Chaudhary A, Gu Q M, Thum O, Profit A A, Qi Y, Jeyakumar L, Fleischer S, Prestwich G D
The University of Utah, Department of Medicinal Chemistry, Salt Lake City, Utah 84112-5820, USA.
J Biol Chem. 1998 Apr 3;273(14):8344-50. doi: 10.1074/jbc.273.14.8344.
The phosphoinositide binding selectivity of Golgi coatomer COPI polypeptides was examined using photoaffinity analogs of the soluble inositol polyphosphates Ins(1,4,5)P3, Ins(1,3,4,5)P4, and InsP6, and of the polyphosphoinositides PtdIns(3,4,5)P3, PtdIns(4,5)P2, and PtdIns(3,4)P2. Highly selective Ins(1,3,4,5)P4-displaceable photocovalent modification of the alpha-COP subunit was observed with a p-benzoyldihydrocinnamide (BZDC)-containing probe, [3H]BZDC-Ins(1,3,4,5)P4. A more highly phosphorylated probe, [3H]BZDC-InsP6 probe labeled six of the seven subunits, with only beta, beta', delta, and epsilon-COP showing competitive displacement by excess InsP6. Importantly, [3H]BZDC-triester-PtdIns(3,4,5)P3, the lipid with the same phosphorylation pattern as Ins(1,3,4,5)P4, showed specific, PtdIns(3,4,5)P3-displaceable labeling of only alpha-COP. Labeling by the PtdIns(4,5)P2 and PtdIns(3,4)P2 photoaffinity probes was less intense and showed no discrimination based on PtdInsPn ligand. Thus, both the D-3 and D-5 phosphates are critical for the alpha-COP-PtdIns(3,4,5)P3 interaction, suggesting an important role for this polyphosphoinositide in vesicular trafficking.
利用可溶性肌醇多磷酸Ins(1,4,5)P3、Ins(1,3,4,5)P4和InsP6以及多磷酸肌醇PtdIns(3,4,5)P3、PtdIns(4,5)P2和PtdIns(3,4)P2的光亲和类似物，研究了高尔基体衣被蛋白COPI多肽的磷酸肌醇结合选择性。使用含对苯甲酰基二氢肉桂酰胺（BZDC）的探针[3H]BZDC-Ins(1,3,4,5)P4，观察到α-COP亚基的高度选择性的、可被Ins(1,3,4,5)P4取代的光共价修饰。一种磷酸化程度更高的探针[3H]BZDC-InsP6标记了七个亚基中的六个，只有β、β'、δ和ε-COP显示出被过量InsP6竞争性取代。重要的是，[3H]BZDC-三酯-PtdIns(3,4,5)P3（与Ins(1,3,4,5)P4具有相同磷酸化模式的脂质）仅显示出对α-COP的特异性、可被PtdIns(3,4,5)P3取代的标记。PtdIns(4,5)P2和PtdIns(3,4)P2光亲和探针的标记强度较低，并且没有基于PtdInsPn配体的区分。因此，D-3和D-5磷酸对于α-COP与PtdIns(3,4,5)P3的相互作用至关重要，表明这种多磷酸肌醇在囊泡运输中具有重要作用。
