title: Point mutations in the extracytosolic loop between transmembrane segments M5 and M6 of the yeast Pma1 H+-ATPase: alanine-scanning mutagenesis.
abstract: Membrane-spanning segments M4, M5, M6, and M8 of the H(+)-, Ca(2+)-, and K(+), Na(+)-ATPases, which belong to the P2-type pumps are the core through which cations are transported. M5 and M6 loop is a short extracytoplasmic stretch of the seven amino acid residues (714-DNSLDID) connecting two of these segments, M5 and M6, where residues involved in the formation of the proton-binding site(s) are located. In the present study, we have used alanine-scanning mutagenesis to explore the structural and functional relationships within this loop of the yeast plasma membrane Pma1 H(+)-ATPase. Of the 7 Ala mutants made, substitution for the most conserved residue (Leu-717) has led to a severe misfolding and complete block in biogenesis of the mutant enzyme. The replacement of Asp-714 has also caused misfolding leading to significant decrease in the expression of the mutant and loss of activity. The remaining mutants were expressed in secretory vesicles at 21-119% of the wild-type level and were active enough to be analyzed in detail. One of these mutants (I719A) showed five- to threefold decrease in both expression and ATP hydrolyzing and H(+) pumping activities and also threefold reduction in the coupling ratio between ATP hydrolysis and H(+) transport. Thus, Ala substitutions at three positions of the seven seriously affected biogenesis, folding, stability and/or functioning of the enzyme. Taken together, these results lead to suggestion that M5 and M6 loop play an important role in the protein stability and function and is responsible for proper arrangement of transmembrane segments M5 and M6 and probably other domains of the enzyme. Results for additional conserved substitutions (Asn and Glu) at Asp-714 and Asp-720 confirmed this suggestion.
authors.affiliation: a Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences , pr. Nauki 5, Pushchino 142290 , Russia.
authors.forename: Valery V
authors.initials: VV
authors.lastname: Petrov
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D006868:Hydrolysis; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D017354:Point Mutation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases; D000409:Alanine
keywords: H+ transport; H+-ATPase; biogenesis; coupling; plasma membrane; secretory vesicles; yeast