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Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion.

James DJ, Khodthong C, Kowalchyk JA, Martin TF - J. Cell Biol. (2008)

Bottom Line: Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles.Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition.To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT
Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

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CAPS acceleration of fusion requires a functional PH domain. (A) CAPS PH domain fails to accelerate fusion but inhibits CAPS stimulation. Fusion reactions with 5 mol% PI 4,5-P2–containing acceptor liposomes were conducted in the absence (PH −CAPS) or presence (PH +CAPS) of 1 μM CAPS, and the indicated concentrations of a CAPS GST-PH domain fusion protein. For clarity, points were selected and plotted every 7.5 min. Exponential fits of time courses are shown. (B) Phosphoinositide binding characteristics of CAPS PH domain. Indicated amounts of CAPS GST-PH domain fusion protein (either wild-type PH or mutant PHDEE) immobilized on glutathione agarose beads were incubated with liposomes containing PI 4-P, PI 4,5-P2, or PI 3,4-P2 as described in Materials and methods. (C) Liposome fusion reactions were conducted with VAMP-2 donor liposomes incubated with syntaxin-1/SNAP-25 acceptor liposomes containing 5 mol% PI 4-P, PI 4,5-P2, or PI 3,4-P2 as indicated. Rate constants were determined by exponential fit to time courses in the absence or presence of 1 μM CAPS. Mean ± SEM values are shown for three independent experiments. (D) The CAPS PH domain mutant R558D/K560E/K561E with impaired phosphoinositide binding fails to accelerate fusion. Fusion reactions with 10 mol% PI 4,5-P2–containing acceptor liposomes were conducted in the absence or presence of 1 μM wild-type CAPS or mutant CAPS (CAPS PHDEE).
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fig4: CAPS acceleration of fusion requires a functional PH domain. (A) CAPS PH domain fails to accelerate fusion but inhibits CAPS stimulation. Fusion reactions with 5 mol% PI 4,5-P2–containing acceptor liposomes were conducted in the absence (PH −CAPS) or presence (PH +CAPS) of 1 μM CAPS, and the indicated concentrations of a CAPS GST-PH domain fusion protein. For clarity, points were selected and plotted every 7.5 min. Exponential fits of time courses are shown. (B) Phosphoinositide binding characteristics of CAPS PH domain. Indicated amounts of CAPS GST-PH domain fusion protein (either wild-type PH or mutant PHDEE) immobilized on glutathione agarose beads were incubated with liposomes containing PI 4-P, PI 4,5-P2, or PI 3,4-P2 as described in Materials and methods. (C) Liposome fusion reactions were conducted with VAMP-2 donor liposomes incubated with syntaxin-1/SNAP-25 acceptor liposomes containing 5 mol% PI 4-P, PI 4,5-P2, or PI 3,4-P2 as indicated. Rate constants were determined by exponential fit to time courses in the absence or presence of 1 μM CAPS. Mean ± SEM values are shown for three independent experiments. (D) The CAPS PH domain mutant R558D/K560E/K561E with impaired phosphoinositide binding fails to accelerate fusion. Fusion reactions with 10 mol% PI 4,5-P2–containing acceptor liposomes were conducted in the absence or presence of 1 μM wild-type CAPS or mutant CAPS (CAPS PHDEE).

Mentions: CAPS contains a PI 4,5-P2–binding PH domain that is essential for CAPS function in vesicle exocytosis (Grishanin et al., 2002). Because CAPS stimulation of SNARE-dependent liposome fusion was largely PI 4,5-P2 dependent, we determined whether PI 4,5-P2 binding by the PH domain was sufficient to stimulate liposome fusion. A PH domain fusion protein failed to stimulate fusion even at high concentrations (Fig. 4 A), which indicates that binding to PI 4,5-P2 per se is not sufficient to accelerate SNARE-dependent fusion. The PH domain fusion protein did inhibit the acceleration of SNARE-dependent fusion by CAPS (Fig. 4 A). A high molar excess of the fusion protein was required for inhibition, but this likely reflects the attenuated PI 4,5-P2–binding affinity of this GST fusion protein compared with CAPS. Nonetheless, the results suggest that binding to PI 4,5-P2 is essential for CAPS stimulation of fusion. PI 4,5-P2 may function by recruiting CAPS via its PH domain to the acceptor liposome membrane. Because the PH domain of CAPS also binds PI 3,4-P2 and, to a lesser extent, PI 4-P (Fig. 4 B), we tested the activity of CAPS in accelerating fusion with acceptor liposomes that contained these phosphoinositides. Consistent with the phosphoinositide-binding properties of the CAPS PH domain, we found that CAPS promoted an eightfold increase in the rate of liposome fusion with 5 mol% PI 4,5-P2 or PI 3,4-P2 in SNAP-25/syntaxin-1 acceptor liposomes but only a 4.7-fold increase with 5 mol% PI 4-P in liposomes (Fig. 4 C). The similar phosphoinositide-binding properties of the CAPS PH domain and the CAPS acceleration of fusion are consistent with a role for the PH domain in mediating the membrane recruitment of CAPS for SNARE-dependent fusion. A lack of headgroup specificity of the CAPS PH domain for inositide bisphosphates may not be relevant in cells where PI 4,5-P2 but not PI 3,4-P2 is highly enriched in the plasma membrane. In contrast, binding to PI 4-P is likely relevant in cells where the priming of vesicle exocytosis by PI 4-P has been characterized (Olsen et al., 2003).


Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion.

