THE C-TERMINUS OF PHOSPHATIDYLINOSITOL TRANSFER PROTEIN MODULATES MEMBRANE INTERACTIONS AND TRANSFER ACTIVITY BUT NOT PHOSPHOLIPID-BINDING

Rat phosphatidylinositol transfer protein (PITP) is a 32kDa protein co ntaining 271 amino acids. It is involved in a number of cell functions including secretion and cell signaling. To further characterize struc ture/activity relationships of PITP, two C-terminal truncated derivati ves, PITP(1-259) and PITP(1-253), were produced in Escherichia coli an d purified to homogeneity. PITP(1-259) had transfer activity equal to 30-40% to that of native PITP in transfer of either phosphatidylcholin e (PC) or phosphatidylinositol (PI) when transfer was measured using 9 5/5mol% PC/PI donor and acceptor vesicles; PITP(1-253) had only slight transfer activity, even under the most favorable assay conditions. Th us, amino acids 254-258 are critical for transfer activity. The transf er activity of PITP(1-259) was strongly dependent on the composition o f the donor and acceptor vesicles. With 100 mol% PC donor and acceptor vesicles, PITP(1-259) transfer activity ranged from 70 to 100% to tha t of PITP. The presence of 2 mol% phosphatidic acid (PA) in either don or or acceptor vesicles reduced transfer activity to between 10 and 20 % that of full-length PITP under the same conditions. If both donor an d acceptor contained 2% PA, PITP(1-259) was essentially inactive, thou gh the activity of PITP was not affected significantly under these con ditions. PITP(1-253) and PITP(1-259) bind much more avidly to vesicles than does PITP, and this enhanced binding reflects increased electros tatic interactions. Thus, the C-terminal residues modulate the affinit y of PITP for vesicles and the efficiency of phospholipid transfer. (C ) 1998 Elsevier Science B.V.