High impact information on AHA2

• Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2 plasma membrane H(+)-ATPase [1].
• All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes [2].
• 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 [3].
• 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 [3].
• To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca(2+)-, Na(+)/K(+)-, H(+)/K(+)-, and H(+)-ATPases and which coordinates Ca(2+) ions in the SERCA1 Ca(2+)-ATPase [4].

Biological context of AHA2

• In comparison with the 16 introns reported in AHA3, AHA2 is missing one intron in the 5'-untranslated region and a second intron in the C-terminal coding region [5].
• We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein [6].
• Binding of 14-3-3 protein to the plasma membrane H(+)-ATPase AHA2 involves the three C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation of Thr(947) [6].
• ATP hydrolytic activity of AHA2 expressed in yeast internal membranes was activated by all tested isoforms of the 14-3-3 protein of yeast and Arabidopsis, but only in the presence of fusicoccin, and activation was prevented by a phosphoserine peptide representing a known 14-3-3 protein binding motif in Raf-1 [7].
• When compared with AHA3, AHA1 and AHA2 had an apparent higher turnover rate for ATP hydrolysis, exhibited a 10-fold higher apparent affinity for ATP, and a 3-fold higher sensitivity toward vanadate [8].

Anatomical context of AHA2

• By testing the fusicoccin binding activity of yeast membranes, the C-terminal regulatory domain of AHA2 was found to be part of a functional fusicoccin receptor, a component of which was the 14-3-3 protein [7].
• AHA2 was expressed mainly in intracellular membranes and only supported very slow growth of transformed yeast cells [9].
• We have used the 2.6 A structure of the rabbit sarcoplasmic reticulum Ca(2+)-ATPase isoform 1a, SERCA1a [Toyoshima, C., Nakasako, M., Nomura, H. and Ogawa, H. (2000) Nature 405, 647-655], to build models by homology modelling of two plasma membrane (PM) H(+)-ATPases, Arabidopsis thaliana AHA2 and Saccharomyces cerevisiae PMA1 [10].

Associations of AHA2 with chemical compounds

• In addition, AHA2 had a slightly lower apparent affinity for H+ and seemed to be more susceptible to activation by lysophosphatidylcholine than did AHA1 and AHA3 [8].
• Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana [11].

Other interactions of AHA2

• The AHA2 gene is most similar to AHA1, with predicted proteins containing 95% amino acid identity [5].

Analytical, diagnostic and therapeutic context of AHA2

• Northern blot analysis indicates that AHA2 mRNA relative to total cellular RNA is expressed at significantly higher levels in root tissue as compared with shoot tissue [5].
• The mRNA start site and 5'-untranslated sequence for AHA2 were determined from cDNA amplified by the polymerase chain reaction [5].
• Following site-directed mutagenesis within the 45 C-terminal residues of AHA2, we conclude that, in addition to the 946YpTV motif, a number of residues located further upstream are required for phosphorylation-independent binding of 14-3-3 [12].
• Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: mapping of residues that when altered give rise to an activated enzyme [11].

References