Repression of photomorphogenesis in Arabidopsis thaliana requires activity of the COP9 signalosome (CSN), CDD, and COP1 complexes, but how these three complexes work in concert to accomplish this important developmental switch has remained unknown. Here, we demonstrate that Arabidopsis CULLIN4 (CUL4) associates with the CDD complex and a common catalytic subunit to form an active E3 ubiquitin ligase both in vivo and in vitro. The partial loss of function of CUL4 resulted in a constitutive photomorphogenic phenotype with respect to morphogenesis and light-regulated gene expression. Furthermore, CUL4 exhibits a synergistic genetic interaction with COP10 and DET1. Therefore, this CUL4-based E3 ligase is essential for the repression of photomorphogenesis. This CUL4-based E3 ligase appears to associate physically with COP1 E3 ligase and positively regulates the COP1-dependent degradation of photomorphogenesis-promoting transcription factors, whereas the CSN controls the biochemical modification of CUL4 essential for E3 activity. Thus, this study suggests a biochemical activity connection between CSN and CDD complexes in their cooperation with COP1 in orchestrating the repression of photomorphogenesis.

Plant phototropism is an adaptive response to changes in light direction, quantity, and quality that results in optimization of photosynthetic light harvesting, as well as water and nutrient acquisition. Though several components of the phototropic signal response pathway have been identified in recent years, including the blue light (BL) receptors phototropin1 (phot1) and phot2, much remains unknown. Here, we show that the phot1-interacting protein NONPHOTOTROPIC HYPOCOTYL3 (NPH3) functions as a substrate adapter in a CULLIN3-based E3 ubiquitin ligase, CRL3 NPH3 . Under low-intensity BL, CRL3 NPH3 mediates the mono/multiubiquitination of phot1, likely marking it for clathrin-dependent internalization from the plasma membrane. In high-intensity BL, phot1 is both mono/multi-and polyubiquitinated by CRL3 NPH3 , with the latter event targeting phot1 for 26S proteasome-mediated degradation. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters.

The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control1. With prevalent hyper-activation of the mTOR pathway in human cancers2, novel strategies to enhance TOR pathway inhibition are highly desirable. We used a yeast-based platform to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor of the SCFMet30 ubiquitin ligase (SMER3). The large SCF (Skp1-Cullin-F-box) family of ubiquitin ligases performs important functions in diverse cellular processes including transcription, cell-cycle control, and immune response3. Accordingly, there would be great value in developing SCF ligase inhibitors that act by a defined mechanism to specifically inactivate ligase activity. We show here that SMER3 selectively inhibits SCFMet30 in vivo and in vitro, but not the closely related SCFCdc4. Our results demonstrate that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes, and suggest new strategies for combination therapy with rapamycin.

Background: KEAP1 is a ubiquitin ligase adaptor that promotes the ubiquitination and degradation of NRF2, a transcription factor that drives the antioxidant response.Results: Wilms tumor gene on the X chromosome (WTX) stabilizes NRF2 by competing with NRF2 for binding to KEAP1.Conclusion: WTX regulates the antioxidant response.Significance: This study reveals a novel regulatory mechanism governing the antioxidant response.

We investigate how state involvement

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The full enzymatic activity of the cullin-RING ubiquitin ligases (CRLs) requires a ubiquitin-like protein (that is, Nedd8) modification. By deamidating Gln40 of Nedd8 to glutamate (Q40E), the bacterial cycle-inhibiting factor (Cif) family is able to inhibit CRL E3 activities, thereby interfering with cellular functions. Despite extensive structural studies on CRLs, the molecular mechanism by which Nedd8 Gln40 deamidation affects CRL functions remains unclear. We apply a new quantitative cross-linking mass spectrometry approach to characterize three different types of full-length human Cul1–Rbx1 complexes and uncover major Nedd8-induced structural rearrangements of the CRL1 catalytic core. More importantly, we find that those changes are not induced by Nedd8(Q40E) conjugation, indicating that the subtle change of a single Nedd8 amino acid is sufficient to revert the structure of the CRL catalytic core back to its unmodified form. Our results provide new insights into how neddylation regulates the conformation and activity of CRLs.

Cell-fate in eukaryotes is regulated by MAP Kinases (MAPKs) that translate external cues to cellular responses. In plants, two MAPKs, MPK3/6, regulate diverse processes of development, environmental response, and immunity. Yet, the mechanism bridging these shared signaling components with a specific target remains unresolved. Focusing on the development of stomata, epidermal valves for gas exchange and transpiration, we report here that the bHLH protein SCREAM functions as a scaffold by recruiting MPK3/6 to downregulate SPEECHLESS, a transcription factor initiating stomatal cell lineages.SCREAM directly binds with MPK3/6 through an evolutionarily-conserved yet unconventional bipartite motif. Mutations in this motif abrogate association, phosphorylation and degradation of SCREAM, unmask hidden non-redundancies between MPK3 and MPK6, and result in uncontrolled stomatal differentiation. Structural analyses of MPK6 at the 2.75Å resolution unraveled bipartite binding of SCREAM with MPK6, that is distinct from an upstream MAPKK. Our findings elucidate, at the atomic resolution, the mechanism directly linking extrinsic signals to transcriptional reprogramming during the establishment of stomatal cell-fate, and highlight a unique substrate-binding mode adopted by plant MAPKs.