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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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Ligands and enzymes which target phosphoinositides, DAG and ergosterol inhibit vacuole fusion. Fusion reactions with vacuoles from BJ3505 and DKY6281 were performed in the absence or presence of the indicated concentrations of GST-FYVE (A), MED (B), ENTH (C), C1b (D), Filipin (E), PSS-380 (F), MTM-1 (G), or SigD (H), added from the start of the reaction. Fusion was assayed by phosphatase activity and expressed in U. Closed arrows indicate inhibitory concentrations used in this study. Open arrows indicate sub-inhibitory concentrations used for determining lipid localization.
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fig1: Ligands and enzymes which target phosphoinositides, DAG and ergosterol inhibit vacuole fusion. Fusion reactions with vacuoles from BJ3505 and DKY6281 were performed in the absence or presence of the indicated concentrations of GST-FYVE (A), MED (B), ENTH (C), C1b (D), Filipin (E), PSS-380 (F), MTM-1 (G), or SigD (H), added from the start of the reaction. Fusion was assayed by phosphatase activity and expressed in U. Closed arrows indicate inhibitory concentrations used in this study. Open arrows indicate sub-inhibitory concentrations used for determining lipid localization.

Mentions: Vacuole fusion requires PI(3)P (Boeddinghaus et al., 2002), PI(4,5)P2 (Mayer et al., 2000), ergosterol (Kato and Wickner, 2001), and DAG (Jun et al., 2005). We now use specific lipid-binding ligands for two different purposes, at either inhibitory concentrations (Fig. 1, closed arrows) or, for probing lipid localization, at subinhibitory concentrations (Fig. 1, open arrows).


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)

Ligands and enzymes which target phosphoinositides, DAG and ergosterol inhibit vacuole fusion. Fusion reactions with vacuoles from BJ3505 and DKY6281 were performed in the absence or presence of the indicated concentrations of GST-FYVE (A), MED (B), ENTH (C), C1b (D), Filipin (E), PSS-380 (F), MTM-1 (G), or SigD (H), added from the start of the reaction. Fusion was assayed by phosphatase activity and expressed in U. Closed arrows indicate inhibitory concentrations used in this study. Open arrows indicate sub-inhibitory concentrations used for determining lipid localization.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172599&req=5

fig1: Ligands and enzymes which target phosphoinositides, DAG and ergosterol inhibit vacuole fusion. Fusion reactions with vacuoles from BJ3505 and DKY6281 were performed in the absence or presence of the indicated concentrations of GST-FYVE (A), MED (B), ENTH (C), C1b (D), Filipin (E), PSS-380 (F), MTM-1 (G), or SigD (H), added from the start of the reaction. Fusion was assayed by phosphatase activity and expressed in U. Closed arrows indicate inhibitory concentrations used in this study. Open arrows indicate sub-inhibitory concentrations used for determining lipid localization.
Mentions: Vacuole fusion requires PI(3)P (Boeddinghaus et al., 2002), PI(4,5)P2 (Mayer et al., 2000), ergosterol (Kato and Wickner, 2001), and DAG (Jun et al., 2005). We now use specific lipid-binding ligands for two different purposes, at either inhibitory concentrations (Fig. 1, closed arrows) or, for probing lipid localization, at subinhibitory concentrations (Fig. 1, open arrows).

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