EVIDENCE FOR PROTEIN DOLICHYLATION

Labeling of human colon carcinoma cells with [H-3]dol, followed by ext ensive delipidation and removal of dol-P oligosaccharides, showed that dol are bound to cellular proteins with sizes of 5, 10, 27, 75 and >1 40 kDa, HPLC purification of proteolytic products of [H-3]dol- and [S- 35]cys-labeled proteins revealed a hydrophobic peak containing both do l and cysteine, The dol/cys-labeled products were clearly separated fr om GG-cys, and exhibited a hydrophobicity between that of dol-P and do l, In another set of experiments delipidated proteins were treated wit h methyl iodide, which cleaves thioether bonds, After HPLC purificatio n of released dol-like lipids, these were subjected to mass spectromet ry, This demonstrated molecular ions with the same mass as that of dol , Taken together our data provide evidence for the existence of protei ns covalently modified with dol. (C) 1997 Federation of European Bioch emical Societies.