Signal sequence cleavage and plasma membrane targeting of the retinal rod NCKX1 and cone NCKX2 Na+/Ca2+ - K+ exchangers.
Retinal rod and cone photoreceptors express two distinct Na(+)/Ca(2+)-K(+) exchanger (NCKX) gene products. Both the rod NCKX1 and cone NCKX2 are polytopic membrane proteins thought to contain a putative cleavable signal peptide. A cleavable signal peptide is unusual for plasma membrane proteins; moreover, predictive algorithms suggest the presence of a cleavable signal peptide for all rod NCKX1 proteins and a noncleavable signal anchor for the cone NCKX2 proteins. In this study we have placed a peptide tag at different positions of the NCKX sequence to examine whether the putative signal sequence is indeed cleaved in either NCKX1 or NCKX2 proteins expressed in heterologous systems. The signal peptide was found to be, at least in part, cleaved in dolphin rod NCKX1 and in chicken and human cone NCKX2 expressed in HEK293 cells; no signal peptide cleavage was observed for chicken rod NCKX1 despite the fact that the SignalP predictive algorithm assigned this sequence to have the highest likelihood for a cleavable signal peptide among the three NCKX sequences tested here. For the two NCKX proteins that contained a cleavable signal peptide, only cleaved NCKX protein was found in the plasma membrane of HEK293 cells. Deletion of the signal sequence in both dolphin rod NCKX1 or cone NCKX2 did not affect NCKX protein synthesis but did disrupt plasma membrane targeting as judged from abolition of NCKX function and from lack of surface biotinylation. These results are consistent with delayed signal peptide cleavage for the rod and cone NCKX proteins.
Kang
KyeongJin
K
Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, N.W. Calgary, Alberta T2N 4N1, Canada.
Schnetkamp
Paul P M
PP
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002645:Chickens / Q000378:metabolism; D004289:Dolphins / Q000378:metabolism; D005453:Fluorescence; D016259:Genes, myc / Q000235:genetics; D006801:Humans; D007313:Insecta / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011188:Potassium / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology*; D017433:Protein Structure, Secondary / Q000235:genetics; D021381:Protein Transport; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D017384:Sequence Deletion; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D021382:Protein Sorting Signals; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D011188:Potassium; D002118:Calcium