Limits...
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.

Show MeSH

Related in: MedlinePlus

Interdependence of the vertex enrichment of regulatory lipids. Docking reactions were labeled with (A) 0.2 μM Cy3-FYVE or (B) 5 μM filipin and incubated with either 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or buffer alone. After 30 min at 27°C, reactions were placed on ice, labeled with MDY-64 (A) or FM4-64 (B), and prepared for fluorescence microscopy. Relative enrichments of specific probes were determined as in Fig. 3. Data are presented as geometric mean values ± 95% confidence intervals of the relative enrichment at vertices.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172599&req=5

fig5: Interdependence of the vertex enrichment of regulatory lipids. Docking reactions were labeled with (A) 0.2 μM Cy3-FYVE or (B) 5 μM filipin and incubated with either 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or buffer alone. After 30 min at 27°C, reactions were placed on ice, labeled with MDY-64 (A) or FM4-64 (B), and prepared for fluorescence microscopy. Relative enrichments of specific probes were determined as in Fig. 3. Data are presented as geometric mean values ± 95% confidence intervals of the relative enrichment at vertices.

Mentions: We monitored PI(3)P distribution when other vertex-enriched lipids were sequestered by ligands. The addition of C1b, MED, ENTH, or filipin inhibited PI(3)P vertex enrichment (P < 0.00001; Fig. 5 A). Lipid ligands also affected ergosterol vertex enrichment. Filipin staining showed that ergosterol was enriched at vertices (Fig. 4, D and G). ENTH blocked ergosterol vertex enrichment (P < 0.01; Fig. 5 B), showing that PI(4,5)P2 is required for ergosterol vertex accumulation. In contrast, the PI(3)P ligand PX augmented ergosterol levels at vertices (P < 0.001; Fig. 5 B), suggesting either that PI(3)P inhibits ergosterol vertex enrichment or that inhibition of fusion by PX domain prevents fusion-triggered dissociation of assembled vertices. Though MED is bound mainly to PI(4,5)P2 in cells (McLaughlin et al., 2002), it can also bind PI(3,4)P2 (Wang et al., 2001a), PI(4)P (Seki et al., 1996) and PI(3)P (unpublished data). Apparently, the positive effect of MED on ergosterol vertex enrichment (by blocking fusion-induced vertex loss) and the negative effect (through sequestration of PI(4,5)P2) are balanced. C1b only had a minor effect on ergosterol accumulation at vertices, suggesting that DAG is not required for ergosterol enrichment. Though C1b blocked PI(3)P localization to vertices (Fig. 4 A), PI(3)P is presumably not needed for ergosterol enrichment at vertices because ergosterol enrichment is not inhibited by PX domain. These effects could occur directly, through disruption of lipid microdomain architecture, or indirectly through lipid binding proteins such as Vam7p.


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)

Interdependence of the vertex enrichment of regulatory lipids. Docking reactions were labeled with (A) 0.2 μM Cy3-FYVE or (B) 5 μM filipin and incubated with either 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or buffer alone. After 30 min at 27°C, reactions were placed on ice, labeled with MDY-64 (A) or FM4-64 (B), and prepared for fluorescence microscopy. Relative enrichments of specific probes were determined as in Fig. 3. Data are presented as geometric mean values ± 95% confidence intervals of the relative enrichment at vertices.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Interdependence of the vertex enrichment of regulatory lipids. Docking reactions were labeled with (A) 0.2 μM Cy3-FYVE or (B) 5 μM filipin and incubated with either 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or buffer alone. After 30 min at 27°C, reactions were placed on ice, labeled with MDY-64 (A) or FM4-64 (B), and prepared for fluorescence microscopy. Relative enrichments of specific probes were determined as in Fig. 3. Data are presented as geometric mean values ± 95% confidence intervals of the relative enrichment at vertices.
Mentions: We monitored PI(3)P distribution when other vertex-enriched lipids were sequestered by ligands. The addition of C1b, MED, ENTH, or filipin inhibited PI(3)P vertex enrichment (P < 0.00001; Fig. 5 A). Lipid ligands also affected ergosterol vertex enrichment. Filipin staining showed that ergosterol was enriched at vertices (Fig. 4, D and G). ENTH blocked ergosterol vertex enrichment (P < 0.01; Fig. 5 B), showing that PI(4,5)P2 is required for ergosterol vertex accumulation. In contrast, the PI(3)P ligand PX augmented ergosterol levels at vertices (P < 0.001; Fig. 5 B), suggesting either that PI(3)P inhibits ergosterol vertex enrichment or that inhibition of fusion by PX domain prevents fusion-triggered dissociation of assembled vertices. Though MED is bound mainly to PI(4,5)P2 in cells (McLaughlin et al., 2002), it can also bind PI(3,4)P2 (Wang et al., 2001a), PI(4)P (Seki et al., 1996) and PI(3)P (unpublished data). Apparently, the positive effect of MED on ergosterol vertex enrichment (by blocking fusion-induced vertex loss) and the negative effect (through sequestration of PI(4,5)P2) are balanced. C1b only had a minor effect on ergosterol accumulation at vertices, suggesting that DAG is not required for ergosterol enrichment. Though C1b blocked PI(3)P localization to vertices (Fig. 4 A), PI(3)P is presumably not needed for ergosterol enrichment at vertices because ergosterol enrichment is not inhibited by PX domain. These effects could occur directly, through disruption of lipid microdomain architecture, or indirectly through lipid binding proteins such as Vam7p.

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