Molecular Analysis of Protein-Protein Interactions in the Ethylene Pathway in the Different Ethylene Receptor Subfamilies

Abstract

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.

High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1

“…The homomeric affinities of the AHK1 osmosensor strongly resemble the affinities observed for other sensor histidine kinases like AtETR1 and AtETR2 [7]. The same experimental setup and method, under which a dissociation constant of 208 nM was observed in this study for AHK1, showed values of 326 nM and 96 nM for AtETR1 and AtETR2 respectively [7]. The in vitro MST results of AHK1 homomerization were confirmed by two independent in vivo approaches, namely yeast mbSUS and in planta FRET-FLIM.…”

“…Remarkably, despite the apparent monomeric state under size exclusion chromatography conditions, the functionality of AHK1 in terms of potential to form homodimers could be shown in MST. The homomeric affinities of the AHK1 osmosensor strongly resemble the affinities observed for other sensor histidine kinases like AtETR1 and AtETR2 [7]. The same experimental setup and method, under which a dissociation constant of 208 nM was observed in this study for AHK1, showed values of 326 nM and 96 nM for AtETR1 and AtETR2 respectively [7].…”

“…Sequence analysis suggests that all members of the HK family have a modular basic structure consisting of an N-terminal transmembrane sensor domain followed by a catalytic transmitter domain and a receiver domain at the C-terminus. Biochemical, biophysical, and cellular studies show that the receptors form dimers and higher-molecular-weight oligomers at the membrane in their functional state [5][6][7][8][9][10][11]. 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).…”

Ethylene signaling in plants

“…It is unclear if dimerization between kinase domains of the other receptor isoforms occurs. It has also been suggested that heterodimers are possible (35,50). Evidence that these are receptors is that all of these proteins bind ethylene with high affinity (41,47,51,52) and specific mutations in any one of these proteins leads to ethyleneinsensitivity (36,(38)(39)(40)53).…”

Diverse and dynamic roles of F-box proteins in plant biology

“…The initiation of ethylene signal transduction begins with the perception of ethylene by the ethylene receptor family: ETHYLENE RESPONSE 1 (ETR1), ETHYLENE RESPONSE SENSOR 1 (ERS1) which are type-I subfamily, ETHYLENE RESPONSE 2 (ETR2), ETHYLENE-INSEN-SITIVE 4 (EIN4), and ETHYLENE RESPONSE SENSOR 2 (ERS2), a type-II subfamily, at the endoplasmic reticulum (ER) where the receptor can form a homomer or heteromer between different subfamilies (Berleth et al 2019). The signalling pathway continues from a transmembrane protein, ETHYLENE-INSENSITIVE 2 (EIN2), to transcription factor, ETHYLENE-INSENSITIVE 3 (EIN3)/ EIN3-LIKE1 (EIL1), to target genes encoding transcription factors such as ERF, WRKY and NAC family genes, or directly regulates gene expression (Dolgikh et al 2019).…”