The Dolichol Pathway of Protein Glycosylation in Rat Liver

Incorporation of mannose into endogenous lipid and protein acceptors of the rat liver endoplasmic reticulum was investigated by incubating rough microsomes with GDP‐ 14 Cmannose in the absence of detergent. Labeled dolichyl monophosphate mannose was readily formed from the endogenous dolichyl monophosphate. In contrast, the label was not detectable in dolichyl diphosphate oligosaccharides and it was incorporated into protein only to a small extent, except when preparations previously treated with pyrophosphate were incubated in the presence of UDP‐ N ‐ acetylglucosamine and GTP. Then, incorporation of label into such lipid and protein derivatives was considerably enhanced. GTP could not be substituted by other nucleotides. The requirement for UDP‐ N ‐acetylglucosamine, and other experimental evidence, demonstrate that mannosylated oligosaccharides which contain internal N ‐acetylglucosamine units are entirely assembled on dolichyl diphosphate from the exogenous nucleotide sugars. The kinetics of mannose incorporation into dolichol‐linked oligosaccharides and into protein suggest that the protein acceptors are mainly mannosylated through transfer of oligosaccharides preassembled on dolichyl diphosphate. In heavy microsomes which have not been treated with PP i , or upon incubation in the absence of GTP, the amount of dolichyl phosphate which can accept N ‐acetylglucosamine 1‐phosphate is less than that which can accept mannose. However, when PP i ‐treated preparations are incubated with UDP‐ N ‐acetylglucosamine and GDP‐mannose in the presence of GTP, a larger amount of dolichyl phosphate is mobilized in the form of various saccharide derivatives of dolichyl diphosphate, and the amount of dolichyl monophosphate mannose is correspondingly decreased. It seems, therefore, that the membranes contain a particular pool of dolichyl monophosphate, which readily forms dolichyl monophosphate mannose, but reacts with UDP‐ N ‐acetylglucosamine only under appropriate conditions. This pool could act as precursor of the glycoside derivatives of dolichyl diphosphate which transfer their saccharide moiety to proteins.