The carboxyl-terminal region of the Na+/H+ exchanger interacts with mammalian heat shock protein.
We expressed the carboxyl-terminal 178 amino acids of the rabbit cardiac Na+/H+ exchanger as a fusion protein with glutathione-S-transferase. The fusion protein (PCR178) was found in the supernatant of extracts of E. coli and was purified using Glutathione-Sepharose affinity chromatography. Affinity-purified antibodies raised against the carboxyl-terminal region of the Na+/H+ exchanger identified the resultant protein. PCR178 copurified with a 70 kDa protein. Amino-terminal sequencing of the 70 kDa protein identified it as dnaK, the bacterial equivalent of the mammalian 70 kDa heat shock protein (hsp70). DnaK was dissociated from the Na+/H+ exchanger fusion protein by the addition of MgATP. When purified PCR178 was coupled to a cyanogen bromide-activated Sepharose column, bovine hsp70 bound to the column and was eluted with MgATP. Nondenaturing polyacrylamide gel electrophoresis showed that, in the absence of MgATP, hsp70 formed a complex with PCR178. The complex was dissociated by the addition of MgATP. GST alone did not form a complex with hsp70. Immunoprecipitation of the Na+/H+ exchanger with antiexchanger antibodies resulted in coprecipitation of hsp70 protein from antiporter containing cells. Cells that overexpress the Na+/H+ exchanger had increased amounts of hsp70 which coprecipitated with antiexchanger antibody. The results show that heat shock protein complexes with the mammalian Na+/H+ exchanger.
Silva
N L
NL
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Haworth
R S
RS
Singh
D
D
Fliegel
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D005982:Glutathione Transferase