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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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Enzymatic modification of regulatory lipids inhibits vertex assembly. Docking reactions (30 min, 27°C), using vacuoles containing GFP-Ypt7p (A) or vacuoles labeled with Cy3-FYVE to mark PI(3)P (B) or filipin to mark ergosterol (C), bore 1 μM MTM-1, 2 μM SigD, 1 U/ml PI-PLC, or 2.7 μM Plc1p. After incubation, reactions were placed on ice, counterstained with FM4-64 (A and C) or MDY-64 (B), and prepared for microscopic analysis. Geometric means ± 95% confidence intervals of the relative vertex enrichment are shown.
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fig8: Enzymatic modification of regulatory lipids inhibits vertex assembly. Docking reactions (30 min, 27°C), using vacuoles containing GFP-Ypt7p (A) or vacuoles labeled with Cy3-FYVE to mark PI(3)P (B) or filipin to mark ergosterol (C), bore 1 μM MTM-1, 2 μM SigD, 1 U/ml PI-PLC, or 2.7 μM Plc1p. After incubation, reactions were placed on ice, counterstained with FM4-64 (A and C) or MDY-64 (B), and prepared for microscopic analysis. Geometric means ± 95% confidence intervals of the relative vertex enrichment are shown.

Mentions: To complement the above studies, we used lipid modifying enzymes. MTM-1, which inhibits fusion (Fig. 1 G), blocked Ypt7p vertex enrichment (P < 0.00001; Fig. 8 A). This mirrors the effect of FYVE domain on Ypt7p enrichment (Fig. 7 A), and confirms that PI(3)P is essential for Ypt7p vertex enrichment. MTM-1-mediated depletion of PI(3)P enhanced ergosterol enrichment (P < 0.01; Fig. 8 C), in accord with our observations using PX (Fig. 5 B). To complement our studies with ENTH on the role of PI(4,5)P2, we used the PI 5-phosphatase SigD and PI(4,5)P2 specific lipase Plc1p (Flick and Thorner, 1993). As for ENTH, SigD and Plc1p inhibited the vertex localization of Ypt7p and PI(3)P (Fig. 8, A and B; P < 0.00001). Interestingly, ergosterol vertex enrichment was inhibited by SigD (P < 0.01) but not by Plc1p (Fig. 8 C); the generation of DAG by Plc1p may slightly stimulate ergosterol enrichment, in accord with the modest decrease caused by C1b (Fig. 5 B). We also tested the role of unmodified PI. We have previously shown that the hydrolysis of PI by PI-PLC inhibits vacuole fusion (Mayer et al., 2000). This inhibition was relieved by the addition of PI(4,5)P2 (ibid), suggesting that vacuolar PI primarily serves as a source for the synthesis of PI(4)P and PI(4,5)P2. PI-PLC inhibited vertex enrichment of Ypt7p and PI(3)P (P < 0.00001; Fig. 8, A and B). The similarity of these results with those seen with Plc1p suggests that PI is needed for PI(4,5)P2 synthesis for vacuole fusion.


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)

Enzymatic modification of regulatory lipids inhibits vertex assembly. Docking reactions (30 min, 27°C), using vacuoles containing GFP-Ypt7p (A) or vacuoles labeled with Cy3-FYVE to mark PI(3)P (B) or filipin to mark ergosterol (C), bore 1 μM MTM-1, 2 μM SigD, 1 U/ml PI-PLC, or 2.7 μM Plc1p. After incubation, reactions were placed on ice, counterstained with FM4-64 (A and C) or MDY-64 (B), and prepared for microscopic analysis. Geometric means ± 95% confidence intervals of the relative vertex enrichment are shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Enzymatic modification of regulatory lipids inhibits vertex assembly. Docking reactions (30 min, 27°C), using vacuoles containing GFP-Ypt7p (A) or vacuoles labeled with Cy3-FYVE to mark PI(3)P (B) or filipin to mark ergosterol (C), bore 1 μM MTM-1, 2 μM SigD, 1 U/ml PI-PLC, or 2.7 μM Plc1p. After incubation, reactions were placed on ice, counterstained with FM4-64 (A and C) or MDY-64 (B), and prepared for microscopic analysis. Geometric means ± 95% confidence intervals of the relative vertex enrichment are shown.
Mentions: To complement the above studies, we used lipid modifying enzymes. MTM-1, which inhibits fusion (Fig. 1 G), blocked Ypt7p vertex enrichment (P < 0.00001; Fig. 8 A). This mirrors the effect of FYVE domain on Ypt7p enrichment (Fig. 7 A), and confirms that PI(3)P is essential for Ypt7p vertex enrichment. MTM-1-mediated depletion of PI(3)P enhanced ergosterol enrichment (P < 0.01; Fig. 8 C), in accord with our observations using PX (Fig. 5 B). To complement our studies with ENTH on the role of PI(4,5)P2, we used the PI 5-phosphatase SigD and PI(4,5)P2 specific lipase Plc1p (Flick and Thorner, 1993). As for ENTH, SigD and Plc1p inhibited the vertex localization of Ypt7p and PI(3)P (Fig. 8, A and B; P < 0.00001). Interestingly, ergosterol vertex enrichment was inhibited by SigD (P < 0.01) but not by Plc1p (Fig. 8 C); the generation of DAG by Plc1p may slightly stimulate ergosterol enrichment, in accord with the modest decrease caused by C1b (Fig. 5 B). We also tested the role of unmodified PI. We have previously shown that the hydrolysis of PI by PI-PLC inhibits vacuole fusion (Mayer et al., 2000). This inhibition was relieved by the addition of PI(4,5)P2 (ibid), suggesting that vacuolar PI primarily serves as a source for the synthesis of PI(4)P and PI(4,5)P2. PI-PLC inhibited vertex enrichment of Ypt7p and PI(3)P (P < 0.00001; Fig. 8, A and B). The similarity of these results with those seen with Plc1p suggests that PI is needed for PI(4,5)P2 synthesis for vacuole fusion.

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