THE INTERACTION OF COATOMER WITH INOSITOL POLYPHOSPHATES IS CONSERVED IN SACCHAROMYCES-CEREVISIAE

Coatomer is an oligomeric complex of coat proteins that regulates vesicular traffic through the Golgi complex and from the Golgi to the endoplasmic reticulum [Pelham (1994) Cell 79, 1125-1127]. We have investigated whether the binding of InsP(6) to mammalian coatomer [Fleischer, Xie, Mayrleitner, Shears and Fleischer (1994) J. Biol. Chem. 269, 17826-17832] is conserved in the genetically amenable model Saccharomyces cerevisiae. We have isolated coatomer from S. cerevisiae and found it to bind InsP, at two apparent classes of binding sites (K-D1 = 0.8+/-0.2nM; K-D2=361+/-102nM). Ligand specificity was studied by displacing 4.5 nM [H-3]InsP(6) from coatomer with various Ins derivatives. The following IC50 values (nM) were obtained: myo InsP(6)=6; bis(diphospho)inositol tetrakisphosphate=6; diphosphoinositol pentakisphosphate=6; scyllo-InsP(6) =12; Ins(1,3,4,5,6)P-5=13; Ins(1,2,4,5,6)P-5=22; Ins(1,3,4,5)P-4=22; 1-0-(1,2-di-0-octanoyl-sn-glycero-3-phospho)- D-Ins(3,4,5)P-3=290. Less than 10% of the H-3 label was displaced by 1 mu M of either Ins(1,4,5)P-3 or inositol hexakis-sulphate. A cell-free lysate of S. cerevisiae synthesized diphosphoinositol polyphosphates (PP-InsP(2)) from InsP(6), but our binding data, plus measurements of the relative levels of inositol polyphosphates in intact yeast [Hawkins, Stephens and Piggott (1993) J. Biol. Chem. 268, 3374-3383], indicate that InsP(6) is the major physiologically relevant ligand. Thus a reconstituted vesicle trafficking system using coatomer and other functionally related components isolated from yeast should be a useful model for elucidating the functional significance of the binding of InsP(6) by coatomer.