SPECIFIC INTERACTION OF GOLGI COATOMER PROTEIN ALPHA-COP WITH PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE

The phosphoinositide binding selectivity of Golgi coatomer COPI polype ptides mas examined using photoaffinity analogs of the soluble inosito l polyphosphates Ins(1,4,5)P-3, Ins(1,3,4,5)P-4, and InsP(6), and of t he polyphosphoinositides PtdIns(3,4,5)P-3, Pt-dIns(4,5)P-2, and PtdIns (3,4)P-2. Highly selective Ins(1,5,4,5)P-4-displaceable photocovalent modification of the alpha-COP subunit was observed with ap-benzoyldihy drocinnamide (BZDC)-containing probe, [3(H)]BZDC-Ins(1,3,4,5)P-4. A mo re highly phosphorylated probe, [H-3]BZDC-InsP(6) probe labeled six of the seven subunits, with only beta, beta', delta and epsilon-COP show ing competitive displacement by excess InsP(6). Importantly, [H-3]BZDC -triester-PtdIns(3,4,5)P-3, the lipid with the same phosphorylation pa ttern as Ins(1,3,4,5)P-4, showed specific, PtdIns(3,4,5)P-3-displaceab le labeling of only a alpha-COP. Labeling by the PtdIns(4,5)P-2 and Pt dIns(3,4)P-2 photoaffinity probes was less intense and showed no discr imination based on PtdInsP(n) ligand. Thus, both the D-3 and D-5 phosp hates are critical for the alpha-COP-PtdIns(3,4,5)P-3 interaction, sug gesting an important role for this polyphosphoinositide in vesicular t rafficking.