Amino acids in the TM4-TM5 loop of Na,K-ATPase are important for biosynthesis

The ten-transmembrane Na,K-ATPase alpha-subunit exposes very few amino acids to the extra membrane space except for an approximately 408 residue-long loop between transmembrane segments four and five. The present paper focuses on the role of this loop in biosynthesis of functional Na,K-ATPase. Expression of 39 mutations in this loop to phylogenetically conserved as well as nonconserved residues showed that only two could be expressed at 30degreesC. By contrast, only five could not be produced in a functional form at 15degreesC. A detailed analysis showed that a number of these mutants are temperature-sensitive folding mutants, as they induce the unfolded protein response at 30degreesC but not at 15degreesC. We used an algorithm to predict that residues (868)ENGFLIPIHLL(878) in the L78 loop exposed to the endoplasmic reticulum lumen constitute the most likely BiP binding site. Correct folding of this sequence may be important in the endoplasmic reticulum quality control, as the same loop is responsible for the alpha-beta-associations required to leave this compartment. On the basis of the Ca-ATPase crystal structure and the presented data, we propose a model to account for the role of the TM4-TM5 loop in Na,K-ATPase biosynthesis.