Truncation of the C terminus of the rat brain Na(+)-Ca(2+) exchanger RBE-1 (NCX1.4) impairs surface expression of the protein.
The C terminus of the rat brain Na(+)-Ca(2+) exchanger (RBE-1; NCX1. 4) (amino acids 875-903) is modeled to contain the last transmembrane alpha 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 Ca(2+) 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(+)-Ca(2+) 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(+)-Ca(2+) 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 [(35)S]methionine-labeled protein derived from wild type Na(+)-Ca(2+) 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.
Kasir
J
J
Department of Biochemistry, Hebrew University Hadassah Medical School Jerusalem 91120, Israel.
Ren
X
X
Furman
I
I
Rahamimoff
H
H
D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009842:Oligopeptides; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010455:Peptides; D051381:Rats; D017384:Sequence Deletion; D012964:Sodium / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D008565:Membrane Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D019831:Sodium-Calcium Exchanger; D014415:Tunicamycin; C085706:FLAG peptide; D012964:Sodium; D006596:Hexosaminidases; D002118:Calcium