Role of phylogenetically conserved amino acids in folding of Na,K-ATPase

This paper focuses on the amino acid sequence 708-TGDGVNDSPALKK in pig kidn ey Na,K-ATPase as one of the best conserved among P-type ATPases. In Ca-ATP ase this sequence forms a strand-loop-helix structure as part of a Rossman fold next to the phosphorylation site. Substitution of polar residues in th e investigated sequence interfered with high-level accumulation of mutant p rotein. Mutant alpha (1)-subunit protein only accumulated in membranes from yeast cells grown at 15 degreesC whereas wild-type protein accumulated at both 15 and 35 degreesC. A systematic screen for the molecular mechanism be hind lack of accumulation of mutant protein at 35 degreesC showed that tran scription and translation were unaffected by the mutations. To demonstrate in vivo protein folding problems, an unfolded protein response reporter sys tem was constructed in yeast. In this strain, only expression of mutant Na, K-ATPase alpha (1)-subunit caused induction of the unfolded protein respons e at 35 degreesC, indicating folding problems in the ER. Lowering the expre ssion temperature to 15 degreesC prevented induction of the unfolded protei n response after mutant protein expression, indicating correct folding at t his temperature. At the permissive temperature mutant proteins were able to escape the endoplasmic reticulum quality control, reach the plasma membran e, and bind ouabain with high affinity. Since mutants in the 708-TGDGVNDSPA LKK segment had a thermo inactivation profile identical to that of wild hyp e, they were classified as temperature-sensitive synthesis mutants. The res ults indicate that this segment contributes side chains of importance for o verall folding and maturation of Na,K-ATPase and all other P-type ATPases.