A systematic mutagenesis study of Ile-282 in transmembrane segment M4 of the plasma membrane H+-ATPase.
Homology models of plasma membrane H(+)-ATPase (Bukrinsky, J. T., Buch-Pedersen, M. J., Larsen, S., and Palmgren, M. G. (2001) FEBS Lett. 494, 6-10) has pointed to residues in transmembrane segment M4 as being important for proton translocation by P-type proton pumps. To test this model, alanine-scanning mutagenesis was carried out through 12 residues in the M4 of the plant plasma membrane H(+)-ATPase AHA2. An I282A mutation showed apparent reduced H(+) affinity, and this residue was subsequently substituted with all other naturally occurring amino acids by saturation mutagenesis. The ability of mutant enzymes to substitute for the yeast proton pump PMA1 was found to correlate with the size of the side chain rather than its chemical nature. Thus, smaller side chains (Gly, Ala, and Ser) at this position resulted in lower H(+) affinity and lowered levels of H(+) transport in vivo, whereas substitution with side chains of similar and larger size resulted in only minor effects. Substitutions of Ile-282 had only minor effects on ATP affinity and sensitivity toward vanadate, ruling out an indirect effect through changes in the enzyme conformational equilibrium. These results are consistent with a model in which the backbone carbonyl oxygen of Ile-282 contributes directly to proton translocation.
Fraysse
A Staffan
AS
Department of Plant Biology, The Royal Danish Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Møller
Anders L B
AL
Poulsen
Lisbeth R
LR
Wollenweber
Bernd
B
Buch-Pedersen
Morten J
MJ
Palmgren
Michael G
MG
D000255:Adenosine Triphosphate / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics*; D002244:Carbon / Q000737:chemistry; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics*; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007532:Isoleucine / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D010944:Plants / Q000201:enzymology; D053499:Plasma Membrane Calcium-Transporting ATPases; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics*; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics*; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology
D027682:Cation Transport Proteins; D010940:Plant Proteins; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins; D007532:Isoleucine; D014638:Vanadates; D002244:Carbon; D000255:Adenosine Triphosphate; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D005947:Glucose; D000409:Alanine; D010100:Oxygen