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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 are required for vacuolar association of Vam7p. Vam7p association with vacuolar membranes was assayed by immunoblot in fusion reactions without inhibitor or with 2 μM GST-FYVE, 10 μM C1b, 19 μM filipin, 38 μM GST-ENTH, 2 μM SigD, or 1 μM MTM-1. Reactions were also performed in the presence of anti-Sec18p Fab to inhibit priming. Fusion reactions were incubated for 90 min at 27°C, then fractionated into membrane pellets and supernatants by centrifugation (13,000 g, 15 min, 4°C). Membranes were resuspended in 30 μl PS buffer with protease inhibitors (1 μM leupeptin, 5 μM pepstatin and 0.1 μM pefabloc-SC). (A) Equal proportions of the pellet and supernatant fractions were mixed with SDS-loading buffer, resolved by SDS-PAGE, then transferred to nitrocellulose. (B and C) Quantitative analysis of membrane-bound Vam7p by Western blots. Data represent mean values ± SEM. Relative band intensities were measured using NIH Image 1.62 (n = 4).
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fig9: Regulatory lipids are required for vacuolar association of Vam7p. Vam7p association with vacuolar membranes was assayed by immunoblot in fusion reactions without inhibitor or with 2 μM GST-FYVE, 10 μM C1b, 19 μM filipin, 38 μM GST-ENTH, 2 μM SigD, or 1 μM MTM-1. Reactions were also performed in the presence of anti-Sec18p Fab to inhibit priming. Fusion reactions were incubated for 90 min at 27°C, then fractionated into membrane pellets and supernatants by centrifugation (13,000 g, 15 min, 4°C). Membranes were resuspended in 30 μl PS buffer with protease inhibitors (1 μM leupeptin, 5 μM pepstatin and 0.1 μM pefabloc-SC). (A) Equal proportions of the pellet and supernatant fractions were mixed with SDS-loading buffer, resolved by SDS-PAGE, then transferred to nitrocellulose. (B and C) Quantitative analysis of membrane-bound Vam7p by Western blots. Data represent mean values ± SEM. Relative band intensities were measured using NIH Image 1.62 (n = 4).

Mentions: Lipids are enriched at the vertices of docked vacuoles. Vacuoles from DKY6281 yeast were incubated under docking conditions for 30 min at 27°C with fluorescent lipid ligands. After incubation, docking reactions were placed on ice and labeled with either FM4-64 or MDY-64. (A) Outer membrane, boundary membrane and vertex ring subdomains of docked vacuoles. (B and E) MDY-64 or (H, K, and N) FM4-64 label the entire membrane. Specific lipids were labeled with (C) 0.2 μM Cy3-FYVE to label PI(3)P, (F) 0.6 μM Cy3-ENTH to label PI(4,5)P2, (I) 5 μM filipin to label ergosterol, (L) 1 μM Alexa488-C1b to label DAG, or (O) 2.5 μM PSS-380 to label PS. For Cy3-ENTH labeling, vacuoles were reisolated (5,220 g, 4°C, 5 min) at the end of the 30-min incubation to eliminate background fluorescence from unbound probe and resuspended in the original volume of PS buffer before labeling with MDY-64 and analysis. D, G, J, M, and P show merged images of nonspecific and specific probes. Closed arrows are examples of vertex sites enriched in the specified regulatory lipid. Open arrows are outer membrane microdomains. Bars, 2 μm. As shown below (Fig. 9), ENTH displaces Vam7p from membranes after 90 min under fusion conditions which are different from the docking conditions and time (30 min) used here.


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 are required for vacuolar association of Vam7p. Vam7p association with vacuolar membranes was assayed by immunoblot in fusion reactions without inhibitor or with 2 μM GST-FYVE, 10 μM C1b, 19 μM filipin, 38 μM GST-ENTH, 2 μM SigD, or 1 μM MTM-1. Reactions were also performed in the presence of anti-Sec18p Fab to inhibit priming. Fusion reactions were incubated for 90 min at 27°C, then fractionated into membrane pellets and supernatants by centrifugation (13,000 g, 15 min, 4°C). Membranes were resuspended in 30 μl PS buffer with protease inhibitors (1 μM leupeptin, 5 μM pepstatin and 0.1 μM pefabloc-SC). (A) Equal proportions of the pellet and supernatant fractions were mixed with SDS-loading buffer, resolved by SDS-PAGE, then transferred to nitrocellulose. (B and C) Quantitative analysis of membrane-bound Vam7p by Western blots. Data represent mean values ± SEM. Relative band intensities were measured using NIH Image 1.62 (n = 4).
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Related In: Results  -  Collection

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fig9: Regulatory lipids are required for vacuolar association of Vam7p. Vam7p association with vacuolar membranes was assayed by immunoblot in fusion reactions without inhibitor or with 2 μM GST-FYVE, 10 μM C1b, 19 μM filipin, 38 μM GST-ENTH, 2 μM SigD, or 1 μM MTM-1. Reactions were also performed in the presence of anti-Sec18p Fab to inhibit priming. Fusion reactions were incubated for 90 min at 27°C, then fractionated into membrane pellets and supernatants by centrifugation (13,000 g, 15 min, 4°C). Membranes were resuspended in 30 μl PS buffer with protease inhibitors (1 μM leupeptin, 5 μM pepstatin and 0.1 μM pefabloc-SC). (A) Equal proportions of the pellet and supernatant fractions were mixed with SDS-loading buffer, resolved by SDS-PAGE, then transferred to nitrocellulose. (B and C) Quantitative analysis of membrane-bound Vam7p by Western blots. Data represent mean values ± SEM. Relative band intensities were measured using NIH Image 1.62 (n = 4).
Mentions: Lipids are enriched at the vertices of docked vacuoles. Vacuoles from DKY6281 yeast were incubated under docking conditions for 30 min at 27°C with fluorescent lipid ligands. After incubation, docking reactions were placed on ice and labeled with either FM4-64 or MDY-64. (A) Outer membrane, boundary membrane and vertex ring subdomains of docked vacuoles. (B and E) MDY-64 or (H, K, and N) FM4-64 label the entire membrane. Specific lipids were labeled with (C) 0.2 μM Cy3-FYVE to label PI(3)P, (F) 0.6 μM Cy3-ENTH to label PI(4,5)P2, (I) 5 μM filipin to label ergosterol, (L) 1 μM Alexa488-C1b to label DAG, or (O) 2.5 μM PSS-380 to label PS. For Cy3-ENTH labeling, vacuoles were reisolated (5,220 g, 4°C, 5 min) at the end of the 30-min incubation to eliminate background fluorescence from unbound probe and resuspended in the original volume of PS buffer before labeling with MDY-64 and analysis. D, G, J, M, and P show merged images of nonspecific and specific probes. Closed arrows are examples of vertex sites enriched in the specified regulatory lipid. Open arrows are outer membrane microdomains. Bars, 2 μm. As shown below (Fig. 9), ENTH displaces Vam7p from membranes after 90 min under fusion conditions which are different from the docking conditions and time (30 min) used here.

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