title: Structure-function analysis of the glucose-6-phosphate transporter deficient in glycogen storage disease type Ib.
abstract: 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.
authors.affiliation: Section on Cellular Differentiation, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892, USA.
authors.forename: Li-Yuan
authors.initials: LY
authors.lastname: Chen
authors.forename: Chi-Jiunn
authors.initials: CJ
authors.lastname: Pan
authors.forename: Jeng-Jer
authors.initials: JJ
authors.lastname: Shieh
authors.forename: Janice Yang
authors.initials: JY
authors.lastname: Chou
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000256:Adenoviridae / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017920:Antiporters / Q000096:biosynthesis / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003546:Cysteine Endopeptidases / Q000502:physiology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000737:chemistry; D005822:Genetic Vectors; D005952:Glucose-6-Phosphatase / Q000235:genetics; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005953:Glycogen Storage Disease Type I / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000502:physiology; D020125:Mutation, Missense; D010455:Peptides / Q000502:physiology; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary / Q000502:physiology; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics
chemical_list: D017920:Antiporters; D015853:Cysteine Proteinase Inhibitors; D009004:Monosaccharide Transport Proteins; D009097:Multienzyme Complexes; D010455:Peptides; C503531:SLC37A4 protein, human; D014764:Viral Proteins; C111261:glucose 6-phosphate(transporter); C067713:lactacystin; D019298:Glucose-6-Phosphate; D005952:Glucose-6-Phosphatase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine