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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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Regulatory lipids are not required for Ypt7p-dependent tethering. For quantitative microscopic assay of docking (Mayer and Wickner, 1997), vacuoles were incubated with PS buffer or treated with 2.8 μM Gdi1p and 11.4 μM Gyp1-46p for 10 min at 27°C in the absence of ATP. Aliquots were then added to chilled tubes containing docking buffer, 2 μM GST-FYVE, 10 μM MED, 30 μM ENTH, 10 μM C1b or 19 μM filipin. Reactions were supplemented with the docking reaction ATP-regenerating system and returned to 27°C for 20 min. After incubation, docking reactions were placed on ice, incubated for 2 min with FM4-64 and mounted on slides for analysis. For each condition, 10 random fields were scored for cluster size. Vacuole clusters (A, black bars) include some vacuoles of enlarged diameter, reflecting fusion (not depicted).
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fig2: Regulatory lipids are not required for Ypt7p-dependent tethering. For quantitative microscopic assay of docking (Mayer and Wickner, 1997), vacuoles were incubated with PS buffer or treated with 2.8 μM Gdi1p and 11.4 μM Gyp1-46p for 10 min at 27°C in the absence of ATP. Aliquots were then added to chilled tubes containing docking buffer, 2 μM GST-FYVE, 10 μM MED, 30 μM ENTH, 10 μM C1b or 19 μM filipin. Reactions were supplemented with the docking reaction ATP-regenerating system and returned to 27°C for 20 min. After incubation, docking reactions were placed on ice, incubated for 2 min with FM4-64 and mounted on slides for analysis. For each condition, 10 random fields were scored for cluster size. Vacuole clusters (A, black bars) include some vacuoles of enlarged diameter, reflecting fusion (not depicted).

Mentions: Even high levels of these lipid ligands do not prevent Ypt7p-dependent tethering (Fig. 2). To assay tethering (Mayer and Wickner, 1997; Wang et al., 2002), vacuoles were incubated with ATP for 30 min to allow docking into clusters. Random microscopic fields were photographed and scored for vacuoles per cluster. Though most vacuoles participate in cluster formation (Fig. 2 A, black bars), clustering was blocked by Ypt7p extraction by Gdi1p, aided by the GAP Gyp1-46 (gray bars). Vacuole docking is followed by fusion, lowering the number of vacuoles per cluster; thus this assay only provides a minimal estimate of docking. Blocking fusion by lipid ligands enhances the number of vacuoles per cluster (Fig. 2 B-E, black bars). Nevertheless, this is authentic docking as it is blocked by Ypt7p extraction. Filipin inhibits at multiple stages of the vacuole fusion pathway including priming (Kato and Wickner, 2001), and this parallel slowing of each reaction stage yielded a distribution of vacuoles per cluster (Fig. 2 F) that resembled the uninhibited reaction (Fig. 2 A). The clustering of filipin treated vacuoles was still inhibited by inactivation of Ypt7p. Thus, we used lipid ligands to quantify the distribution of relevant proteins and lipids on vacuoles which were tethered via the physiological pathway.


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)

Regulatory lipids are not required for Ypt7p-dependent tethering. For quantitative microscopic assay of docking (Mayer and Wickner, 1997), vacuoles were incubated with PS buffer or treated with 2.8 μM Gdi1p and 11.4 μM Gyp1-46p for 10 min at 27°C in the absence of ATP. Aliquots were then added to chilled tubes containing docking buffer, 2 μM GST-FYVE, 10 μM MED, 30 μM ENTH, 10 μM C1b or 19 μM filipin. Reactions were supplemented with the docking reaction ATP-regenerating system and returned to 27°C for 20 min. After incubation, docking reactions were placed on ice, incubated for 2 min with FM4-64 and mounted on slides for analysis. For each condition, 10 random fields were scored for cluster size. Vacuole clusters (A, black bars) include some vacuoles of enlarged diameter, reflecting fusion (not depicted).
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Related In: Results  -  Collection

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

fig2: Regulatory lipids are not required for Ypt7p-dependent tethering. For quantitative microscopic assay of docking (Mayer and Wickner, 1997), vacuoles were incubated with PS buffer or treated with 2.8 μM Gdi1p and 11.4 μM Gyp1-46p for 10 min at 27°C in the absence of ATP. Aliquots were then added to chilled tubes containing docking buffer, 2 μM GST-FYVE, 10 μM MED, 30 μM ENTH, 10 μM C1b or 19 μM filipin. Reactions were supplemented with the docking reaction ATP-regenerating system and returned to 27°C for 20 min. After incubation, docking reactions were placed on ice, incubated for 2 min with FM4-64 and mounted on slides for analysis. For each condition, 10 random fields were scored for cluster size. Vacuole clusters (A, black bars) include some vacuoles of enlarged diameter, reflecting fusion (not depicted).
Mentions: Even high levels of these lipid ligands do not prevent Ypt7p-dependent tethering (Fig. 2). To assay tethering (Mayer and Wickner, 1997; Wang et al., 2002), vacuoles were incubated with ATP for 30 min to allow docking into clusters. Random microscopic fields were photographed and scored for vacuoles per cluster. Though most vacuoles participate in cluster formation (Fig. 2 A, black bars), clustering was blocked by Ypt7p extraction by Gdi1p, aided by the GAP Gyp1-46 (gray bars). Vacuole docking is followed by fusion, lowering the number of vacuoles per cluster; thus this assay only provides a minimal estimate of docking. Blocking fusion by lipid ligands enhances the number of vacuoles per cluster (Fig. 2 B-E, black bars). Nevertheless, this is authentic docking as it is blocked by Ypt7p extraction. Filipin inhibits at multiple stages of the vacuole fusion pathway including priming (Kato and Wickner, 2001), and this parallel slowing of each reaction stage yielded a distribution of vacuoles per cluster (Fig. 2 F) that resembled the uninhibited reaction (Fig. 2 A). The clustering of filipin treated vacuoles was still inhibited by inactivation of Ypt7p. Thus, we used lipid ligands to quantify the distribution of relevant proteins and lipids on vacuoles which were tethered via the physiological pathway.

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