LIMITED PROTEOLYSIS OF RAT PHOSPHATIDYLINOSITOL TRANSFER PROTEIN BY TRYPSIN CLEAVES THE C-TERMINUS, ENHANCES BINDING TO LIPID VESICLES, ANDREDUCES PHOSPHOLIPID TRANSFER ACTIVITY

Rat phosphatidylinositol transfer protein (PITP) is a 32-kDa protein o f 271 amino acids that transfers phosphatidylinositol and phosphatidyl choline between membranes. The alpha isoform of rat PITP was expressed in Escherichia coli and purified in high yields, The purified protein contained 1 mol of phosphatidylglycerol and had a transfer activity f or phosphatidylinositol and phosphatidylcholine equal to or greater th an that of PITP purified from mammalian brain, Limited protease digest ion was used to further define structure, activity, and function relat ionships in PITP. PITP alone is relatively resistant to digestion by c hymotrypsin, trypsin, and Staphylococcus V8 protease but is readily cl eaved by subtilisin. Phospholipid vesicles containing phosphatidic aci d enhance susceptibility to digestion by all four proteases, In the pr esence of vesicles, PITP, which migrates as a 36-kDa protein in SDS-po lyacrylamide gel electrophoresis, is cleaved rapidly by trypsin to a f orm that appears to be 2-3 kDa smaller than the native form, The trypt ic fragment retains partial phospholipid transfer activity and shows a n enhanced affinity for phospholipid vesicles containing phosphatidic acid. Analysis of the tryptic digestion products by immunoblotting, N- terminal sequencing, and electrospray mass spectrometry showed that tr ypsin cleaves the C terminus of PITP at Arg(253) and Arg(259), Thus, r emoval of the C terminus enhances the affinity of PITP for vesicles an d results in a dimunition of transfer activity, Overall, the data show that PITP undergoes conformation changes and that the C terminus beco mes more accessible to trypsin when bound to vesicles. Hence, the C te rminus is not an essential component of the membrane binding site and may be located distal to it.