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Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles.

Fratti RA, Jun Y, Merz AJ, Margolis N, Wickner W - J. Cell Biol. (2004)

Bottom Line: Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment.Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles.Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

ABSTRACT
Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein-protein interactions (e.g., coated vesicles). During docking, yeast vacuoles assemble "vertex" ring-shaped microdomains around the periphery of their apposed membranes. Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)-VpsC Rab effector complex, SNAREs, and actin. Membrane fusion initiates at vertex microdomains. We now find that the "regulatory lipids" ergosterol, diacylglycerol and 3- and 4-phosphoinositides accumulate at vertices in a mutually interdependent manner. Regulatory lipids are also required for the vertex enrichment of SNAREs, Ypt7p, and HOPS. Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment. Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles. Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.

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Regulatory lipids control the vertex enrichment of Ypt7p, SNAREs, and HOPS. Docking reactions using vacuoles from strains expressing GFP fusions to Ypt7p (A), Vam7p (B), Vam3p (C), Vti1p (D), Vps33p (E), or Pho8p (F) were treated with 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or 2 μM GST-FYVE and assayed for vertex enrichment. Reactions were incubated for 30 min at 27°C, placed on ice and labeled with FM4-64. Geometric mean values ± 95% confidence intervals of relative vertex enrichment are shown. (G–R) Fluorescent images of docked vacuoles containing GFP-Vti1p (G–L), or GFP-Ypt7p (M–R). Docking reactions bore no inhibitor (G–I and M–O) or 30 μM ENTH (J–L and P–R). G, J, M, and P show membrane staining with FM4-64. H, K, N, and Q show the distribution of GFP-Vti1p (H and K) or GFP-Ypt7p (N and Q). Merged images illustrate the enrichment of GFP-Vti1p (I) and Ypt7p (O) at vertices relative to outer membrane. ENTH treatment abolished the vertex enrichment of these proteins (L and R). Arrows are examples of vertices enriched in GFP-Vti1 (H and I) and GFP-Ypt7 (N and O) relative to outer membrane. Bars, 5 μm.
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fig7: Regulatory lipids control the vertex enrichment of Ypt7p, SNAREs, and HOPS. Docking reactions using vacuoles from strains expressing GFP fusions to Ypt7p (A), Vam7p (B), Vam3p (C), Vti1p (D), Vps33p (E), or Pho8p (F) were treated with 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or 2 μM GST-FYVE and assayed for vertex enrichment. Reactions were incubated for 30 min at 27°C, placed on ice and labeled with FM4-64. Geometric mean values ± 95% confidence intervals of relative vertex enrichment are shown. (G–R) Fluorescent images of docked vacuoles containing GFP-Vti1p (G–L), or GFP-Ypt7p (M–R). Docking reactions bore no inhibitor (G–I and M–O) or 30 μM ENTH (J–L and P–R). G, J, M, and P show membrane staining with FM4-64. H, K, N, and Q show the distribution of GFP-Vti1p (H and K) or GFP-Ypt7p (N and Q). Merged images illustrate the enrichment of GFP-Vti1p (I) and Ypt7p (O) at vertices relative to outer membrane. ENTH treatment abolished the vertex enrichment of these proteins (L and R). Arrows are examples of vertices enriched in GFP-Vti1 (H and I) and GFP-Ypt7 (N and O) relative to outer membrane. Bars, 5 μm.

Mentions: Because regulatory lipids depend on each other and on fusion proteins for vertex enrichment (Figs. 5 and 6), we asked whether fusion protein vertex enrichment may depend on regulatory lipids. We examined the effect of lipid ligands on vertex enrichment of Ypt7p, the SNAREs Vam7p, Vam3p, and Vti1p, and the HOPS subunit Vps33p. Ypt7p vertex enrichment is impervious to several protein-targeted inhibitors of fusion (Wang et al., 2003), yet was blocked by ENTH, C1b, FYVE, or filipin (P < 0.0001; Fig. 7 A). The inhibition of Ypt7p enrichment by FYVE contrasts with the previous finding that the PI(3)P ligand PX domain did not affect Ypt7p vertex accumulation (Wang et al., 2003). This may reflect a greater affinity of dimeric FYVE than monomeric PX for PI(3)P.


Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles.

