Abstract
Plants employ a number of phosphorylation cascades in response to a wide range of environmental stimuli. Previous studies in Arabidopsis and yeast indicate that histidine kinase AHK1 is a positive regulator of drought and osmotic stress responses. Based on these studies AHK1 was proposed a plant osmosensor, although the molecular basis of plant osmosensing still remains unknown. To understand the molecular role and signaling mechanism of AHK1 in osmotic stress, we have expressed and purified full-length AHK1 from Arabidopsis in a bacterial host to allow for studies on the isolated transmembrane receptor. Purification of the recombinant protein solubilized from the host membranes was achieved in a single step by metal-affinity chromatography. Analysis of the purified AHK1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting show a single band indicating that the preparation is highly pure and devoid of contaminants or degradation products. In addition, gel filtration experiments indicate that the preparation is homogenous and monodisperse. Finally, CD-spectroscopy, phosphorylation activity, dimerization studies, and protein–protein interaction with plant phosphorylation targeting AHP2 demonstrate that the purified protein is functionally folded and acts as phospho-His or phospho-Asp phosphatase. Hence, the expression and purification of recombinant AHK1 reported here provide a basis for further detailed functional and structural studies of the receptor, which might help to understand plant osmosensing and osmosignaling on the molecular level.
Highlights
Plants are sessile organisms that have to adapt to fluctuating environmental conditions during their life cycle
AHK5output (Figure 7, lane 3) nor the background-caused and AHK5output -caused enhanced labelling of AHP2 (Figure 7, lanes 4 and 6) were observed anymore. These results indicate that AHK1 is able to empty the AHK5output -to-AHP2 phosphorelay system from phosphoryl residues and, acts as
Despite participatinginintwo-component two-component signal transfer processesand andtheir theircounterparts counterparts in in plants plants involved ourour processes involved in in multi-step multi-stepphosphorelay phosphorelaysignaling, signaling, informationon onthermodynamics thermodynamics and and kinetics kinetics that ofof these sensor kinases information thatdetermine determinethe theinteraction interaction these sensor kinases with theirdownstream downstreamhistidine histidine phosphotransfer phosphotransfer proteins changes with their proteinsasaswell wellasasthe theconformational conformational changes triggering signal transfer activity in the phoshorelay is still sparse
Summary
Plants are sessile organisms that have to adapt to fluctuating environmental conditions during their life cycle. In contrast to the typical prokaryotic two-component phosphotransfer systems (TCS) involving a single His-Asp phosphotransfer process from a soluble or membrane-bound sensor histidine kinase to the receiver domain of a cognate response regulator protein, plants employ multi-step His-Asp phosphotransfer cascades (phosphorelays). Further studies demonstrate that five members of the Arabidopsis HK family encode receptors sensing the plant hormone ethylene (ETR1, ERS1, ETR2, ERS2, and EIN4) [12,13,14], whereas the other six encode non-ethylene receptor kinases (AHK1, AHK2, AHK3, CRE1/AHK4, CKI1, and CKI2/AHK5). Three of the non-ethylene receptors (AHK2, AHK3, and CRE1) have been identified as receptors for the plant hormone cytokinin [15,16,17]. The residual three HK receptors (AHK1, CKI1, and AHK5) show neither ethylene nor cytokinin-related activity and have been attributed to a variety of plant processes including osmoregulation [18], megagametogenesis [19], salt sensitivity [20], or stomatal responses [21], the molecular trigger or ligand stimulating these activities is not known for all of these processes yet
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