Signal Sequence Cleavage and Plasma Membrane Targeting of the Retinal Rod NCKX1 and Cone NCKX2 Na⁺/Ca²⁺−K⁺Exchangers

Abstract

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.

Residues Contributing to the Ca2+ and K+ Binding Pocket of the NCKX2 Na+/Ca2+-K+ Exchanger

“…Ref. 24) confirmed that only the small amount of mutant NCKX2 protein represented by the lower band was present in the plasma membrane (data not shown). Fig.…”

“…This is a highly uncommon phenotype among the ϳ150 single residue NCKX2 mutants we have analyzed in the High Five cell system (also observed for the S185C and S185T mutant NCKX2 proteins in some, but not all experiments). As signal peptide cleavage appears essential for plasma membrane targeting (24), strongly reduced transport observed for the S545T NCKX2 mutant proteins may be caused by the lack of cleaved mutant NCKX2 protein present in the plasma membrane. Surface biotinylation of the S545T mutant NCKX2 protein (e.g.…”

“…control represents cells transfected with empty vector. In most blots wild-type NCKX2 displays a doublet, the lower band representing NCKX2 from which the signal peptide is cleaved (24). In some cases, an additional ghost band is observed at a slightly lower molecular size (e.g.…”

Na+-dependent Inactivation of the Retinal Cone/Brain Na+/Ca2+-K+ Exchanger NCKX2

“…The NCKX proteins studied to date share a common predicted structure of two sets of five and six hydrophobic domains; the current topological model suggests an arrangement of five and five transmembrane segments separated by a large cytosolic loop (19). They also have a cleaved signal peptide that is essential for plasma membrane targeting, leaving a large extracellular loop at the N terminus (the C-terminal loop is also extracellular) (20). NCKX and NCX proteins share sequence homology in only two transmembrane regions forming internal repeats termed the ␣1 and ␣2 repeats.…”

K+-Dependent Na+/Ca2+Exchangers: Key Contributors to Ca2+Signaling

“…The various NCKX isoforms share the same overall predicted membrane topology, with 11 transmembrane segments (TMs 0-10), the first of which (TM 0) is thought to be cleaved by a signal peptidase after protein translation (20) (FIGURE 2). Glycosylation-scan- 19).…”