Plasma membrane H(+)-ATPase and 14-3-3 isoforms of Arabidopsis leaves: evidence for isoform specificity in the 14-3-3/H(+)-ATPase interaction.
The plasma membrane H(+)-ATPase is activated by binding of 14-3-3 protein to the phosphorylated C terminus. Considering the large number of 14-3-3 and H(+)-ATPase isoforms in Arabidopsis (13 and 11 expressed genes, respectively), specificity in binding may exist between 14-3-3 and H(+)-ATPase isoforms. We now show that the H(+)-ATPase is the main target for 14-3-3 binding at the plasma membrane, and that all twelve 14-3-3 isoforms tested bind to the H(+)-ATPase in vitro. Using specific antibodies for nine of the 14-3-3 isoforms, we show that GF14epsilon, mu, lambda, omega, chi, phi, nu, and upsilon are present in leaves, but that isolated plasma membranes lack GF14chi, phi and upsilon. Northern blots using isoform-specific probes for all 14-3-3 and H(+)-ATPase isoforms showed that transcripts were present for most of the isoforms. Based on mRNA levels, GF14epsilon, mu, lambda and chi are highly expressed 14-3-3 isoforms, and AHA1, 3, and 11 highly expressed H(+)-ATPase isoforms in leaves. However, mass peptide fingerprinting identified AHA1 and 2 with the highest score, and their presence could be confirmed by MS/MS. It may be calculated that under 'unstressed' conditions less than one percent of total 14-3-3 is attached to the H(+)-ATPase. However, during a condition requiring full activation of H+ pumping, as induced here by the presence of the fungal toxin fusicoccin, several percent of total 14-3-3 may be engaged in activation of the H(+)-ATPase.
Alsterfjord
Magnus
M
Department of Plant Biochemistry, Lund University, PO Box 124, SE-22100 Lund, Sweden. magnus.alsterfjord@plantbio.lu.se
Sehnke
Paul C
PC
Arkell
Annika
A
Larsson
Håkan
H
Svennelid
Fredrik
F
Rosenquist
Magnus
M
Ferl
Robert J
RJ
Sommarin
Marianne
M
Larsson
Christer
C
D048948:14-3-3 Proteins / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013379:Substrate Specificity
D048948:14-3-3 Proteins; D007527:Isoenzymes; D020033:Protein Isoforms; D006180:Proton-Translocating ATPases