© 2004 Oxford University Press
Plasma Membrane H+-ATPase and 14-3-3 Isoforms of Arabidopsis Leaves: Evidence for Isoform Specificity in the 14-3-3/H+-ATPase Interaction
1 Department of Plant Biochemistry, Lund University, PO Box 124, SE-22100 Lund, Sweden
2 Program in Plant Cellular and Molecular Biology, Department of Horticultural Sciences, University of Florida, Gainesville, FL 32611, U.S.A.
3 Department of Plant Biology, Swedish University of Agricultural Sciences, PO Box 7080, SE-75007 Uppsala, Sweden
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.
4 Corresponding author: E-mail, magnus.alsterfjord{at}plantbio.lu.se; Fax, +46-462-224-116.
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