Role of a mannosyl lipid intermediate in the synthesis of Neurospora crassa glycoproteins

Abstract

Particulate membrane preparations from N. crassa incorporated mannose from GDP-[14C]mannose into endogenous lipid and particulate protein acceptors. Synthesis of the mannosyl lipid is reversible in the presence of GDP. Chemical and chromatographic characterization of the mannosyl lipid suggest that it is a mannosylphosphorylpolyisoprenol. The other endogenous acceptor was precipitated by trichloroacetic acid. Gel filtration and electrophoresis studies before and after treatment with proteolytic enzymes indicate that the 2nd acceptor is a glycoprotein(s). .beta.-Elimination studies on the mannosyl protein formed from GDP-[14C]mannose with Mg2+ in the reaction mixture or formed from mannosyl lipid indicate that a single mannose unit is transferred directly to form an O-glycosidic bond with the peptide chain. Several lines of evidence indicate that in N. crassa the mannosyl lipid is an obligatory intermediate in the in vitro mannosylation of the protein. At 15.degree. C the initial formation of the mannosyl lipid is faster than the initial formation of the mannosyl protein. Exogenous partially purified mannosyl lipid can function as a mannosyl donor for the synthesis of the mannosyl protein. This reaction was also dependent on a divalent metal. The rate of this reaction was optimal at a concentration of Triton X-100 which effectively inhibited the transfer of mannose from GDP-[14C]mannose to lipid and protein, indicating that GDP-mannose was not an intermediate in the transfer of mannose from lipid to protein. The mannosyl protein formed in this reaction was indistinguishable by several criteria from the mannosyl protein formed from GDP-[14C]mannose and Mg2+. The effect of a chase with an excess of unlabeled GDP-mannose on the incorporation of mannose into endogenous acceptors was immediate cessation of the synthesis and subsequent turnover of the mannosyl lipid; in contrast, incorporation of mannose into protein continued and was proportional to the loss of mannose from the mannosyl lipid.