Structural requirements for the stability and microsomal transport activity of the human glucose 6-phosphate transporter.
Deficiencies in glucose 6-phosphate (G6P) transporter (G6PT), a 10-helical endoplasmic reticulum transmembrane protein of 429 amino acids, cause glycogen storage disease type 1b. To date, only three missense mutations in G6PT have been shown to abolish microsomal G6P transport activity. Here, we report the results of structure-function studies on human G6PT and demonstrate that 15 missense mutations and a codon deletion (delta F93) mutation abolish microsomal G6P uptake activity and that two splicing mutations cause exon skipping. While most missense mutants support the synthesis of G6PT protein similar to that of the wild-type transporter, immunoblot analysis shows that G20D, delta F93, and I278N mutations, located in helix 1, 2, and 6, respectively, destabilize the G6PT. Further, we demonstrate that G6PT mutants lacking an intact helix 10 are misfolded and undergo degradation within cells. Moreover, amino acids 415-417 in the cytoplasmic tail of the carboxyl-domain, extending from helix 10, also play a critical role in the correct folding of the transporter. However, the last 12 amino acids of the cytoplasmic tail play no essential role(s) in functional integrity of the G6PT. Our results, for the first time, elucidate the structural requirements for the stability and transport activity of the G6PT protein.
Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
L Y
LY
Chen
B
B
Lin
C J
CJ
Pan
H
H
Hiraiwa
J Y
JY
Chou
D000595:Amino Acid Sequence; D000818:Animals; D017920:Antiporters; D001483:Base Sequence; D019556:COS Cells; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D004795:Enzyme Stability; D006801:Humans; D008661:Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018807:Polymorphism, Single-Stranded Conformational; D011487:Protein Conformation; D017510:Protein Folding
D017920:Antiporters; D017931:DNA Primers; D009004:Monosaccharide Transport Proteins; C503531:SLC37A4 protein, human; C111261:glucose 6-phosphate(transporter); D010770:Phosphotransferases