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Characterization of yeast plasma membrane H(+)-ATPase mutant pma1-A135V and its revertants
S Na1, D S Perlin, D Seto-Young
1Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts 02254.
Abstract:
An A135V substitution in the first transmembrane segment of the yeast plasma membrane H(+)-ATPase (PMA1) confers cellular resistance to hygromycin B, exhibits growth sensitivity to low external pH, and results in a defective enzyme that hydrolyzes ATP at 33% of wild type level. The importance of the A135 residue was probed genetically by analysis involving both site-directed mutagenesis and randomly generated second-site intragenic suppressor mutations. No other amino acid at position 135 gave either the wild type phenotype or the normal enzyme activity of A135. Substitutions with the bulkier amino acid residues A135L, A135I, and A135F produced more severe cellular phenotypes than the original A135V mutation. The substitution of the smaller side chain residue Gly was also a mutant, although not as severe as the A135V mutant. The introduction of a bulky Trp or a polar Ser residue produced dominant lethality, while charged amino acids produced recessive lethality. Reduced rates of proton transport measured by acidification of the medium by whole cells correlate closely with the severity of cellular phenotype. Some of the mutant enzymes exhibit an apparent instability in vitro. Thus, the localized structure around A135 is highly constrained. The cellular sensitivity to low external pH of the A135V mutant was used to select intragenic revertants. Most full revertants (low pHR, HygS) restored A135, but second-site mutations in putative transmembrane segments 2 (V146I and V157F) and 4 (L327V) were also observed. Two partial revertants (low pHR, HygR) have secondary mutations at S660C or a double change at F611L-S660F in the putative ATP binding domain. These results provide additional evidence for functional coupling between the cytoplasmic domain catalyzing ATP hydrolysis and transmembrane helices 1 and 2.
Insights
Altering residue 135 in yeast plasma membrane H(+)-ATPase (PMA1) affects proton transport and hygromycin B resistance. Suppressor mutations reveal functional coupling between the ATP-binding domain and transmembrane helices.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- The yeast plasma membrane H(+)-ATPase (PMA1) is crucial for maintaining cellular proton gradients.
- Specific amino acid residues within transmembrane segments are critical for enzyme function and cellular phenotypes.
- Understanding structure-function relationships in PMA1 provides insights into proton pump mechanisms.
Purpose of the Study:
- To investigate the functional importance of residue 135 in the first transmembrane segment of PMA1.
- To characterize the effects of various amino acid substitutions at position 135 on enzyme activity and cellular phenotypes.
- To identify second-site mutations that suppress the observed defects and elucidate functional coupling within PMA1.
Main Methods:
- Site-directed mutagenesis was employed to introduce specific amino acid substitutions at position 135.
- Randomly generated intragenic suppressor mutations were selected based on cellular phenotypes (pH sensitivity, hygromycin B resistance).
- Enzyme activity (ATP hydrolysis) and proton transport rates (medium acidification) were measured in vitro and in whole cells.
Main Results:
- The A135V substitution resulted in reduced ATP hydrolysis (33% of wild type), hygromycin B resistance, and sensitivity to low external pH.
- Substitutions with bulkier or smaller amino acids at position 135 generally exacerbated cellular phenotypes, indicating a constrained local structure.
- Suppressor mutations were identified in transmembrane segments 2 and 4, as well as the ATP-binding domain, suggesting functional communication.
Conclusions:
- Residue 135 plays a critical role in PMA1 structure and function, influencing proton transport and drug resistance.
- The identified suppressor mutations provide evidence for functional coupling between the cytoplasmic ATP-hydrolyzing domain and transmembrane helices.
- This study highlights the intricate interplay between different domains of the H(+)-ATPase for its overall activity.
