PubMed1994
Our laboratory recently identified a sodium-dependent transport system for phosphate from rat kidney cortex (NaPi-2; Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5979-5983). In the present study we have investigated whether or not this cotransporter is glycosylated and the role of N-glycosylation in determining its function. Glycosidase digestion of the NaPi-2 protein from rat brush border membranes, in vitro translation studies, or oocyte expression of the NaPi-2 cRNA indicate that the mature protein is glycosylated. Glycosidase treatment reduces the size of the protein from approximately 70-110 kDa to approximately 60-65 kDa. We therefore used site-directed mutagenesis to identify which of the putative consensus sites for N-linked glycosylation are utilized in the mature NaPi-2 protein. Altering the nucleotide sequences encoding both of the Asn-298 and Asn-328 residues to Gln produced mutants that are completely devoid of glycosylation, whereas mutants in which each of these sites were mutated separately are glycosylated when expressed in oocytes. These results suggest that both of these sites are modified by N-linked glycosylation in the mature protein. Surface expression of glycosylated and unglycosylated NaPi-2-related proteins was documented by biotinylation experiments. In contrast to the wild-type (fully glycosylated) transporter, immunocytochemistry provides evidence for a partial intracellular localization of mutant unglycosylated cotransporters. Na/Pi cotransport was studied in oocytes expressing wild-type or mutagenized NaPi-2 proteins using tracer or electrophysiological techniques. Although the transport rates are lower (by a factor of 2-3) after expression of the unglycosylated NaPi-2 protein, the Pi transport characteristics (pH dependence, apparent affinity for Pi or Na+) are similar in oocytes expressing either wild-type or glycosylation-deficient proteins.