Abstract
We have constructed a mutant Na,K-ATPase alpha1-subunit with all native cysteine residues replaced. Using the baculovirus system, this cysteine-less alpha1-subunit and wild-type beta1-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 alpha-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 alphabeta assembly appeared reduced in the cysteine-less mutant. Cells harvested after more than 3 days of infection showed extensive degradation of the cysteine-less alpha-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.
Highlights
Na,K-ATPase (EC 3.6.1.3) is a heterodimeric membrane protein that utilizes the energy of hydrolysis of one ATP molecule to transport three sodium ions and two potassium ions against their electrochemical potential gradients
The distribution of ␣-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
Expression and Enzymatic Characterization of Expressed Na,K-ATPases—The wild-type and cysteine-less versions of the sheep Na,K-ATPase ␣1-subunits were co-expressed with the wild-type 1-subunit in baculovirus-infected High Five cells [19] for 3 or 5 days, and the ER, Golgi apparatus, and plasma membrane fractions of the infected cells were isolated by sucrose gradient fractionation
Summary
Plasmids and Construction of Mutants—A sheep ␣1-subunit cDNA was cloned into the pOCUS-2 vector (Novagen) as a NotI and Sse8387I fragment. Recombinant baculovirus containing the sheep 1-subunit and the wild-type or cysteine-less ␣1-subunit cDNA was produced by following the protocols described previously [19]. The genomic DNA of the recombinant baculoviruses was isolated by using the Easy-DNA Kit (Invitrogen) and was sequenced to ensure the appropriate cysteine mutations in the ␣-subunit. Protein Expression and Purification—Log-phase high viability High Five cells were infected with the recombinant baculoviruses for 3 or 5 days and were harvested for protein purification [19]. ATPase Assays—Na,K-ATPase assays of High Five membrane proteins were carried out as described [19]. For determining Kϩ activation, the KCl concentration was varied from 0 to 30 mM. For ATP activation, the ATP concentration was varied from 0 to 1.2 mM. Curve fitting of the ligand-activation data was performed using the computer software Sigma Plot as described previously [22]
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