Insertion of red fluorescent protein into the amino terminus of Na,K‐ATPase catalytic subunit does not disrupt post‐translational processing

The first five amino acids of the catalytic α1 isoform from the Na,K‐ATPase undergo proteolytic cleavage during or after translation. Previous work suggests that amino acids downstream of the first nine are not required for proteolysis. To explore this inference, I tested whether increasing the distance between the amino terminus and the main body of the protein would influence cleavage. A chimeric cDNA was prepared in which sequence encoding a variant of DS‐red, mCherry, was inserted into a highly‐conserved leucine‐lysine pair just downstream from the amino terminus of the nascent polypeptide. The resulting mutant was introduced into opossum kidney cells (OK) by DNA‐mediated gene transfer, and stable recipients were examined by fluorescence microscopy and immunoblotting. Cells exhibited both surface and intracellular fluorescence, indicating that the chimeric catalytic subunit was targeted successfully to the plasmalemma. A site‐directed antibody raised against the first nine residues of the α1 nascent chain (anti‐VGR) did not bind to membranes from the transfected cells. However, a band with a mobility appropriate for the chimeric α was detected with an antibody that is specific for mCherry and a second site‐directed antibody specific for rat α1, further confirming expression of the chimeric protein. Because anti‐VGR does not recognize cleaved α1, the absence of binding suggests that the α1‐mCherry construct underwent normal processing, despite the insertion of irrelevant sequence in the amino terminus. Consistent with earlier work, these results suggest strongly that the determinants of amino terminal processing in the catalytic subunit of the Na,K‐ATPase reside within the first few residues of the nascent polypeptide. [Supported by NIH RR‐19799.]