Abstract
The activity of many P-type ATPases is found to be regulated by interacting proteins or autoinhibitory elements located in N- or C-terminal extensions. An extended C terminus of fungal and plant P-type plasma membrane H(+)-ATPases has long been recognized to be part of a regulatory apparatus involving an autoinhibitory domain. Here we demonstrate that both the N and the C termini of the plant plasma membrane H(+)-ATPase are directly involved in controlling the pump activity state and that N-terminal displacements are coupled to secondary modifications taking place at the C-terminal end. This identifies the first group of P-type ATPases for which both ends of the polypeptide chain constitute regulatory domains, which together contribute to the autoinhibitory apparatus. This suggests an intricate mechanism of cis-regulation with both termini of the protein communicating to obtain the necessary control of the enzyme activity state.
Highlights
Autoinhibition of proteins is a widespread occurrence of enzymatic regulation identified among a variety of different protein structures and families [4]
In an attempt to obtain three-dimensional structural information of the autoinhibited Plasma membrane (PM) Hϩ-ATPase, we constructed a mutant of the PM Hϩ-ATPase with a cleavable affinity tag in the N terminus, inserted at position 11 (Fig. 2)
The plant plasma membrane Hϩ-ATPase has long been known to be autoinhibited through a regulatory domain located at its C terminus
Summary
Construction of Mutants—The multicopy vector Yep-351 [20] containing the full-length cDNA of the Arabidopsis thaliana AHA2 PM Hϩ-ATPase isoform under the control of the PMA1 promoter [21] was used as in Ref. 22. Yeast complementation assays were performed on solid minimal media containing either galactose or glucose as described previously [8]. The yeast cells were harvested, and plasma membranes were purified essentially as described [8], except that yeast cells were homogenized in 29% glycerol (v/v), 50 mM Mes-KOH, pH 6.5, 10 mM EDTA, 1.3 mM ATP, 50 mM KCl, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, and 0.4 g/ml pepstatin A. Immunological detection of bound 14-3-3 protein was performed by a so-called 14-3-3 overlay assay as described [12]. ATPase Assay—An ATPase assay was performed as described previously [8] on plasma-enriched fractions, and unless otherwise stated, the assay buffer contained 3 mM ATP
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