Glycolipid intermediates involved in the transfer of N-acetylglucosamine to endogenous proteins in a yeast membrane preparation

Abstract

A particulate membrane fraction from Saccharomyces cerevisiae contains transferases which catalyze the incorporation of N-acetylglucosamine from UDP- N-acetylglucosamine into a lipid fraction as well as into a protein fraction. The lipid fraction contains two alkali-stable lipids which can be separated on a silica G-60 column. The sugar moieties of these polyprenoid lipids are: N-acetyiglucosamine and di- N-acetylchitobiose. The transfer of carbohydrate from isolated glycolipids to endogenous protein has been examined. After separation of protein and saccharide by hydrazinolysis and reacetylation only di- N-acetylchitobiose is found, and also when glycolipid containing only one N-acetylglucosamine is used as substrate. Maximum transfer of saccharides from glycolipids to protein is obtained at a Triton X-100 concentration of 1%. At this Triton X-100 concentration there is practically no transfer of N-acetylglucosamine from UDP- N-acetylglucosamine to the phosphorylated lipid. Therefore, when polyprenyl diphosphate N- acetyl[ 3H]- glucosamine is incubated together with UDP-N- acetyl[ 14C] glucosamine with the membrane fraction in the presence of 1% Triton X-100, a doubly labelled di- N-acetylchitobiose linked to lipid is formed with N- acetyl[ 14C] glucosamine at the non-reducing end of the chain.