RESUMO
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.
RESUMO
Signal perception and transmission of the plant hormone ethylene are mediated by a family of receptor histidine kinases located at the Golgi-ER network. Similar to bacterial and other plant receptor kinases, these receptors work as dimers or higher molecular weight oligomers at the membrane. Sequence analysis and functional studies of different isoforms suggest that the ethylene receptor family is classified into two subfamilies. In Arabidopsis, the type-I subfamily has two members (ETR1 and ERS1) and the type-II subfamily has three members (ETR2, ERS2, and EIN4). Whereas subfamily-I of the Arabidopsis receptors and their interactions with downstream elements in the ethylene pathway has been extensively studied in the past; related information on subfamily-II is sparse. In order to dissect the role of type-II receptors in the ethylene pathway and to decode processes associated with this receptor subfamily on a quantitative molecular level, we have applied biochemical and spectroscopic studies on purified recombinant receptors and downstream elements of the ethylene pathway. To this end, we have expressed purified ETR2 as a prototype of the type-II subfamily, ETR1 for the type-I subfamily and downstream ethylene pathway proteins CTR1 and EIN2. Functional folding of the purified receptors was demonstrated by CD spectroscopy and autokinase assays. Quantitative analysis of protein-protein interactions (PPIs) by microscale thermophoresis (MST) revealed that ETR2 has similar affinities for CTR1 and EIN2 as previously reported for the subfamily-I prototype ETR1 suggesting similar roles in PPI-mediated signal transfer for both subfamilies. We also used in planta fluorescence studies on transiently expressed proteins in Nicotiana benthamiana leaf cells to analyze homo- and heteromer formation of receptors. These studies show that type-II receptors as well as the type-I receptors form homo- and heteromeric complexes at these conditions. Notably, type-II receptor homomers and type-II:type-I heteromers are more stable than type-I homomers as indicated by their lower dissociation constants obtained in microscale thermophoresis studies. The enhanced stability of type-II complexes emphasizes the important role of type-II receptors in the ethylene pathway.
