title: The interaction of coatomer with inositol polyphosphates is conserved in Saccharomyces 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 [Pelham (1994) Cell 79, 1125-1127]. We have investigated whether the binding of InsP6 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 InsP6 at two apparent classes of binding sites (KD1 = 0.8 +/- 0.2 nM; KD2 = 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 microM of either Ins(1,4,5)P3 or inositol hexakis-sulphate. A cell-free lysate of S. cerevisiae synthesized diphosphoinositol polyphosphates (PP-InsPn) from InsP6, 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 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.
authors.affiliation: Inositol Lipid Section, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
authors.forename: N
authors.initials: N
authors.lastname: Ali
authors.forename: R
authors.initials: R
authors.lastname: Duden
authors.forename: M E
authors.initials: ME
authors.lastname: Bembenek
authors.forename: S B
authors.initials: SB
authors.lastname: Shears
mesh_terms: D001665:Binding Sites; D020756:Coatomer Protein; D005656:Fungal Proteins / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D020756:Coatomer Protein; D005656:Fungal Proteins; D007295:Inositol Phosphates; D008565:Membrane Proteins