Biophys J, August 2001, p. 994-1005, Vol. 81, No. 2

ADP Ribosylation Factor 6 Binding to Phosphatidylinositol 4,5-Bisphosphate-Containing Vesicles Creates Defects in the Bilayer Structure: An Electron Spin Resonance Study

Mingtao Ge,^* Jared S. Cohen, H. Alex Brown, and Jack H. Freed^*

 ^*Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853, and  Department of Molecular Medicine, Cornell University, Ithaca, New York 15853 USA

The effects of binding of myristoylated ADP ribosylation factor 6 (myr-ARF6), an activator of phospholipase D (PLD), to a model membrane were investigated using an electron spin resonance (ESR) labeling technique. Initial studies were conducted in vesicles composed of 1-palmitoyl-2-oleoyl phosphatidylethanolamine, dipalmitoylphosphatidylcholine, phosphatidylinositol 4,5-biphosphate (PIP₂), and cholesterol. Recombinant ARF6 binding significantly enhances defects in both the headgroup and acyl-chain regions of the membrane, which are revealed by the emergence of sharp components in the spectra from a headgroup label, 1,2-dipalmitoylphosphatidyl-2,2,6,6-tetramethyl-1-piperidinyloxy-choline (DPPTC), and a chain label, 10PC, after myr-ARF6 binding. Binding of non-myristoylated ARF6 (non-ARF6) shows markedly reduced effects. Interestingly, no change in spectra from DPPTC was observed upon myr-ARF6 binding when PIP₂ in the vesicles was replaced by other negatively charged lipids, including phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol, even when normalized for charge. The production of the sharp peak appears to be a specific event, because another GTP binding protein, CDC42, which binds PIP₂ and activates PLD, fails to induce changes in vesicle structure. These results suggest a previously unappreciated role for ARF in mediating a protein/lipid interaction that produces defects in lipid bilayers. This function may serve as an initial event in destabilizing membrane structure for subsequent membrane fusion or biogenesis of vesicles.

Biophys J, August 2001, p. 994-1005, Vol. 81, No. 2
© 2001 by the Biophysical Society   0006-3495/01/08/994/12  $2.00

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