Expression of an active NA,K-ATPase with an α-subunit lacking all twenty-three native cysteine residues

Abstract

We have constructed a mutant Na,K-ATPase α1-subunit with all native cysteine residues replaced. Using the baculovirus system, this cysteine-less α1-subunit and wild-type β1-subunit were expressed in High Five cells. After 3 days of infection, cells were fractionated, and endoplasmic reticulum, Golgi apparatus, and plasma membranes were isolated. The molecular activity of the cysteine-less mutant in the plasma membranes was close to the wild-type protein (8223 min-1 versus 6655 min-1). Cation and ATP activation of Na,K-ATPase activities revealed that replacing all 23 cysteines resulted in only a 50% reduction of K(m) for Na+, a 2-fold increase in K(m) for K+, and no changes in K(m) for ATP. The distribution of α-subunits among the membranes showed a high percentage of cysteine-less protein in the endoplasmic reticulum and Golgi apparatus compared with the wild-type protein. Furthermore, the cellular stability of the αβ assembly appeared reduced in the cysteine-less mutant. Cells harvested after more than 3 days of infection showed extensive degradation of the cysteine-less α-subunit, which is not observed with the wild-type enzyme. Thus the Na,K-ATPase contains no cysteine residues that are critical for function, but the folding and/or assembly pathway of this enzyme is affected by total cysteine substitution.