeneous for their enrichment of each protein (Fig. 4; Wang et al., 2002). If this represents varying degrees of activation of the assembly hierarchy we describe herein, then the HOPS complex (at the end of the hierarchy) should be most enriched at those vertices which show the greatest enrichment for Ypt7p (at the start of the hierarchy). The recent availability of a fast-maturing monomeric red fluorescent protein variant (Campbell et al., 2002) allowed us to ask whether various vertex markers colocalize. We generated a yeast strain carrying both the HOPS subunit Vps33p fused to mRFP and Ypt7p fused to GFP. The blue fluorescent lipid probe TMA-DPH was used instead of the red dye FM4–64 to monitor lipid distribution on the vacuole. In a standard docking reaction, these three fluorophores were monitored on the same vacuole clusters (Fig. 7 A). GFP-Ypt7p and Vps33-mRFP significantly colocalized (P < 0.0001) at vertex sites (Fig. 7 B). As shown above using single GFP fusions, addition of PX domain did not significantly change GFP-Ypt7p localization at vertices. However, added PX domain (Fig. 7 B) significantly decreased the amount of Vps33-mRFP at the same sites (P < 0.0001). Interestingly, Vps33p and Ypt7p intensity were similarly correlated in both the presence and absence of PX domain (r2 = 0.58 and 0.62, respectively). Although less Vps33p accumulates at vertex sites in the presence of PX domain, this result suggests that the Vps33p that does accumulate is recruited through a Ypt7p-dependent mechanism. Regression models incorporating lipid intensity effects indicate that colocalization of GFP-Ypt7p and Vps33-mRFP did not depend on TMA-DPH intensity (unpublished data). A simpler regression model that incorporates effects due to GFP-Ypt7p localization and treatment (absence or presence of PX domain) accounts for almost 70% of the variation in Vps33-mRFP localization at vertex sites (r2 = 0.68; F[3,605] = 436.0; P < 0.0001). Thus, only ∼30% of the variability in the vertex localization of Vps33p localization is explained by experimental noise and other factors. These experiments corroborate and extend our findings that HOPS localization depends on both Ypt7p function and Vam7p function, and support the interpretation that Ypt7p acts upstream of HOPS and other factors (such as Vam7p) to specify the assembly of vertex sites.

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