Sugars in space, N-glycosylation mutants of the erythrocyte anion exchanger

grantor: University of TorontoScanning N-glycosylation mutagenesis was used to determine the topology of a polytopic membrane protein, the human erythrocyte anion exchanger 1 (AE1), using cell-free translation and transfected cells. N-glycosylation acceptor sites located near the middle of large EC loops were efficiently N-glycosylated, however no N-glycosylation occurred at sites located within sharply defined regions close to the adjacent TM segments. To be efficiently N-glycosylated, EC loops in polytopic membrane proteins must be larger than 25 residues in size, with acceptor sites located greater than 12 residues away from the preceding TM segment and greater than 14 residues away from the following TM segment. Application of this requirement allowed a significant refinement of the topology of AE1 including a more accurate mapping of the ends of TM segments. The strict distance dependence for N-glycosylation of loops suggests that TM segments in polytopic membrane proteins are held quite precisely within the translocation machinery during the N-glycosylation process. A novel 12 transmembrane segment model for AE1 was proposed, with a T-loop structure connecting TM segments 9 and 10. Mutations designed to disrupt the folding of T-loop suggest that this loop may be transiently exposed to the lumen of the endoplasmic reticulum during biosynthesis, but normally folds rapidly, precluding N-glycosylation. The results indicate that the folding of the carboxyl-terminal region of AE1 is more complex than originally proposed and that this region of the transporter may have a dynamic aspect. Calnexin is a lectin-like ER chaperone, considered a dissociable part of the translocon complex. The spatial requirements for calnexin interaction with AE1 were characterized. The results show that the interaction of calnexin with the polytopic membrane glycoprotein AE1 was dependent on the presence but not the location of the oligosaccharide. Furthermore, calnexin was associated with AE1 after its release from the translocation machinery. A lectin gel-shift assay was developed, which allowed an enhanced resolution of glycoproteins by SDS gel electrophoresis. The use of N-linked glycosylation has proven a valuable tool to study the topology of a membrane protein in a fine detail and the dynamics of early stages of protein folding in the membrane protein biosynthesis.Ph.D