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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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Protein ligands affect PI(3)P localization. (A and B) Docking reactions using DKY6281 vacuoles labeled with 0.2 μM Cy3-FYVE (as described in Figs. 2 and 3) were treated with 750 nM (excess) Sec17p, 1.2 μM Sec18p, both excess Sec17p and Sec18p, 190 nM anti-Vam3 Fab, 0.5 mM jasplakinolide, 0.8 mM LatB, 11.4 μM Gyp1-46p (Gyp), or 250 μM 3NC. After 30 min at 27°C, reactions were placed on ice and prepared for fluorescence microscopy. Geometric mean values ± 95% confidence intervals of the relative enrichment at vertices are shown. (C) To determine whether the effects of LatB were due to alterations in actin polymerization, vacuoles were harvested from isogenic DDY182 yeast containing Act1 or the latrunculin resistant mutant act1-113 and used in docking experiments. Vacuoles were labeled with Cy3-FYVE and incubated during docking reactions with 0.8 mM LatB or 30 μM ENTH where indicated. Samples were prepared and analyzed as in Figs. 2 and 3.
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fig6: Protein ligands affect PI(3)P localization. (A and B) Docking reactions using DKY6281 vacuoles labeled with 0.2 μM Cy3-FYVE (as described in Figs. 2 and 3) were treated with 750 nM (excess) Sec17p, 1.2 μM Sec18p, both excess Sec17p and Sec18p, 190 nM anti-Vam3 Fab, 0.5 mM jasplakinolide, 0.8 mM LatB, 11.4 μM Gyp1-46p (Gyp), or 250 μM 3NC. After 30 min at 27°C, reactions were placed on ice and prepared for fluorescence microscopy. Geometric mean values ± 95% confidence intervals of the relative enrichment at vertices are shown. (C) To determine whether the effects of LatB were due to alterations in actin polymerization, vacuoles were harvested from isogenic DDY182 yeast containing Act1 or the latrunculin resistant mutant act1-113 and used in docking experiments. Vacuoles were labeled with Cy3-FYVE and incubated during docking reactions with 0.8 mM LatB or 30 μM ENTH where indicated. Samples were prepared and analyzed as in Figs. 2 and 3.

Mentions: We next examined whether protein fusion catalysts affect the vertex enrichment of regulatory lipids. To test the effect of SNAREs, we exploited recombinant Sec17p which, at high concentrations, drives unpaired SNAREs into cis complexes (Wang et al., 2000) and competes with HOPS for binding to SNARE complexes (K. Collins, personal communication). Excess Sec17p reduced Cy3-FYVE enrichment at vertices (P < 0.0001; Fig. 6 A). This was reversed when excess Sec18p was present (Fig. 6 A), establishing the specificity of Sec17p action. Anti-Vam3p, which blocks SNARE vertex enrichment (Wang et al., 2003), also prevented PI(3)P vertex enrichment (P < 0.00001; Fig. 6 A), confirming the role of SNAREs in PI(3)P function.


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)

Protein ligands affect PI(3)P localization. (A and B) Docking reactions using DKY6281 vacuoles labeled with 0.2 μM Cy3-FYVE (as described in Figs. 2 and 3) were treated with 750 nM (excess) Sec17p, 1.2 μM Sec18p, both excess Sec17p and Sec18p, 190 nM anti-Vam3 Fab, 0.5 mM jasplakinolide, 0.8 mM LatB, 11.4 μM Gyp1-46p (Gyp), or 250 μM 3NC. After 30 min at 27°C, reactions were placed on ice and prepared for fluorescence microscopy. Geometric mean values ± 95% confidence intervals of the relative enrichment at vertices are shown. (C) To determine whether the effects of LatB were due to alterations in actin polymerization, vacuoles were harvested from isogenic DDY182 yeast containing Act1 or the latrunculin resistant mutant act1-113 and used in docking experiments. Vacuoles were labeled with Cy3-FYVE and incubated during docking reactions with 0.8 mM LatB or 30 μM ENTH where indicated. Samples were prepared and analyzed as in Figs. 2 and 3.
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getmorefigures.php?uid=PMC2172599&req=5

fig6: Protein ligands affect PI(3)P localization. (A and B) Docking reactions using DKY6281 vacuoles labeled with 0.2 μM Cy3-FYVE (as described in Figs. 2 and 3) were treated with 750 nM (excess) Sec17p, 1.2 μM Sec18p, both excess Sec17p and Sec18p, 190 nM anti-Vam3 Fab, 0.5 mM jasplakinolide, 0.8 mM LatB, 11.4 μM Gyp1-46p (Gyp), or 250 μM 3NC. After 30 min at 27°C, reactions were placed on ice and prepared for fluorescence microscopy. Geometric mean values ± 95% confidence intervals of the relative enrichment at vertices are shown. (C) To determine whether the effects of LatB were due to alterations in actin polymerization, vacuoles were harvested from isogenic DDY182 yeast containing Act1 or the latrunculin resistant mutant act1-113 and used in docking experiments. Vacuoles were labeled with Cy3-FYVE and incubated during docking reactions with 0.8 mM LatB or 30 μM ENTH where indicated. Samples were prepared and analyzed as in Figs. 2 and 3.
Mentions: We next examined whether protein fusion catalysts affect the vertex enrichment of regulatory lipids. To test the effect of SNAREs, we exploited recombinant Sec17p which, at high concentrations, drives unpaired SNAREs into cis complexes (Wang et al., 2000) and competes with HOPS for binding to SNARE complexes (K. Collins, personal communication). Excess Sec17p reduced Cy3-FYVE enrichment at vertices (P < 0.0001; Fig. 6 A). This was reversed when excess Sec18p was present (Fig. 6 A), establishing the specificity of Sec17p action. Anti-Vam3p, which blocks SNARE vertex enrichment (Wang et al., 2003), also prevented PI(3)P vertex enrichment (P < 0.00001; Fig. 6 A), confirming the role of SNAREs in PI(3)P function.

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