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Vol. 10, Issue 4, 1019-1030, April 1999

Uridine Diphosphate-Glucose Transport into the Endoplasmic Reticulum of Saccharomyces cerevisiae: In Vivo and In Vitro Evidence

Olga Castro,* Ling Yun Chen,dagger Armando J. Parodi,* and Claudia Abeijóndagger Dagger

 *Instituto de Investigaciones Bioquímicas Fundación Campomar, 1405 Buenos Aires, Argentina; and  dagger Department of Molecular and Cell Biology, Boston University School of Dental Medicine, Boston, Massachusetts 02118-2392

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 µM 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.

Dagger    Corresponding author. E-mail address: cabeijon{at}bu.edu.

Molecular Biology of the Cell
Vol. 10, 1019-1030, April 1999
Copyright © 1999 by The American Society for Cell Biology


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