Membrane protein sequestering by ionic protein-lipid interactions.
Neuronal exocytosis is catalysed by the SNAP receptor protein syntaxin-1A, which is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis. However, how syntaxin-1A is sequestered is unknown. Here we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Using super-resolution stimulated-emission depletion microscopy on the plasma membranes of PC12 cells, we found that PIP2 is the dominant inner-leaflet lipid in microdomains about 73 nanometres in size. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the carboxy-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independently of cholesterol or lipid phases.
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Geert
G
van den Bogaart
Karsten
K
Meyenberg
H Jelger
HJ
Risselada
Hayder
H
Amin
Katrin I
KI
Willig
Barbara E
BE
Hubrich
Markus
M
Dier
Stefan W
SW
Hell
Helmut
H
Grubmüller
Ulf
U
Diederichsen
Reinhard
R
Jahn
D000818:Animals; D002784:Cholesterol; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal; D056004:Molecular Dynamics Simulation; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016716:PC12 Cells; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry* / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D055672:Static Electricity; D050827:Syntaxin 1 / Q000737:chemistry* / Q000378:metabolism*; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism
D009419:Nerve Tissue Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D053835:Unilamellar Liposomes; D002784:Cholesterol; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases