Abstract
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.
Highlights
Yolk-free homogenates were prepared from oocytes 1 3 days afterinjection with water or cRNA (5 ng). 20-25 oocytes were washed twice in Barth's solution (mM 88 mM NaCl, 1mM KCl, 0.82 mM MgSO, 0.41 m~ CaCl, 0.33 m~ Ca(NO,), 2.4 mM NaHCO, 10 rn Hepems-HC1, pH 7.4, and 20 mgfliter gentamycin) and once in oocyte homogenization bufferbefore homogenization in 4 pl of homogenization buffedoocyte by 10 passages through a 25-gauge needle
A goat anti-rabbit IgG conjugated with horseradish peroxidase and an enhanced chemiluminescence (ECL) detection system (Amersham) was used to detect Napi-2 in immunoblots prepared from cRNA-injected oocytes
endoglycosidase H (Endo-H) Digestion of in Vitro Danslated Napi-2ProteinsNap,2-related in vitro translation products were synthesized from the Nap,2 cRNA in thepresence of [35SlMet,separated by
Summary
Be hereby marked “advertisement”in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. Of oocytes have been described in detail elsewhere (11). 11 To whom correspondence should beaddressed:Institute of Physiol- coding Nap,-2and mutant cDNAs were linearized usingXbaI and used ogy, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, for the in vitro synthesis of cRNA, including capping, using T7 RNA. 50 nl of water or water containing cRNA (native or Glycosylation of the Rat Kidney NalPi Cotransporter mutagenized NaP,-2) was injected into oocytes using a semi-automatic injector(Inject+Matic system, J. Membrane Purification Rat kidney brush-border membranes were isolated according to a magnesium-precipitation technique as described (13). Yolk-free homogenates were prepared from oocytes 1 3 days afterinjection with water or cRNA (5 ng). Homogenates were centrifuged twice a t 100 x g for 10 min. Protein determinationswere obtained using the Bradford assay (14)
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