Truncation of the C Terminus of the Rat Brain Na+-Ca2+ Exchanger RBE-1 (NCX1.4) Impairs Surface Expression of the Protein

• Published in: The Journal of biological chemistry 1999-08, Vol.274 (35), p.24873-24880
• Main Authors: Kasir, Judith, Ren, Xiaoyan, Furman, Ian, Rahamimoff, Hannah
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.35.24873

The C terminus of the rat brain Na+-Ca2+ exchanger (RBE-1; NCX1.4) (amino acids 875–903) is modeled to contain the last transmembrane α helix (amino acids 875–894) and an intracellular extramembraneous tail of 9 amino acids (895–903). Truncation of the last 9 C-terminal amino acids, Glu-895 to stop, did not significantly impair functional expression in HeLa or HEK 293 cells. Truncation, however, of 10 amino acids (Leu-894 to stop; mutant C10) reduced Na+gradient-dependent Ca2+ uptake to 35–39% relative to the wild type parent exchanger, and further truncation of 13 or more amino acids resulted in expression of trace amounts of transport activity. Western analysis indicated that Na+-Ca2+ exchanger protein was produced whether transfection was carried out with functional or non-functional mutants. Immunofluorescence studies of HEK 293 cells expressing N-Flag epitope-tagged wild type and mutant Na+-Ca2+exchangers revealed that transport activity in whole cells correlated with surface expression. All cells expressing the wild type exchanger or C9 exhibited surface expression of the protein. Only 39% of the cells expressing C10 exhibited surface expression, and none was detected in cells transfected with non-functional mutants C13 and C29. Since functional and non-functional mutants were glycosylated, the C terminus is not mandatory to translocation into the endoplasmic reticulum (ER). Endoglycosidase H digestion of [35S]methionine-labeled protein derived from wild type Na+-Ca2+ exchanger and from C10 indicated that resistance to the digestion was acquired after 1 and 5 h of chase, respectively. C29 did not acquire detectable resistance to endoglycosidase H digestion even after 10 h of chase. Taken together, these results suggest that the “cellular quality control machinery” can tolerate the structural change introduced by truncation of the C terminus up to Ser-893 albeit with reduced rate of ER→Golgi transfer and reduced surface expression of the truncated protein. Further truncation of C-terminal amino acids leads to retention of the truncated protein in the ER, no transfer to the Golgi, and no surface expression.