Transmembrane topology of human glucose 6-phosphate transporter

Glycogen storage disease type 1b is caused by a deficiency in a glucose 6-p hosphate transporter (G6PT) that translocates glucose 6-phosphate from the cytoplasm to the endoplasmic reticulum lumen where the active site of gluco se 6-phosphatase is situated. Using amino- and carboxyl-terminal tagged G6P T, we demonstrate that proteolytic digestion of intact microsomes resulted in the cleavage of both tags, indicating that both termini of G6PT face the cytoplasm. This is consistent with ten and twelve transmembrane domain mod els for G6PT predicted by hydropathy analyses. A region of G6PT correspondi ng to amino acid residues 50-71, which constitute a transmembrane segment i n the twelve-domain model, are situated in a 51-residue luminal loop in the ten-domain model. To determine which of these two models is correct, we ge nerated two G6PT mutants, T53N and S55N, that created a potential Asn-linke d glycosylation site at residues 53-55 ((NSS)-S-53) Or 55-57 (N(55)QS), res pectively. (NSS)-S-53 Or N(55)QS would be glycosylated only if it is situat ed in a luminal loop larger than 33 residues as predicted by the ten-domain model. Whereas wild-type G6PT is not a glycoprotein, both T53N and S55N mu tants are glycosylated, strongly supporting the ten-helical model for G6PT.