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PMID: 11867768 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 99 ·No. 5 ·2002-03-05 ·Pages 2842-7

Kinuta M, Yamada H, Abe T, Watanabe M, Li SA, Kamitani A, Yasuda T, Matsukawa T, Kumon H, Takei K

Abstract

As a step toward the elucidation of mechanisms in vesicle budding, a cell-free assay that measures cytosol-induced vesicle generation from liposomes was established. This assay then was used to explore the role of phosphoinositides in vesicle formation. Liposomes incubated with brain cytosol in the presence of ATP and GTP massively generated small vesicles, as assessed both quantitatively and qualitatively by a dynamic light-scattering assay. Both ATP and GTP were required. Vesicle formation was inhibited greatly by the immunodepletion of dynamin 1 from the cytosol, indicating a major contribution of this GTPase in this reaction and suggesting that it mimics endocytic vesicle fission. Increasing the concentration of l-alpha-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] but not of l-alpha-phosphatidylinositol 4-monophosphate or l-alpha-phosphatidylinositol in the lipid membranes enhanced vesicle formation. Lipid analysis revealed rapid degradation of PtdIns(4,5)P2 to l-alpha-phosphatidylinositol during the incubation with the reaction reaching a maximum within 5 sec, whereas vesicle formation proceeded with a longer time course. PtdIns(4,5)P2 degradation was independent of vesicle formation and occurred also in the presence of guanosine 5'-O-(thiotriphosphate), where few vesicle formations occurred. These results suggest that PtdIns(4,5)P2 plays a critical role in the early step of vesicle formation, possibly in the recruitment of coats and fission factors to membranes.

MeSH Terms
Adenosine Triphosphate/metabolism Animals Brain/metabolism Cattle Cell-Free System Cytosol/metabolism Dynamin I Dynamins GTP Phosphohydrolases/metabolism Guanosine Triphosphate/metabolism Liposomes/metabolism Phosphatidylinositol 4,5-Diphosphate/metabolism
Chemicals
Liposomes Phosphatidylinositol 4,5-Diphosphate Guanosine Triphosphate Adenosine Triphosphate Dynamin I GTP Phosphohydrolases Dynamins
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Kinuta Masahiro
Department of Neuroscience, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Yamada Hiroshi
Abe Tadashi
Watanabe Masami
Li Shun-Ai
Kamitani Akihiro
Yasuda Tatsuji
Matsukawa Takashi
Kumon Hiromi
Takei Kohji
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.261715599
Published
2002-03-05
Epub
2002-00-26
Pages
2842-7
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC122435
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.261715599 PMC Full Text View journal details
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