Journal Article

Plasma Membrane H<sup>+</sup>-ATPase and 14-3-3 Isoforms of <i>Arabidopsis</i> Leaves: Evidence for Isoform Specificity in the 14-3-3/H<sup>+</sup>-ATPase Interaction

Magnus Alsterfjord, Paul C. Sehnke, Annika Arkell, Håkan Larsson, Fredrik Svennelid, Magnus Rosenquist, Robert J. Ferl, Marianne Sommarin and Christer Larsson

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 45, issue 9, pages 1202-1210
Published in print September 2004 | ISSN: 0032-0781
Published online September 2004 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pch136
Plasma Membrane H+-ATPase and 14-3-3 Isoforms of Arabidopsis Leaves: Evidence for Isoform Specificity in the 14-3-3/H+-ATPase Interaction

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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.

Keywords: Keywords: Arabidopsis — Isoforms — Plasma membrane H+-ATPase — 14-3-3 protein.; Abbreviations: AHA, Arabidopsis H+-ATPase; BSA, bovine serum albumin; DTT, dithiothreitol; EST, expressed sequence tag; FC, fusicoccin; GF14, G box factor 14-3-3, the designation used for Arabidopsis 14-3-3 proteins; MS, mass spectrometry; NCBI, National Center for Biotechnology Information; PM, plasma membrane; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; PVPP, polyvinylpolypyrrolidone; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TAIR, The Arabidopsis Information Resource.

Journal Article.  6437 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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