Fratti RA, Jun Y, Merz AJ, Margolis N, Wickner W - J. Cell Biol. (2004)

Regulatory lipids control the vertex enrichment of Ypt7p, SNAREs, and HOPS. Docking reactions using vacuoles from strains expressing GFP fusions to Ypt7p (A), Vam7p (B), Vam3p (C), Vti1p (D), Vps33p (E), or Pho8p (F) were treated with 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or 2 μM GST-FYVE and assayed for vertex enrichment. Reactions were incubated for 30 min at 27°C, placed on ice and labeled with FM4-64. Geometric mean values ± 95% confidence intervals of relative vertex enrichment are shown. (G–R) Fluorescent images of docked vacuoles containing GFP-Vti1p (G–L), or GFP-Ypt7p (M–R). Docking reactions bore no inhibitor (G–I and M–O) or 30 μM ENTH (J–L and P–R). G, J, M, and P show membrane staining with FM4-64. H, K, N, and Q show the distribution of GFP-Vti1p (H and K) or GFP-Ypt7p (N and Q). Merged images illustrate the enrichment of GFP-Vti1p (I) and Ypt7p (O) at vertices relative to outer membrane. ENTH treatment abolished the vertex enrichment of these proteins (L and R). Arrows are examples of vertices enriched in GFP-Vti1 (H and I) and GFP-Ypt7 (N and O) relative to outer membrane. Bars, 5 μm.
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fig7: Regulatory lipids control the vertex enrichment of Ypt7p, SNAREs, and HOPS. Docking reactions using vacuoles from strains expressing GFP fusions to Ypt7p (A), Vam7p (B), Vam3p (C), Vti1p (D), Vps33p (E), or Pho8p (F) were treated with 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or 2 μM GST-FYVE and assayed for vertex enrichment. Reactions were incubated for 30 min at 27°C, placed on ice and labeled with FM4-64. Geometric mean values ± 95% confidence intervals of relative vertex enrichment are shown. (G–R) Fluorescent images of docked vacuoles containing GFP-Vti1p (G–L), or GFP-Ypt7p (M–R). Docking reactions bore no inhibitor (G–I and M–O) or 30 μM ENTH (J–L and P–R). G, J, M, and P show membrane staining with FM4-64. H, K, N, and Q show the distribution of GFP-Vti1p (H and K) or GFP-Ypt7p (N and Q). Merged images illustrate the enrichment of GFP-Vti1p (I) and Ypt7p (O) at vertices relative to outer membrane. ENTH treatment abolished the vertex enrichment of these proteins (L and R). Arrows are examples of vertices enriched in GFP-Vti1 (H and I) and GFP-Ypt7 (N and O) relative to outer membrane. Bars, 5 μm.
Mentions: Because regulatory lipids depend on each other and on fusion proteins for vertex enrichment (Figs. 5 and 6), we asked whether fusion protein vertex enrichment may depend on regulatory lipids. We examined the effect of lipid ligands on vertex enrichment of Ypt7p, the SNAREs Vam7p, Vam3p, and Vti1p, and the HOPS subunit Vps33p. Ypt7p vertex enrichment is impervious to several protein-targeted inhibitors of fusion (Wang et al., 2003), yet was blocked by ENTH, C1b, FYVE, or filipin (P < 0.0001; Fig. 7 A). The inhibition of Ypt7p enrichment by FYVE contrasts with the previous finding that the PI(3)P ligand PX domain did not affect Ypt7p vertex accumulation (Wang et al., 2003). This may reflect a greater affinity of dimeric FYVE than monomeric PX for PI(3)P.

Bottom Line: Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment.Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles.Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

ABSTRACT
Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein-protein interactions (e.g., coated vesicles). During docking, yeast vacuoles assemble "vertex" ring-shaped microdomains around the periphery of their apposed membranes. Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)-VpsC Rab effector complex, SNAREs, and actin. Membrane fusion initiates at vertex microdomains. We now find that the "regulatory lipids" ergosterol, diacylglycerol and 3- and 4-phosphoinositides accumulate at vertices in a mutually interdependent manner. Regulatory lipids are also required for the vertex enrichment of SNAREs, Ypt7p, and HOPS. Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment. Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles. Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.

Show MeSH
Related in: MedlinePlus