title: Post-translational modification of plant plasma membrane H(+)-ATPase as a requirement for functional complementation of a yeast transport mutant.
abstract: Many heterologous membrane proteins expressed in the yeast Saccharomyces cerevisiae fail to reach their normal cellular location and instead accumulate in stacked internal membranes. Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 (AHA2) is expressed predominantly in yeast internal membranes and fails to complement a yeast strain devoid of its endogenous H(+)-ATPase Pma1. We observed that phosphorylation of AHA2 in the heterologous host and subsequent binding of 14-3-3 protein is crucial for the ability of AHA2 to substitute for Pma1. Thus, mutants of AHA2, complementing pma1, showed increased phosphorylation at the penultimate residue (Thr(947)), which creates a binding site for endogenous 14-3-3 protein. Only a pool of ATPase in the plasma membrane is phosphorylated. Double mutants carrying in addition a T947A substitution lost their ability to complement pma1. However, mutants affected in both autoinhibitory regions of the C-terminal regulatory domain complemented pma1 irrespective of their ability to become phosphorylated at Thr(947). This demonstrates that it is the activity status of the mutant enzyme and neither redirection of trafficking nor 14-3-3 binding per se that determines the ability of H(+)-pumps to rescue pma1.
authors.affiliation: Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Jahn
authors.forename: Alexander
authors.initials: A
authors.lastname: Schulz
authors.forename: Jan
authors.initials: J
authors.lastname: Taipalensuu
authors.forename: Michael Gjedde
authors.initials: MG
authors.lastname: Palmgren
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D002462:Cell Membrane / Q000201:enzymology*; D003001:Cloning, Molecular; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics; D007700:Kinetics; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D006180:Proton-Translocating ATPases / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D007527:Isoenzymes; D006180:Proton-Translocating ATPases