Specific interaction of Golgi coatomer protein α-COP with phosphatidylinositol 3,4,5-trisphosphate

The phosphoinositide binding selectivity of Golgi coatomer COPI polypeptides mas examined using photoaffinity analogs of the soluble inositol polyphosphates Ins(1,4,5)P-3, Ins(1,3,4,5)P-4, and InsP(6), and of the 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-benzoyldihydrocinnamide (BZDC)-containing probe, [3(H)]BZDC-Ins(1,3,4,5)P-4. A more highly phosphorylated probe, [H-3]BZDC-InsP(6) probe labeled six of the seven subunits, with only beta, beta', delta and epsilon-COP showing competitive displacement by excess InsP(6). Importantly, [H-3]BZDC-triester-PtdIns(3,4,5)P-3, the lipid with the same phosphorylation pattern as Ins(1,3,4,5)P-4, showed specific, PtdIns(3,4,5)P-3-displaceable labeling of only a alpha-COP. Labeling by the PtdIns(4,5)P-2 and PtdIns(3,4)P-2 photoaffinity probes was less intense and showed no discrimination based on PtdInsP(n) ligand. Thus, both the D-3 and D-5 phosphates are critical for the alpha-COP-PtdIns(3,4,5)P-3 interaction, suggesting an important role for this polyphosphoinositide in vesicular trafficking.