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Eukaryotic Cell, June 2003, p. 534-541, Vol. 2, No. 3
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.3.534-541.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

A Saccharomyces cerevisiae Mutant Unable To Convert Glucose to Glucose-6-Phosphate Accumulates Excessive Glucose in the Endoplasmic Reticulum due to Core Oligosaccharide Trimming

Attila Miseta,¹ Margit Tökés-Füzesi,¹ David P. Aiello,² and David M. Bedwell^2*

Department of Clinical Chemistry, University Medical School, Peçs, Hungary,¹ Department of Microbiology, The University of Alabama at Birmingham, Birmingham, Alabama 35294²

Received 15 August 2002/ Accepted 14 February 2003

D-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu₃Man₉GlcNac₂ effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5 or alg6, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER). Consistent with this conclusion, both [¹⁴C]glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P. The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway. Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca²⁺ uptake. This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.

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* Corresponding author. Mailing address: Department of Microbiology, BBRB 432/Box 8, 1530 Third Ave. S., University of Alabama at Birmingham, Birmingham, AL 35294-2170. Phone: (205) 934-6593. Fax: (205) 975-5482. E-mail: dbedwell{at}uab.edu.

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Eukaryotic Cell, June 2003, p. 534-541, Vol. 2, No. 3
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.3.534-541.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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