Evidence for protein dolichylation

Labeling of human colon carcinoma cells with [H-3]dol, followed by extensive delipidation and removal of dol-P oligosaccharides, showed that dol are bound to cellular proteins with sizes of 5, 10, 27, 75 and >140 kDa, HPLC purification of proteolytic products of [H-3]dol- and [S-35]cys-labeled proteins revealed a hydrophobic peak containing both dol and cysteine, The dol/cys-labeled products were clearly separated from GG-cys, and exhibited a hydrophobicity between that of dol-P and dol, In another set of experiments delipidated proteins were treated with methyl iodide, which cleaves thioether bonds, After HPLC purification of released dol-like lipids, these were subjected to mass spectrometry, This demonstrated molecular ions with the same mass as that of dol, Taken together our data provide evidence for the existence of proteins covalently modified with dol. (C) 1997 Federation of European Biochemical Societies.