Membrane protein sequestering by ionic protein–lipid interactions

• Published in: Nature (London) Vol. 479; no. 7374; pp. 552 - 555
• Main Authors: van den Bogaart, Geert, Meyenberg, Karsten, Risselada, H. Jelger, Amin, Hayder, Willig, Katrin I., Hubrich, Barbara E., Dier, Markus, Hell, Stefan W., Grubmüller, Helmut, Diederichsen, Ulf, Jahn, Reinhard
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 24-11-2011; Nature Publishing Group
• Subjects: 631/378/548/2589; 631/45/612/1237; 631/80; 639/638/563/981; Animals; Cholesterol; Humanities and Social Sciences; letter; Lipids; Membrane Microdomains - chemistry; Membrane Microdomains - metabolism; Microscopy, Confocal; Molecular Dynamics Simulation; multidisciplinary; Nerve Tissue Proteins - metabolism; PC12 Cells; Phosphatidylinositol 4,5-Diphosphate - chemistry; Phosphatidylinositol 4,5-Diphosphate - metabolism; Phosphoric Monoester Hydrolases - metabolism; Protein Binding; Proteins; Rats; Science; Science (multidisciplinary); Simulation; Static Electricity; Syntaxin 1 - chemistry; Syntaxin 1 - metabolism; Unilamellar Liposomes - chemistry; Unilamellar Liposomes - metabolism
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature10545

Electrostatic factors in membrane organization
Exocytosis in neuronal cells requires the SNARE protein syntaxin-1A, which is clustered at sites where synaptic vesicles are poised to undergo exocytosis. Reinhard Jahn and colleagues use super-resolution stimulated-emission depletion (STED) microscopy to show that syntaxin clusters in the membrane through electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2) into 70-nanometre microdomains. The results demonstrate that electrostatic protein–lipid interactions can result in the formation of microdomains independent of cholesterol or lipid phases and have important implications for the organization of the plasma membrane.
Neuronal exocytosis is catalysed by the SNAP receptor protein syntaxin-1A
1
, which is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis
2
,
3
. 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.