Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S.cerevisiae

Elena L. Rudashevskaya, Juanying Ye, Ole Nørregaard Jensen, Michael G. Palmgren, Anja T. Fuglsang

Publikation: Konferencebidrag uden forlag/tidsskriftPosterForskning

Abstrakt

 It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H+-ATPases are app. 60 amino acid residues longer than their yeast homologous. Yeast is found to phosphorylate at least one residue within the plant C-terminus. At the same time a wide range of investigations on structure, function, regulation and interaction of H+-ATPase is carried out with implication of heterologous system of yeast cells, expressing plant proton pump. Therefore identification of possible regulatory effects by phosphorylation events in plant H+-ATPase in the system is significant.

A number of putative phosphorylation sites at regulatory C-domain of H+-ATPase (AHA2) have been point-mutated to alanine residues (to prevent possible phosphorylation) or aspartate residues (to mimic phosphorylation of residue) and the mutated aha2 enzyme expressed in the yeast strain RS-72. Most of the mutations show positive or negative effect on yeast growth in functional complementation assays. It shows in vivo functioning of the residues and suggests, that plant H+-ATPase could be regulated by phosphorylation at several sites being in yeast cells.

Plant H+-ATPase purified from yeast cells by his-tag affinity chromatography was subjected to IMAC and TiO2 for enrichment of phosphopeptides. The phosphopeptides were analyzed by LC/MS/MS. Phosphorylation sites identified in vivo in the heterologous host are compared to the functional analysis of the aha2 point mutations.
OriginalsprogEngelsk
Publikationsdato2008
StatusUdgivet - 2008
Begivenhed12th International ATPase Conference - Aarhus, Danmark
Varighed: 5. aug. 200810. aug. 2008

Konference

Konference12th International ATPase Conference
LandDanmark
ByAarhus
Periode05/08/200810/08/2008

    Fingerprint

Citationsformater

L. Rudashevskaya, E., Ye, J., Jensen, O. N., G. Palmgren, M., & T. Fuglsang, A. (2008). Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S.cerevisiae. Poster session præsenteret på 12th International ATPase Conference, Aarhus, Danmark.