ADP ribosylation factor 6 binding to phosphatidylinositol 4,5-bisphosphate-containing vesicles creates defects in the bilayer structure: An electron spin resonance study

The effects of binding of myristoylated ADP ribosylation factor 6 (myr-ARF6 ), an activator of phospholipase D (PLD), to a model membrane were investig ated using an electron spin resonance (ESR) labeling technique. Initial stu dies were conducted in vesicles composed of 1-palmitoyl-2-oleoyl phosphatid ylethanolamine, dipalmitoyiphosphatidylcholine, phosphatidylinositol 4,5-bi phosphate (PIP2), and cholesterol. Recombinant ARF6 binding significantly e nhances defects in both the headgroup and aryl-chain regions of the membran e, which are revealed by the emergence of sharp components in the spectra f rom a headgroup label, 1,2-dipalmitoylphosphatidyl-2,2,6,6-tetramethyl-1-pi peridinyloxy-choline (DPPTC), and a chain label, 10PC, after myr-ARF6 bindi ng. Binding of non-myristoylated ARF6 (non-ARF6) shows markedly reduced eff ects. Interestingly, no change in spectra from DPPTC was observed upon myr- ARF6 binding when PIP2 in the vesicles was replaced by other negatively cha rged lipids, including phosphatidylinositol, phosphatidylserine, and phosph atidylglycerol, even when normalized for charge. The production of the shar p peak appears to be a specific event, because another GTP binding protein, CDC42, which binds PIP2 and activates PLD, fails to induce changes in vesi cle structure. These results suggest a previously unappreciated role for AR F in mediating a protein/lipid interaction that produces defects in lipid b ilayers. may serve as an initial event in destabilizing membrane structure for subsequent membrane fusion or biogenesis of vesicles.