Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region

The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capa ble of functionally replacing the H+-ATPase genes of the yeast Saccharomyce s cerevisiae, provided that the external pH is kept above 5.0, Single point mutations within the pma2 gene were previously identified that improved H-ATPase activity and allowed yeast growth at pH 4.0. The aim of the present study was to identify most of the PMA2 positions, the mutation of which wo uld lead to improved growth and to determine whether all these mutations re sult in similar enzymatic and structural modifications. We selected additio nal mutants in total 42 distinct point mutations localized in 30 codons, Th ey were distributed in 10 soluble and membrane regions of the enzyme. Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lo wer inhibition by ADP, and lower stimulation by lysophosphatidylcholine tha n wild-type PMA2. The mutants thus seem to be constitutively activated. Par tial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible. Thes e data therefore support the hypothesis that point mutations in various H+- ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation. The high density of mutations within the first half of the C-t erminal region suggests that this part is involved in the interaction betwe en the inhibitory C-terminal region and the rest of the enzyme.