Georg Groth

CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis

The gaseous phytohormone ethylene C2H4 mediates numerous aspects of growth and development. Genetic analysis has identified a number of critical elements in ethylene signaling, but how these elements interact biochemically to transduce the signal from the ethylene receptor complex at the endoplasmic reticulum (ER) membrane to transcription factors in the nucleus is unknown. To close this gap in our understanding of the ethylene signaling pathway, the challenge has been to identify the target of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) Raf-like protein kinase, as well as the molecular events surrounding ETHYLENE-INSENSITIVE2 (EIN2), an ER membrane-localized Nramp homolog that positively regulates ethylene responses. Here we demonstrate that CTR1 interacts with and directly phosphorylates the cytosolic C-terminal domain of EIN2. Mutations that block the EIN2 phosphorylation sites result in constitutive nuclear localization of the EIN2 C terminus, concomitant with constitutive activation of ethylene responses in Arabidopsis. Our results suggest that phosphorylation of EIN2 by CTR1 prevents EIN2 from signaling in the absence of ethylene, whereas inhibition of CTR1 upon ethylene perception is a signal for cleavage and nuclear localization of the EIN2 C terminus, allowing the ethylene signal to reach the downstream transcription factors. These findings significantly advance our understanding of the mechanisms underlying ethylene signal transduction.

EIN2, the central regulator of ethylene signalling, is localized at the ER membrane where it interacts with the ethylene receptor ETR1.

Genetic studies have identified the membrane protein EIN2 (ethylene insensitive 2) as a central component of ethylene signalling in Arabidopsis. In addition, EIN2 might take part in multiple hormone signalling pathways and in response to pathogens as demonstrated by recent genetic and biochemical studies. Here we show, by an integrated approach using in vivo and in vitro fluorescence techniques, that EIN2 is localized at the ER (endoplasmic reticulum) membrane where it shows specific interaction with the ethylene receptor protein ETR1.

Structure of the c14 rotor ring of the proton translocating chloroplast ATP synthase.

The structure of the membrane integral rotor ring of the proton translocating F1F0 ATP synthase from spinach chloroplasts was determined to 3.8 Å resolution by x-ray crystallography. The rotor ring consists of 14 identical protomers that are symmetrically arranged around a central pore. Comparisons with the c11 rotor ring of the sodium translocating ATPase from Ilyobacter tartaricus show that the conserved carboxylates involved in proton or sodium transport, respectively, are 10.6–10.8 Å apart in both c ring rotors. This finding suggests that both ATPases have the same gear distance despite their different stoichiometries. The putative proton-binding site at the conserved carboxylate Glu61 in the chloroplast ATP synthase differs from the sodium-binding site in Ilyobacter. Residues adjacent to the conserved carboxylate show increased hydrophobicity and reduced hydrogen bonding. The crystal structure reflects the protonated form of the chloroplast c ring rotor. We propose that upon deprotonation, the conformation of Glu61 is changed to another rotamer and becomes fully exposed to the periphery of the ring. Reprotonation of Glu61 by a conserved arginine in the adjacent a subunit returns the carboxylate to its initial conformation.

New Insight in Ethylene Signaling: Autokinase Activity of ETR1 Modulates the Interaction of Receptors and EIN2

Ethylene insensitive 2 (EIN2), an integral membrane protein of the ER network, has been identified as the central regulator of the ethylene signaling pathway. Still, the mechanism by which the ethylene signal is transferred from the receptors to EIN2 has not been solved yet. Here, we show that protein phosphorylation is a key mechanism to control the interaction of EIN2 and the receptors. In vivo and in vitro fluorescence studies reveal that the kinase domain of the receptors is essential for the interaction. Cyanide, an ethylene agonist, which is known to reduce auto-phosphorylation of the ethylene receptor ethylene resistant 1 (ETR1) or a mutation in the kinase domain of ETR1 that prevents auto-phosphorylation (H353A), increases the affinity of the receptors for EIN2. On the other hand, mimicking permanent auto-phosphorylation of ETR1 as in the mutant H353E releases the EIN2-ETR1 interaction from the control by the plant hormone. Based on our data, we propose a novel model on the integration of EIN2 in the ethylene signaling cascade.

Molecular association of the Arabidopsis ETR1 ethylene receptor and a regulator of ethylene signaling, RTE1

The plant hormone ethylene plays important roles in growth and development. Ethylene is perceived by a family of membrane-bound receptors that actively repress ethylene responses. When the receptors bind ethylene, their signaling is shut off, activating responses. REVERSION-TO-ETHYLENE SENSITIVITY (RTE1) encodes a novel membrane protein conserved in plants and metazoans. Genetic analyses in Arabidopsis thaliana suggest that RTE1 promotes the signaling state of the ethylene receptor ETR1 through the ETR1 N-terminal domain. RTE1 and ETR1 have been shown to co-localize to the endoplasmic reticulum (ER) and Golgi apparatus in Arabidopsis. Here, we demonstrate a physical association of RTE1 and ETR1 using in vivo and in vitro methods. Interaction of RTE1 and ETR1 was revealed in vivo by bimolecular fluorescence complementation (BiFC) in a tobacco cell transient assay and in stably transformed Arabidopsis. The association was also observed using a truncated version of ETR1 comprising the N terminus (amino acids 1–349). Interaction of RTE1 and ETR1 was confirmed by co-immunoprecipitation from Arabidopsis. The interaction occurs with high affinity (Kd, 117 nm) based on tryptophan fluorescence spectroscopy using purified recombinant RTE1 and a tryptophan-less version of purified recombinant ETR1. An amino acid substitution (C161Y) in RTE1 that is known to confer an ETR1 loss-of-function phenotype correspondingly gives a nearly 12-fold increase in the dissociation constant (Kd, 1.38 μm). These findings indicate that a high affinity association of RTE1 and ETR1 is important in the regulation of ETR1.

