It has been proposed that synthesis of beta-1,6-glucan, one of Saccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose-dependent reaction in the lumen of the endoplasmic reticulum (ER). 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 the 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 temperature dependent and saturable with an apparent Km of 46 microM and a Vmax of 200 pmol/mg protein/3 min. Transport was substrate specific because UDP-N-acetylglucosamine did not enter these vesicles. Demonstration of UDP-glucose transport into the ER lumen in vivo was accomplished by functional expression 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 Man9GlcNAc2 to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Delta, lack cell wall beta-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Delta mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.
Monitoring Editor: Suzanne R. Pfeffer CWH41, a gene involved in the assembly of cell wall -1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal ␣-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of -1,6-glucan, we constructed a double mutant, alg5⌬ (lacking dolichol-P-glucose synthase) cwh41⌬, and found that it has the same phenotype as the alg5⌬ single mutant. It contains wild-type levels of cell wall -1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41⌬ single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the -1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5⌬). The triple mutant alg5⌬cwh41⌬kre6⌬ is viable, whereas the double mutant cwh41⌬kre6⌬ in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall -1,6-glucan, characteristic of the cwh41⌬kre1⌬ double mutant, are not observed in the triple mutant alg5⌬cwh41⌬kre1⌬. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41⌬ strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall -1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.
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