Phospholipase C β2 Association with Phospholipid Interfaces Assessed by Fluorescence Resonance Energy Transfer

Abstract

Association with lipid bilayers is essential for activity and G protein regulation of phospholipase C β. To determine the physical and biochemical parameters for membrane association we have developed a fluorescence resonance energy transfer assay using fluorescently labeled phospholipase C β2 and vesicles containing 1% of a phosphatidylethanolamine-rhodamine conjugate (PE-rho). Phospholipase C β2 purified from Sf9 insect cells was labeled with fluorescein at a 1:1 stoichiometry. The resulting protein was active and regulated by G protein βγ subunits. Vesicle binding was detected as quenching of fluorescein-PLC β2 fluorescence at 520 nm. The assay conditions were identical to conditions where activation of F-PLC β2 by βγ subunits and Ca++ could be demonstrated. F-PLC β2 selectively bound to phosphatidylinositol 4,5 bisphosphate (PIP2):PE (1=4) vesicles (Kd=2 μM) when compared to PS:PE (1:4) or all PC vesicles. When vesicles were prepared consisting solely of PS (with 1% PE-rho) to mimic the highly charged nature of PIP2 vesicles, binding was similar to what was observed for PIP2:PE vesicles. This suggests that much of the binding, under these conditions, can be accounted for by ionic interactions without a strong selectivity for the PIP2 head group. The effects of G protein βγ subunits on vesicle association were measured to determine if these subunits activate PLC via a translocation dependent mechanism.