The Activation of Exocytotic Sites by the Formation of Phosphatidylinositol 4,5-Bisphosphate Microdomains at Syntaxin Clusters

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of the lipid bilayer but plays an important role in various cellular functions, including exocytosis and endocytosis. Recently, PI(4,5)P2 was shown to form microdomains in the plasma membrane. In this study, we investigated the r...

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Bibliographic Details
Published in:The Journal of biological chemistry 2005-04, Vol.280 (17), p.17346-17352
Main Authors: Aoyagi, Kyota, Sugaya, Tsukiko, Umeda, Masato, Yamamoto, Seiji, Terakawa, Susumu, Takahashi, Masami
Format: Article
Language:English
Subjects:
Animals
Cell Membrane - metabolism
Electrophoresis, Polyacrylamide Gel
Endocytosis
Enzyme Activation
Ethylmaleimide - pharmacology
Exocytosis
Immunoblotting
Lipid Bilayers - chemistry
Membrane Proteins - chemistry
Microscopy, Fluorescence
PC12 Cells
Phosphatidylinositol 3-Kinases - metabolism
Phosphatidylinositol 4,5-Diphosphate - chemistry
Phosphotransferases (Alcohol Group Acceptor) - chemistry
Phosphotransferases (Alcohol Group Acceptor) - physiology
Potassium - chemistry
Protein Structure, Tertiary
Qa-SNARE Proteins
Rats
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Summary:Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of the lipid bilayer but plays an important role in various cellular functions, including exocytosis and endocytosis. Recently, PI(4,5)P2 was shown to form microdomains in the plasma membrane. In this study, we investigated the relationship between the spatial organization of PI(4,5)P2 microdomains and exocytotic machineries in clonal rat pheochromocytoma PC12 cells. Both PI(4,5)P2 and syntaxin, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein essential for exocytosis, exhibited punctate clusters in isolated plasma membranes. The number of PI(4,5)P2 microdomains colocalizing with syntaxin clusters and large dense core vesicles (LDCVs) was decreased after catecholamine release. Alternatively, the expression of type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) increased the number of PI(4,5)P2 microdomains at syntaxin clusters with docked LDCVs and enhanced exocytotic activity, possibly by increasing the number of release sites. About half of the PI(4,5)P2 microdomains were not colocalized with Thy-1, a specific marker of lipid rafts, and the colocalization of transfected PIP5KI with syntaxin clusters was observed. These results suggest that the formation of PI(4,5)P2 microdomains at syntaxin clusters with docked LDCVs is essential for Ca2+-dependent exocytosis.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M413307200