Catalytic and regulatory sites of yeast plasma membrane H +-ATPase studied by directed mutagenesis

Abstract

More than 35 site-directed mutants of the plasma membrane H +-ATPase of the yeast Saccharomyces cerevisiae have been constructed and expressed to investigate the function of N- and C-termini and of conserved amino acids. Conserved motif TGES seems to form part of both the catalytic machinery for the hydrolysis of the phosphorylated intermediate and the vanadate binding site. In addition, it is involved in the coupling of ATP hydrolysis to H + transport. The phosphorylated intermediate is also essential for this coupling, but not for ATP hydrolysis. The aspartate residues of conserved motifs DPPR, TGD and TGDGVND (the last one) seem to form part of the ATP binding site. The positive charge of the conserved motif KGAP is important for the kinase or phosphorylating activity. A conserved proline and a conserved aspartate predicted to have a transmembrane location are essential for activity. The N-terminus contains a conserved acidic region which may be involved in assembly into the plasma membrane. All the hydrophobic stretches at the C-terminus are also required for assembly. The last 11 amino acids constitute a non-essential inhibitory domain involved in regulation of the enzyme by glucose metabolism.