Expression of a functionally active human renal sodium-calcium exchanger lacking a signal sequence.
The Na+-Ca2+ exchanger is an unusual membrane transport protein as it contains an NH2-terminal signal sequence which is co-translationally removed in the endoplasmic reticulum during synthesis. To determine if the signal sequence was essential for biosynthesis, mutations were introduced in the NH2 terminus of the cDNA coding for the human renal Na+-Ca2+ exchanger in order to alter processing of the protein. To prevent cleavage of the signal sequence during biosynthesis, the last residue of the consensus signal sequence, Ala-1, was changed to Phe. Deletion mutants were also constructed to encode for exchangers which lacked the signal sequence, the signal sequence and the first extracellular loop, or all of the NH2 terminus including the first transmembrane segment of the mature protein. These mutants were expressed in HEK 293 cells and assayed for Na+-Ca2+ exchange activity. Mutants lacking either a signal sequence or containing a noncleavable signal sequence were still targeted to the plasma membrane, where they exhibited Na+-Ca2+ exchange activity. By contrast, the mutants which had more than the signal sequence deleted did not demonstrate any exchange activity. These mutants were, however, still integrated into the membrane and were resistant to alkali extraction. These results show that the signal sequence is not essential for biogenesis of the Na+-Ca2+ exchanger and suggests that the molecule contains one or more internal signal sequences for insertion into the membrane during biosynthesis.
Loo
T W
TW
Department of Medicine, University of Toronto, Ontario, Canada.
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Clarke
D M
DM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium