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 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.
Castro
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O
Instituto de Investigaciones Bioquímicas Fundación Campomar, 1405 Buenos Aires, Argentina.
Chen
L Y
LY
Parodi
A J
AJ
Abeijón
C
C
D002240:Carbohydrate Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics; D015398:Signal Transduction; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D009844:Oligosaccharides; D011994:Recombinant Proteins; D014537:Uridine Diphosphate N-Acetylglucosamine; D005964:Glucosyltransferases; C072423:mannosylglycoprotein 1,3-glucosyltransferase; D014532:Uridine Diphosphate Glucose