ROLE OF N-LINKED GLYCOSYLATION IN RAT RENAL NA P-I-COTRANSPORT/

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 stud y we have investigated whether or not this cotransporter is glycosylat ed and the role of N-glycosylation in determining its function. Glycos idase 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 treatme nt reduces the size of the protein from similar to 70-110 kDa to simil ar to 60-65 kDa. We therefore used site-directed mutagenesis to identi fy which of the putative consensus sites for N-linked glycosylation ar e utilized in the mature NaPi-2 protein. Altering the nucleotide seque nces 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 si tes are modified by N-linked glycosylation in the mature protein. Surf ace expression of glycosylated and unglycosylated NaPi-2-related prote ins was documented by biotinylation experiments. In contrast to the wi ld-type (fully glycosylated) transporter, immunocytochemistry provides evidence for a partial intracellular localization of mutant unglycosy lated cotransporters. Na/P-i cotransport was studied in oocytes expres sing wild-type or mutagenized NaPi-2 proteins using tracer or electrop hysiological techniques. Although the transport rates are lower (by a factor of 2-3) after expression of the unglycosylated NaPi-2 protein, the P-i transport characteristics (pH dependence, apparent affinity fo r P-i or Na+) are similar in oocytes expressing either wild-type or gl ycosylation-deficient proteins.