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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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Quantitation of lipid enrichment at vertices. Docking reactions were performed as in Fig. 3. After 30 min at 27°C with fluorescent lipid ligands, reactions were placed on ice, labeled with either FM4-64 or MDY-64, and analyzed. Cumulative distribution plots depict the percentile values of each specific lipid ligand/nonspecific lipophilic dye ratio for each of the three microdomains. Each curve is comprised of measurements from ≥10 vacuole clusters where the maximum intensity was determined for every vertex and midpoint of boundary and outer membrane. Intensities were measured in both fluorescence channels at each subdomain and are expressed as a ratios of specific to nonspecific label. Outer membrane ratios were normalized to a value of 1 and the enrichment of specific label at vertices and boundaries were expressed relative to outer membrane intensities. Each ratio in a dataset is ordered and plotted versus the percentile rank of the values. Lipids were labeled with the same concentrations of ligands as in Fig. 3. (G) Geometric means with their 95% confidence intervals for the data from the cumulative distribution plots in A–F.
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fig4: Quantitation of lipid enrichment at vertices. Docking reactions were performed as in Fig. 3. After 30 min at 27°C with fluorescent lipid ligands, reactions were placed on ice, labeled with either FM4-64 or MDY-64, and analyzed. Cumulative distribution plots depict the percentile values of each specific lipid ligand/nonspecific lipophilic dye ratio for each of the three microdomains. Each curve is comprised of measurements from ≥10 vacuole clusters where the maximum intensity was determined for every vertex and midpoint of boundary and outer membrane. Intensities were measured in both fluorescence channels at each subdomain and are expressed as a ratios of specific to nonspecific label. Outer membrane ratios were normalized to a value of 1 and the enrichment of specific label at vertices and boundaries were expressed relative to outer membrane intensities. Each ratio in a dataset is ordered and plotted versus the percentile rank of the values. Lipids were labeled with the same concentrations of ligands as in Fig. 3. (G) Geometric means with their 95% confidence intervals for the data from the cumulative distribution plots in A–F.

Mentions: The ratio data for each treatment, probe, and morphological location are shown in cumulative distribution plots where individual measurements are ranked, then each is plotted at its rank percentile (Fig. 4, A–F). Cy3-FYVE, and thus PI(3)P, was enriched at vertices (Fig. 4 A). Similarly, rhodamine-MED and Cy3-ENTH, and thus PI(4,5)P2, were also vertex enriched (Fig. 4, B and C). Filipin, an ergosterol-binding drug, and Alexa488-C1b, a DAG probe, were also enriched at vertices (Fig. 4, D and E). In contrast, PSS-380 (Koulov et al., 2003) was not enriched at vertex or boundary regions (Fig. 4 F), showing the selectivity of vertex lipid enrichment. In sum, PI(3)P, PI(4,5)P2, DAG, and ergosterol, but not PS, accumulate at vertex sites. Fig. 4 G shows the geometric means and 95% confidence intervals for these means for the data in Fig. 4 (A–F). As shown below, the recruitment of these lipids is subject to complex regulation.


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)

Quantitation of lipid enrichment at vertices. Docking reactions were performed as in Fig. 3. After 30 min at 27°C with fluorescent lipid ligands, reactions were placed on ice, labeled with either FM4-64 or MDY-64, and analyzed. Cumulative distribution plots depict the percentile values of each specific lipid ligand/nonspecific lipophilic dye ratio for each of the three microdomains. Each curve is comprised of measurements from ≥10 vacuole clusters where the maximum intensity was determined for every vertex and midpoint of boundary and outer membrane. Intensities were measured in both fluorescence channels at each subdomain and are expressed as a ratios of specific to nonspecific label. Outer membrane ratios were normalized to a value of 1 and the enrichment of specific label at vertices and boundaries were expressed relative to outer membrane intensities. Each ratio in a dataset is ordered and plotted versus the percentile rank of the values. Lipids were labeled with the same concentrations of ligands as in Fig. 3. (G) Geometric means with their 95% confidence intervals for the data from the cumulative distribution plots in A–F.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172599&req=5

fig4: Quantitation of lipid enrichment at vertices. Docking reactions were performed as in Fig. 3. After 30 min at 27°C with fluorescent lipid ligands, reactions were placed on ice, labeled with either FM4-64 or MDY-64, and analyzed. Cumulative distribution plots depict the percentile values of each specific lipid ligand/nonspecific lipophilic dye ratio for each of the three microdomains. Each curve is comprised of measurements from ≥10 vacuole clusters where the maximum intensity was determined for every vertex and midpoint of boundary and outer membrane. Intensities were measured in both fluorescence channels at each subdomain and are expressed as a ratios of specific to nonspecific label. Outer membrane ratios were normalized to a value of 1 and the enrichment of specific label at vertices and boundaries were expressed relative to outer membrane intensities. Each ratio in a dataset is ordered and plotted versus the percentile rank of the values. Lipids were labeled with the same concentrations of ligands as in Fig. 3. (G) Geometric means with their 95% confidence intervals for the data from the cumulative distribution plots in A–F.
Mentions: The ratio data for each treatment, probe, and morphological location are shown in cumulative distribution plots where individual measurements are ranked, then each is plotted at its rank percentile (Fig. 4, A–F). Cy3-FYVE, and thus PI(3)P, was enriched at vertices (Fig. 4 A). Similarly, rhodamine-MED and Cy3-ENTH, and thus PI(4,5)P2, were also vertex enriched (Fig. 4, B and C). Filipin, an ergosterol-binding drug, and Alexa488-C1b, a DAG probe, were also enriched at vertices (Fig. 4, D and E). In contrast, PSS-380 (Koulov et al., 2003) was not enriched at vertex or boundary regions (Fig. 4 F), showing the selectivity of vertex lipid enrichment. In sum, PI(3)P, PI(4,5)P2, DAG, and ergosterol, but not PS, accumulate at vertex sites. Fig. 4 G shows the geometric means and 95% confidence intervals for these means for the data in Fig. 4 (A–F). As shown below, the recruitment of these lipids is subject to complex regulation.

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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Related in: MedlinePlus