Ethylene Controls Autophosphorylation of the Histidine Kinase Domain in Ethylene Receptor ETR1

Perception of the phytohormone ethylene is accomplished by a small family of integral membrane receptors. In Arabidopsis, five ethylene receptor proteins are known, including ethylene resistant 1 (ETR1). The hydrophobic amino-terminal domain of these receptors contains the ethylene-binding site while the carboxyl-terminal part consists of a histidine kinase domain and a response regulator domain, which are well known elements found in bacterial two-component signaling. The soluble membrane-extrinsic carboxyl-terminal part of the receptor, which is likely to play an important role in signal transduction, showed intrinsic kinase activity when expressed and purified on its own. However, a correlation between signal input and autokinase activity was not established in these studies, as receptors were missing the transmembrane amino-terminal sensor domain. Thus, it is still unclear whether autophosphorylation occurs in response to perception of the ethylene signal. Here, we report on autophosphorylation studies of purified full-length ETR1. Autokinase activity of the purified receptor is controlled by ethylene or by ethylene agonists like the pi-acceptor compound cyanide. In fact, both signal molecules were able to completely turn off the intrinsic kinase activity. Furthermore, the observed inhibition of autophosphorylation in ETR1 by both molecules could be prevented when the ethylene antagonist 1-methyl-cyclopropene (MCP) was applied.

New Insights into the Unique Structure of the F0F1-ATP Synthase from the Chlamydomonad Algae Polytomella sp. and Chlamydomonas reinhardtii

In this study, we investigate the structure of the mitochondrial F0F1-ATP synthase of the colorless alga Polytomella sp. with respect to the enzyme of its green close relative Chlamydomonas reinhardtii. It is demonstrated that several unique features of the ATP synthase in C. reinhardtii are also present in Polytomella sp. The α- and β-subunits of the ATP synthase from both algae are highly unusual in that they exhibit extensions at their N- and C-terminal ends, respectively. Several subunits of the Polytomella ATP synthase in the range of 9 to 66 kD have homologs in the green alga but do not have known equivalents as yet in mitochondrial ATP synthases of mammals, plants, or fungi. The largest of these so-called ASA (ATP Synthase-Associated) subunits, ASA1, is shown to be an extrinsic protein. Short heat treatment of isolated Polytomella mitochondria unexpectedly dissociated the otherwise highly stable ATP synthase dimer of 1,600 kD into subcomplexes of 800 and 400 kD, assigned as the ATP synthase monomer and F1-ATPase, respectively. Whereas no ASA subunits were found in the F1-ATPase, all but two were present in the monomer. ASA6 (12 kD) and ASA9 (9 kD), predicted to be membrane bound, were not detected in the monomer and are thus proposed to be involved in the formation or stabilization of the enzyme. A hypothetical configuration of the Chlamydomonad dimeric ATP synthase portraying its unique features is provided to spur further research on this topic.

Structure of spinach chloroplast F1-ATPase complexed with the phytopathogenic inhibitor tentoxin

Tentoxin, a natural cyclic tetrapeptide produced by phytopathogenic fungi from the Alternaria species affects the catalytic function of the chloroplast F1-ATPase in certain sensitive species of plants. In this study, we show that the uncompetitive inhibitor tentoxin binds to the αβ-interface of the chloroplast F1-ATPase in a cleft localized at βAsp-83. Most of the binding site is located on the noncatalytic α-subunit. The crystal structure of the tentoxin-inhibited CF1-complex suggests that the inhibitor is hydrogen bonded to Asp-83 in the catalytic β-subunit but forms hydrophobic contacts with residues Ile-63, Leu-65, Val-75, Tyr-237, Leu-238, and Met-274 in the adjacent α-subunit. Except for minor changes around the tentoxin-binding site, the structure of the chloroplast α3β3-core complex is the same as that determined with the native chloroplast ATPase. Tentoxin seems to act by inhibiting inter-subunit contacts at the αβ-interface and by blocking the interconversion of binding sites in the catalytic mechanism.

Ethylene signaling: Identification of a putative ETR1–AHP1 phosphorelay complex by fluorescence spectroscopy

The plant ethylene receptor ETR1, which shows substantial sequence homology to typical bacterial histidine kinases, is involved in the coordination of several growth and development processes. Fluorescence polarization studies presented here demonstrate a specific interaction of ETR1 with the histidine-containing transfer protein AHP1, supporting the idea that a phosphorelay module is involved in ethylene signaling. The sensitive assay employed in our studies allows analysis of protein-protein interactions in a homogenous aqueous environment, exact control of external parameters, and quantitative analysis of the affinity and stability of the complex. Thereby it provides the basics for a more quantitative elucidation of phosphorelay modules acquired in phytohormone signaling.

Model of the c-subunit oligomer in the membrane domain of F-ATPases

A model is described of a dodecameric complex consisting of the integral membrane component subunit c of the H+‐transporting Fo domain of Escherichia coli F‐ATPase. A high‐resolution partial structure of monomeric subunit c resulting from 1H‐NMR studies [1] was used for constructing the model. The validity of the proposed arrangement of protomers in the dodecameric complex was tested by amino acid substitution analysis and chemical, biochemical and genetic data on subunit c.