The Dolichol Pathway of Protein Glycosylation in Rat Liver. Evidence that GTP Promotes Transformation of Endogenous Dolichyl Phosphate into Dolichylpyrophosphoryl-N-acetylglucosamine in Stripped Rough Microsomes

Tunicamycin, an antibiotic which inhibits the transfer of N-acetylglucosamine 1-phosphate to dolichyl phosphate, has been used to decide whether or not, in stripped rough microsomes incubated with UDP-N-acetylglucosamine and GDP-mannose in the absence of detergent, the earliest effect of GTP in core glycosylation of proteins is to enhance synthesis of dolichylpyrophosphoryl-N-acetylglucosamine from endogenous dolichyl phosphate, or to transform this monoglycoside derivative into dolichylpyrophosphorylchitobiose. It has been found that the presence of tunicamycin in the reaction medium annihilates incorporation of N-acetylglucosamine and mannose into all kinds of glycoside derivative of dolichyl pyrophosphate, whereas dolichylphosphorylmannose is then formed in greater amount. Incorporation of N-acetylglucosamine into protein was also abolished; that of mannose was considerably reduced. Other experiments showed that transfer of N-acetylglucosamine 1-phosphate is the only reaction of the lipid intermediates pathway that becomes limiting after addition of tunicamycin in our GTP-stimulated system. Taking these and previous results from this laboratory [Godelaine et al. (1979) Eur. J. Biochem. 96, 17-26 and 27-34] into account, we conclude that GTP enhances the transformation of endogenous dolichyl phosphate into dolichylpyrophosphoryl-N-acetylglucosamine and leads to the complete assembly of dolichol-linked oligosaccharides which become ultimately transferred to protein. Triton X-100 increased the amount of dolichol glycosylated and markedly raised the ratio of labelled dolichylpyrophosphorylchitobiose to dolichylpyrophosphoryl-N-acetylglucosamine. Such changes being induced by GTP, we suggest that this nucleotide makes it possible to overcome a structural barrier of rough microsomes.