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

Phospholipase C β2 (PLC β2) is activated by G protein βγ subunits and calcium. The enzyme is soluble and its substrate, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), is present in phospholipid membranes. A potential mechanism for regulation of this enzyme is through influencing the equilibrium association of the enzyme with membrane surfaces. In this paper we describe a fluorescence resonance energy transfer (FRET) method for measuring the association of PLC β2 with phospholipid bilayers. The method allows equilibrium measurements to be made under a variety of conditions, including those that support enzymatic activity and ability to be regulated by G proteins. Using this method it was found that PLC β2 bound to vesicles containing anionic lipids and demonstrated a selective and unique interaction with PIP 2 -containing vesicles. The FRET data were corroborated with a centrifugation based method for estimating the affinity of PLC β2 for vesicles. Apparently different modes of association of PLC β2 with vesicles of different composition can be distinguished based on alterations in resonance energy transfer efficiency. Association of PLC β2 with PIP 2 vesicles requires an intact lipid bilayer, is blocked by neomycin, and is not affected by D - myo -inositol 1,4,5-trisphosphate ( D -IP 3 ). G protein βγ subunits do not alter the affinity of PLC β2 for lipid bilayers and at the PIP 2 concentrations used to measure βγ-dependent stimulation of PLC activity, the majority of the PLC β2 is already associated with the vesicle surface. Furthermore, under conditions where βγ subunits strongly activate PLC activity, the extent of association with vesicles is unaffected by βγ subunits or calcium. These results indicate that activation of PLC β2 by G protein βγ subunits or Ca 2+ in vitro does not involve translocation to the vesicle surface.