An In vitro Assay to Recapitulate Hormone-Triggered and SCF-Mediated Protein Ubiquitylation.

Signaling proteins trigger a sequence of molecular switches in the cell, which permit development, growth, and rapid adaptation to changing environmental conditions. SCF-type E3 ubiquitin ligases recognize signaling proteins prompting changes in their fate, one of these being ubiquitylation followed by degradation by the proteasome. SCFs together with their ubiquitylation targets (substrates) often serve as phytohormone receptors, responding and/or assembling in response to fluctuating intracellular hormone concentrations.

Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies.

Cullin RING-type E3 ubiquitin ligases SCF and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons.

Regulation of SCF E3 ligase assembly by S-nitrosylation of Arabidopsis SKP1-like1 impacts on auxin signaling.

The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1-cullin-F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCF complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCF assembly.

The ALF4 protein is a regulator of SCF E3 ligases.

The cullin-RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin-conjugating enzyme. Here, we show that ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN The mutant exhibits a phenotype that suggests defects in plant hormone response.

Variation in auxin sensing guides AUX/IAA transcriptional repressor ubiquitylation and destruction.

Auxin is a small molecule morphogen that bridges SCF-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCF-IAA6 and SCF-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA.

Radioligand Binding Assays for Determining Dissociation Constants of Phytohormone Receptors.

In receptor-ligand interactions, dissociation constants provide a key parameter for characterizing binding. Here, we describe filter-based radioligand binding assays at equilibrium, either varying ligand concentrations up to receptor saturation or outcompeting ligand from its receptor with increasing concentrations of ligand analogue. Using the auxin coreceptor system, we illustrate how to use a saturation binding assay to determine the apparent dissociation constant (K D (') ) for the formation of a ternary TIR1-auxin-AUX/IAA complex.

Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response.

The plant hormone auxin activates primary response genes by facilitating proteolytic removal of auxin/indole-3-acetic acid (AUX/IAA)-inducible repressors, which directly bind to transcriptional auxin response factors (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like β-grasp fold with homologies to the Phox and Bem1p (PB1) domain.

Auxin-induced degradation dynamics set the pace for lateral root development.

Auxin elicits diverse cell behaviors through a simple nuclear signaling pathway initiated by degradation of Aux/IAA co-repressors. Our previous work revealed that members of the large Arabidopsis Aux/IAA family exhibit a range of degradation rates in synthetic contexts. However, it remained an unresolved issue whether differences in Aux/IAA turnover rates played a significant role in plant responses to auxin.

A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin.

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding the F-box protein transport inhibitor response 1 (TIR1) and promotes the degradation of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors. Here we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein.

Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor.

Previous studies have demonstrated that auxin (indole-3-acetic acid) and nitric oxide (NO) are plant growth regulators that coordinate several plant physiological responses determining root architecture. Nonetheless, the way in which these factors interact to affect these growth and developmental processes is not well understood. The Arabidopsis thaliana F-box proteins TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) are auxin receptors that mediate degradation of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to induce auxin-regulated responses.

Plant hormones are versatile chemical regulators of plant growth.

The plant hormones are a structurally unrelated collection of small molecules derived from various essential metabolic pathways. These compounds are important regulators of plant growth and mediate responses to both biotic and abiotic stresses. During the last ten years there have been many exciting advances in our understanding of plant hormone biology, including new discoveries in the areas of hormone biosynthesis, transport, perception and response.

Characterization of the VIER F-BOX PROTEINE genes from Arabidopsis reveals their importance for plant growth and development.

E3 ubiquitin ligases (E3s) target proteins for degradation by the 26S proteasome. In SKP1/CDC53/F-box protein-type E3s, substrate specificity is conferred by the interchangeable F-box protein subunit. The vast majority of the 694 F-box proteins encoded by the Arabidopsis thaliana genome remain to be understood.

Mechanism of auxin perception by the TIR1 ubiquitin ligase.

Auxin is a pivotal plant hormone that controls many aspects of plant growth and development. Perceived by a small family of F-box proteins including transport inhibitor response 1 (TIR1), auxin regulates gene expression by promoting SCF ubiquitin-ligase-catalysed degradation of the Aux/IAA transcription repressors, but how the TIR1 F-box protein senses and becomes activated by auxin remains unclear. Here we present the crystal structures of the Arabidopsis TIR1-ASK1 complex, free and in complexes with three different auxin compounds and an Aux/IAA substrate peptide.

The evolutionarily conserved Arabidopsis thaliana F-box protein AtFBP7 is required for efficient translation during temperature stress.

In eukaryotes, E3 ubiquitin ligases (E3s) mediate the ubiquitylation of proteins that are destined for degradation by the ubiquitin-proteasome system. In SKP1/CDC53/F-box protein (SCF)-type E3 complexes, the interchangeable F-box protein confers specificity to the E3 ligase through direct physical interactions with the degradation substrate. The vast majority of the approximately 700 F-box proteins from the plant model organism Arabidopsis thaliana remain to be characterized.

LucTrap vectors are tools to generate luciferase fusions for the quantification of transcript and protein abundance in vivo.

Proper plant growth and development strongly rely on the plant's ability to respond dynamically to signals and cues from the intra- and extracellular environment. Whereas many of these responses require specific changes at the level of gene expression, in recent years it has become increasingly clear that many plant responses are at least in part also controlled at the level of protein turnover. It is a challenge for signal transduction research to understand how distinct incoming signals are integrated to generate specific changes at the transcript or protein level.

The evolutionarily conserved TOUGH protein is required for proper development of Arabidopsis thaliana.

In this study, we characterize the evolutionarily conserved TOUGH (TGH) protein as a novel regulator required for Arabidopsis thaliana development. We initially identified TGH as a yeast two-hybrid system interactor of the transcription initiation factor TATA-box binding protein 2. TGH has apparent orthologs in all eukaryotic model organisms with the exception of the budding yeast Saccharomyces cerevisiae.

Cullin-containing E3 ubiquitin ligases in plant development.

In eukaryotes, the ubiquitin-proteasome system participates in the control of signal transduction events by selectively eliminating regulatory proteins. E3 ubiquitin ligases specifically bind degradation substrates and mediate their poly-ubiquitylation, a prerequisite for their degradation by the 26S proteasome. On the basis of the analysis of the Arabidopsis genome sequence, it is predicted that there are more than 1000 E3 ubiquitin ligases in plants.