Sorting of rat SPNT in renal epithelium is independent of N-glycosylation.
OBJECTIVE
The sodium-dependent, purine-selective nucleoside transporter, SPNT, has a unique steady-state expression pattern in renal epithelial cells. In comparison with the concentrative nucleoside transporter, CNTI, which is confined to the apical membrane, SPNT is expressed predominantly on the apical membrane but with significant expression on the basolateral membrane as well. Alternate surface expression indicates that SPNT likely has different sorting and trafficking mechanisms from CNTI. Because glycosylation has been reported to be essential for apical targeting of other transporters, and SPNT contains three unique glycosylation sites, we examined the importance of glycosylation in sorting of SPNT. Preliminary studies suggested that glycosylation affects surface expression of SPNT but not CNT1.


METHODS
All three unique glycosylation sites were mutated alone and in tandem. Wild-type and mutant SPNT, tagged with green fluorescence protein, were stably transfected into MDCK. Positive clones were assayed for polarized surface expression by immunofluorescence and functional analysis.


RESULTS
Mutation at all three sites alone or in tandem resulted in functional proteins. Removal of sites N606 and N625 resulted in proteins of reduced molecular mass. None of the unglycosylated mutants localized differently than wild-type SPNT.


CONCLUSIONS
N-linked glycosylation is not essential for polarized sorting.
Mangravite
Lara M
LM
Department of Biopharmaceutical Sciences, University of California, San Francisco, San Francisco, California 94143-0446, USA.
Giacomini
Kathleen M
KM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004285:Dogs; D004848:Epithelium / Q000378:metabolism*; D006031:Glycosylation; D007668:Kidney / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011684:Purine Nucleosides / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid
D026901:Membrane Transport Proteins; D011684:Purine Nucleosides; C065171:cif nucleoside transporter