James DJ, Khodthong C, Kowalchyk JA, Martin TF - J. Cell Biol. (2008)

CAPS acceleration of fusion requires a functional PH domain. (A) CAPS PH domain fails to accelerate fusion but inhibits CAPS stimulation. Fusion reactions with 5 mol% PI 4,5-P2–containing acceptor liposomes were conducted in the absence (PH −CAPS) or presence (PH +CAPS) of 1 μM CAPS, and the indicated concentrations of a CAPS GST-PH domain fusion protein. For clarity, points were selected and plotted every 7.5 min. Exponential fits of time courses are shown. (B) Phosphoinositide binding characteristics of CAPS PH domain. Indicated amounts of CAPS GST-PH domain fusion protein (either wild-type PH or mutant PHDEE) immobilized on glutathione agarose beads were incubated with liposomes containing PI 4-P, PI 4,5-P2, or PI 3,4-P2 as described in Materials and methods. (C) Liposome fusion reactions were conducted with VAMP-2 donor liposomes incubated with syntaxin-1/SNAP-25 acceptor liposomes containing 5 mol% PI 4-P, PI 4,5-P2, or PI 3,4-P2 as indicated. Rate constants were determined by exponential fit to time courses in the absence or presence of 1 μM CAPS. Mean ± SEM values are shown for three independent experiments. (D) The CAPS PH domain mutant R558D/K560E/K561E with impaired phosphoinositide binding fails to accelerate fusion. Fusion reactions with 10 mol% PI 4,5-P2–containing acceptor liposomes were conducted in the absence or presence of 1 μM wild-type CAPS or mutant CAPS (CAPS PHDEE).
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fig4: CAPS acceleration of fusion requires a functional PH domain. (A) CAPS PH domain fails to accelerate fusion but inhibits CAPS stimulation. Fusion reactions with 5 mol% PI 4,5-P2–containing acceptor liposomes were conducted in the absence (PH −CAPS) or presence (PH +CAPS) of 1 μM CAPS, and the indicated concentrations of a CAPS GST-PH domain fusion protein. For clarity, points were selected and plotted every 7.5 min. Exponential fits of time courses are shown. (B) Phosphoinositide binding characteristics of CAPS PH domain. Indicated amounts of CAPS GST-PH domain fusion protein (either wild-type PH or mutant PHDEE) immobilized on glutathione agarose beads were incubated with liposomes containing PI 4-P, PI 4,5-P2, or PI 3,4-P2 as described in Materials and methods. (C) Liposome fusion reactions were conducted with VAMP-2 donor liposomes incubated with syntaxin-1/SNAP-25 acceptor liposomes containing 5 mol% PI 4-P, PI 4,5-P2, or PI 3,4-P2 as indicated. Rate constants were determined by exponential fit to time courses in the absence or presence of 1 μM CAPS. Mean ± SEM values are shown for three independent experiments. (D) The CAPS PH domain mutant R558D/K560E/K561E with impaired phosphoinositide binding fails to accelerate fusion. Fusion reactions with 10 mol% PI 4,5-P2–containing acceptor liposomes were conducted in the absence or presence of 1 μM wild-type CAPS or mutant CAPS (CAPS PHDEE).
Mentions: CAPS contains a PI 4,5-P2–binding PH domain that is essential for CAPS function in vesicle exocytosis (Grishanin et al., 2002). Because CAPS stimulation of SNARE-dependent liposome fusion was largely PI 4,5-P2 dependent, we determined whether PI 4,5-P2 binding by the PH domain was sufficient to stimulate liposome fusion. A PH domain fusion protein failed to stimulate fusion even at high concentrations (Fig. 4 A), which indicates that binding to PI 4,5-P2 per se is not sufficient to accelerate SNARE-dependent fusion. The PH domain fusion protein did inhibit the acceleration of SNARE-dependent fusion by CAPS (Fig. 4 A). A high molar excess of the fusion protein was required for inhibition, but this likely reflects the attenuated PI 4,5-P2–binding affinity of this GST fusion protein compared with CAPS. Nonetheless, the results suggest that binding to PI 4,5-P2 is essential for CAPS stimulation of fusion. PI 4,5-P2 may function by recruiting CAPS via its PH domain to the acceptor liposome membrane. Because the PH domain of CAPS also binds PI 3,4-P2 and, to a lesser extent, PI 4-P (Fig. 4 B), we tested the activity of CAPS in accelerating fusion with acceptor liposomes that contained these phosphoinositides. Consistent with the phosphoinositide-binding properties of the CAPS PH domain, we found that CAPS promoted an eightfold increase in the rate of liposome fusion with 5 mol% PI 4,5-P2 or PI 3,4-P2 in SNAP-25/syntaxin-1 acceptor liposomes but only a 4.7-fold increase with 5 mol% PI 4-P in liposomes (Fig. 4 C). The similar phosphoinositide-binding properties of the CAPS PH domain and the CAPS acceleration of fusion are consistent with a role for the PH domain in mediating the membrane recruitment of CAPS for SNARE-dependent fusion. A lack of headgroup specificity of the CAPS PH domain for inositide bisphosphates may not be relevant in cells where PI 4,5-P2 but not PI 3,4-P2 is highly enriched in the plasma membrane. In contrast, binding to PI 4-P is likely relevant in cells where the priming of vesicle exocytosis by PI 4-P has been characterized (Olsen et al., 2003).

Bottom Line: Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles.Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition.To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT
Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

Show MeSH
Related in: MedlinePlus