The interaction of coatomer with inositol polyphosphates is conserved in Saccaromyces cerevisiae

Abstract

Coatomer is an oligomeric complex of coat proteins that regulates vesicular traffic through the Golgi complex and from the Golgi to the endoplasmic reticulum. We have investigated whether the binding of InsP6 to mammalian coatomer is conserved in the genetically amenable model Saccharomyces cerevisiae. We have isolated coatomer from S. cerevisiae and found it to bind InsP6 at two apparent classes of binding sites (K(D1) = 0.8 ± 0.2 nM; K(D2) = 361 ± 102 nM). Ligand specificity was studied by displacing 4.5 nM [3H]InsP6 from coatomer with various Ins derivatives. The following IC50 values (nM) were obtained: myo-InsP6 = 6; bis(diphospho)inositol tetrakisphosphate = 6; diphosphoinositol pentakisphosphate = 6; scyllo-InsP6 = 12; Ins(1,3,4,5,6)P5 = 13; Ins(1,2,4,5,6)P5 = 22; Ins(1,3,4,5)P4 = 22; 1-O-(1,2-di-O-octanoyl-sn-glycero-3-phospho)- D-Ins(3,4,5)P3 = 290. Less than 10% of the 3H label was displaced by 1 μM of either Ins(1,4,5)P3 or inositol hexakis-sulphate. A cell-free lysate of S. cerevisiae synthesized diphosphoinositol polyphosphates (PP-InsP3) from InsP6, but our binding data, plus measurements of the relative levels of inositol polyphosphates in intact yeast, indicate that InsP6 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 InsP6 by coatomer.