Human Molecular Genetics, 2002, Vol. 11, No. 25 3199-3207
© 2002 Oxford University Press
Structure–function analysis of the glucose-6-phosphate transporter deficient in glycogen storage disease type Ib
Section on Cellular Differentiation, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892
Received August 10, 2002; Revised September 25, 2002; Accepted September 30, 2002
Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), a 10 transmembrane domain endoplasmic reticulum protein. To date, 69 G6PT mutations, including 28 missenses and 2 codon deletions, have been identified in GSD-Ib patients. We previously characterized 15 of the missense and one codon deletion mutations using a pSVL-based expression assay. A lack of sensitivity in this assay limited the discrimination between mutations that lead to loss of function and mutations that leave a low residual activity. We now report an improved G6PT assay, based on an adenoviral vector-mediated expression system and its use in the functional characterization of all 30 codon mutations found in GSD-Ib patients. Twenty of the naturally occurring mutations completely abolish microsomal G6P uptake activity while the other 10 mutations, including 5 previously characterized ones, partially inactivate the transporter. This information should greatly facilitate genotype–phenotype correlation. We also report a structure–function analysis of G6PT. In addition to the 3 destabilizing mutations reported previously, we now show that the G50R, C176R, V235del, G339C and G339D mutations also compromise the G6PT stability. Mutation analysis of the amino-terminal domain of G6PT shows that it is required for optimal G6P uptake activity. Finally, we show that degradation of both wild-type and mutant G6PT is inhibited by a potent proteasome inhibitor, lactacystin, demonstrating that G6PT is a substrate for proteasome-mediated degradation.
* To whom correspondence should be addressed at: Building 10, Room 9S241, NIH, Bethesda, MD 20892-1830, USA. Tel: +3014961094; Fax: +3014026035; Email: chou{at}helix.nih.gov
The authors wish it to be known that, in their opinion, these authors should be considered as joint First Authors.
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