Uridine diphosphate-glucose transport into the endoplasmic reticulum of Saccharomyces cerevisiae: In vivo and in vitro evidence

It has been proposed that synthesis of beta-1,6-glucan, one of Saccharomyce s cerevisiae cell wall components, is initiated by a uridine diphosphate (U DP)-glucose-dependent reaction in the lumen of the endoplasmic reticulum (E R). Because this sugar nucleotide is not synthesized in the lumen of the ER , we have examined whether or not UDP- glucose can be transported across th e ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER-containing microsomes in vitro. Experiments with ER -containing microsomes showed that transport of UDP- glucose was temperatur e dependent and saturable with an apparent K-m of 46 mu M and a V-max of 20 0 pmol/mg protein/3 min. Transport was substrate specific because UDP-N-ace tylglucosamine did not enter these vesicles. Demonstration of UDP-glucose t ransport into the ER lumen in vivo was accomplished by functional expressio n of Schizosaccharomyces pombe UDP-glucose:glycoprotein glucosyltransferase (GT) in S. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected in alg6 or alg5 mutant cells, which transfer Man(9)GlcNAc(2) to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding thi s enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein wit h significant homology and the same size and subcellular location as GT. De letion mutants, kre5 Delta, lack cell wall beta-1,6 glucan and grow very sl owly. Expression of S. pombe GT in kre5 Delta mutants did not complement th e slow-growth phenotype, indicating that both proteins have different funct ions in spite of their similarities.