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Expression of a functionally active human renal sodium-calcium exchanger lacking a signal sequence
1Department of Medicine, University of Toronto, Ontario, Canada.
The Journal of Biological Chemistry
|August 18, 1995
Summary
The NH2-terminal signal sequence is not essential for the biosynthesis of the sodium-calcium exchanger (Na+-Ca2+). This suggests internal sequences guide membrane insertion for this vital transport protein.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The Na+-Ca2+ exchanger is a membrane transport protein.
- It possesses an NH2-terminal signal sequence removed during synthesis in the endoplasmic reticulum.
Purpose of the Study:
- To investigate the essentiality of the NH2-terminal signal sequence for Na+-Ca2+ exchanger biosynthesis.
- To determine if mutations affecting signal sequence processing impact protein function.
Main Methods:
- Mutagenesis of the NH2-terminal signal sequence of the human renal Na+-Ca2+ exchanger cDNA.
- Expression of wild-type and mutant proteins in HEK 293 cells.
- Assay of Na+-Ca2+ exchange activity and membrane integration.
Main Results:
- Mutants lacking a cleavable signal sequence retained Na+-Ca2+ exchange activity and plasma membrane targeting.
- Mutants with extensive NH2-terminal deletions, including the signal sequence, lost exchange activity but remained membrane-integrated.
- Alkali extraction confirmed membrane integration of deleted mutants.
Conclusions:
- The NH2-terminal signal sequence is not essential for the biogenesis of the Na+-Ca2+ exchanger.
- Internal signal sequences likely mediate membrane insertion during biosynthesis.