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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 accelerates liposome fusion in a PI 4,5-P2– and SNARE-dependent manner. (A) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 (indicated by t for t-SNAREs) in 90:10 mol% PC/PI 4,5-P2 were incubated with donor liposomes with 100 copies of VAMP-2 in 85:15 mol% PC/PS in the absence or presence of 1 μM CAPS. Similar incubations with donor and acceptor liposomes in 85:15 mol% PC/PS in the absence or presence of 1 μM CAPS were conducted. NBD fluorescence in parallel reactions with Pf liposomes was used to correct all data. Mean values ± SEM for five independent experiments are shown. (B) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 in 90:10 mol% PC/PI 4,5-P2 were incubated with VAMP-2–containing donor liposomes in the absence or presence of 1 μM CAPS. Parallel incubations used botulinum neurotoxin B–treated donor liposomes incubated without or with 1 μM CAPS. (C) Syntaxin-1/SNAP-25 acceptor liposomes (indicated by t for t-SNAREs) with 10 mol% PI 4,5-P2 or with 15% PS were incubated with VAMP-2 donor liposomes in the presence of indicated CAPS concentrations. Exponential fits of time courses were used to derive rate constants (k). Mean ± SEM values are shown for three independent experiments. (D) Rate constants were determined for fusion reactions without or with 1 μM CAPS in incubations that contained syntaxin-1/SNAP-25 acceptor liposomes with the indicated mole percentage PI 4,5-P2. Data in inset show the percentage of maximal NBD fluorescence at 120 min in incubations without or with CAPS. Mean ± SEM values are shown for four independent experiments. (E) Extent of fusion in 120 min was determined in the absence or presence of 1 μM CAPS with donor VAMP-2 or acceptor syntaxin-1/SNAP-25 liposomes that contained 85:15 mol% PC/PS or 90:10 mol% PC/PI 4,5-P2 as indicated. Mean ± SEM values are shown for three independent experiments (*, P < 0.01 for PC/PIP2 compared with PC/PS; †, P = 0.05 compared with parallel incubations without CAPS).
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fig3: CAPS accelerates liposome fusion in a PI 4,5-P2– and SNARE-dependent manner. (A) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 (indicated by t for t-SNAREs) in 90:10 mol% PC/PI 4,5-P2 were incubated with donor liposomes with 100 copies of VAMP-2 in 85:15 mol% PC/PS in the absence or presence of 1 μM CAPS. Similar incubations with donor and acceptor liposomes in 85:15 mol% PC/PS in the absence or presence of 1 μM CAPS were conducted. NBD fluorescence in parallel reactions with Pf liposomes was used to correct all data. Mean values ± SEM for five independent experiments are shown. (B) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 in 90:10 mol% PC/PI 4,5-P2 were incubated with VAMP-2–containing donor liposomes in the absence or presence of 1 μM CAPS. Parallel incubations used botulinum neurotoxin B–treated donor liposomes incubated without or with 1 μM CAPS. (C) Syntaxin-1/SNAP-25 acceptor liposomes (indicated by t for t-SNAREs) with 10 mol% PI 4,5-P2 or with 15% PS were incubated with VAMP-2 donor liposomes in the presence of indicated CAPS concentrations. Exponential fits of time courses were used to derive rate constants (k). Mean ± SEM values are shown for three independent experiments. (D) Rate constants were determined for fusion reactions without or with 1 μM CAPS in incubations that contained syntaxin-1/SNAP-25 acceptor liposomes with the indicated mole percentage PI 4,5-P2. Data in inset show the percentage of maximal NBD fluorescence at 120 min in incubations without or with CAPS. Mean ± SEM values are shown for four independent experiments. (E) Extent of fusion in 120 min was determined in the absence or presence of 1 μM CAPS with donor VAMP-2 or acceptor syntaxin-1/SNAP-25 liposomes that contained 85:15 mol% PC/PS or 90:10 mol% PC/PI 4,5-P2 as indicated. Mean ± SEM values are shown for three independent experiments (*, P < 0.01 for PC/PIP2 compared with PC/PS; †, P = 0.05 compared with parallel incubations without CAPS).

Mentions: Two potential mechanisms for inhibition were considered. The first involves the intrinsic positive curvature–promoting properties of PI 4,5-P2 as an inverted cone lipid with a large charged hydrophilic head group. Inverted cone lipids inhibit membrane fusion because packing in the contacting leaflets of bilayers (positive curvature) is energetically unfavorable for the formation of a hemifused stalk intermediate (negative curvature) necessary for full fusion (Chernomordik and Zimmerberg, 1995). This mechanism would predict that PI 3,4-P2 was equally inhibitory, whereas PI 4-P was less inhibitory than PI 4,5-P2 as we had shown (Fig. 2 C). Favoring this mechanism, we found that inverted cone lipids, such as lysophosphatidylcholine (LPC), inhibited SNARE-dependent fusion of PC/PS liposomes to a similar extent as inclusion of PI 4,5-P2, and that inhibition by LPC and PI 4,5-P2 was nonadditive (Fig. 2 D). Other inverted cone lipids have similarly been found to inhibit SNARE-dependent liposome fusion (Chen et al., 2006; Melia et al., 2006). A mechanism for inhibition of fusion by PI 4,5-P2 involving curvature promotion would not exhibit donor or acceptor membrane specificity, which is what we observed (see Fig. 3 E).


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

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

CAPS accelerates liposome fusion in a PI 4,5-P2– and SNARE-dependent manner. (A) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 (indicated by t for t-SNAREs) in 90:10 mol% PC/PI 4,5-P2 were incubated with donor liposomes with 100 copies of VAMP-2 in 85:15 mol% PC/PS in the absence or presence of 1 μM CAPS. Similar incubations with donor and acceptor liposomes in 85:15 mol% PC/PS in the absence or presence of 1 μM CAPS were conducted. NBD fluorescence in parallel reactions with Pf liposomes was used to correct all data. Mean values ± SEM for five independent experiments are shown. (B) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 in 90:10 mol% PC/PI 4,5-P2 were incubated with VAMP-2–containing donor liposomes in the absence or presence of 1 μM CAPS. Parallel incubations used botulinum neurotoxin B–treated donor liposomes incubated without or with 1 μM CAPS. (C) Syntaxin-1/SNAP-25 acceptor liposomes (indicated by t for t-SNAREs) with 10 mol% PI 4,5-P2 or with 15% PS were incubated with VAMP-2 donor liposomes in the presence of indicated CAPS concentrations. Exponential fits of time courses were used to derive rate constants (k). Mean ± SEM values are shown for three independent experiments. (D) Rate constants were determined for fusion reactions without or with 1 μM CAPS in incubations that contained syntaxin-1/SNAP-25 acceptor liposomes with the indicated mole percentage PI 4,5-P2. Data in inset show the percentage of maximal NBD fluorescence at 120 min in incubations without or with CAPS. Mean ± SEM values are shown for four independent experiments. (E) Extent of fusion in 120 min was determined in the absence or presence of 1 μM CAPS with donor VAMP-2 or acceptor syntaxin-1/SNAP-25 liposomes that contained 85:15 mol% PC/PS or 90:10 mol% PC/PI 4,5-P2 as indicated. Mean ± SEM values are shown for three independent experiments (*, P < 0.01 for PC/PIP2 compared with PC/PS; †, P = 0.05 compared with parallel incubations without CAPS).
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fig3: CAPS accelerates liposome fusion in a PI 4,5-P2– and SNARE-dependent manner. (A) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 (indicated by t for t-SNAREs) in 90:10 mol% PC/PI 4,5-P2 were incubated with donor liposomes with 100 copies of VAMP-2 in 85:15 mol% PC/PS in the absence or presence of 1 μM CAPS. Similar incubations with donor and acceptor liposomes in 85:15 mol% PC/PS in the absence or presence of 1 μM CAPS were conducted. NBD fluorescence in parallel reactions with Pf liposomes was used to correct all data. Mean values ± SEM for five independent experiments are shown. (B) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 in 90:10 mol% PC/PI 4,5-P2 were incubated with VAMP-2–containing donor liposomes in the absence or presence of 1 μM CAPS. Parallel incubations used botulinum neurotoxin B–treated donor liposomes incubated without or with 1 μM CAPS. (C) Syntaxin-1/SNAP-25 acceptor liposomes (indicated by t for t-SNAREs) with 10 mol% PI 4,5-P2 or with 15% PS were incubated with VAMP-2 donor liposomes in the presence of indicated CAPS concentrations. Exponential fits of time courses were used to derive rate constants (k). Mean ± SEM values are shown for three independent experiments. (D) Rate constants were determined for fusion reactions without or with 1 μM CAPS in incubations that contained syntaxin-1/SNAP-25 acceptor liposomes with the indicated mole percentage PI 4,5-P2. Data in inset show the percentage of maximal NBD fluorescence at 120 min in incubations without or with CAPS. Mean ± SEM values are shown for four independent experiments. (E) Extent of fusion in 120 min was determined in the absence or presence of 1 μM CAPS with donor VAMP-2 or acceptor syntaxin-1/SNAP-25 liposomes that contained 85:15 mol% PC/PS or 90:10 mol% PC/PI 4,5-P2 as indicated. Mean ± SEM values are shown for three independent experiments (*, P < 0.01 for PC/PIP2 compared with PC/PS; †, P = 0.05 compared with parallel incubations without CAPS).
Mentions: Two potential mechanisms for inhibition were considered. The first involves the intrinsic positive curvature–promoting properties of PI 4,5-P2 as an inverted cone lipid with a large charged hydrophilic head group. Inverted cone lipids inhibit membrane fusion because packing in the contacting leaflets of bilayers (positive curvature) is energetically unfavorable for the formation of a hemifused stalk intermediate (negative curvature) necessary for full fusion (Chernomordik and Zimmerberg, 1995). This mechanism would predict that PI 3,4-P2 was equally inhibitory, whereas PI 4-P was less inhibitory than PI 4,5-P2 as we had shown (Fig. 2 C). Favoring this mechanism, we found that inverted cone lipids, such as lysophosphatidylcholine (LPC), inhibited SNARE-dependent fusion of PC/PS liposomes to a similar extent as inclusion of PI 4,5-P2, and that inhibition by LPC and PI 4,5-P2 was nonadditive (Fig. 2 D). Other inverted cone lipids have similarly been found to inhibit SNARE-dependent liposome fusion (Chen et al., 2006; Melia et al., 2006). A mechanism for inhibition of fusion by PI 4,5-P2 involving curvature promotion would not exhibit donor or acceptor membrane specificity, which is what we observed (see Fig. 3 E).

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