Biology subjects

Bennett, E. J.

Publications and source records attributed to Bennett, E. J..

18 recordsLinked to original sources

bioRxiv2026

Proteostasis sustains T cell differentiation potential and tumor-infiltrating lymphocyte function

Tumor-infiltrating lymphocytes (TIL) often fail to restrain tumor growth due to progressive differentiation to an exhausted state. In healthy tissues, tissue-resident memory T cells (TRM) maintain protection for years, and patient tumors that contain TIL with TRM features are associated with better prognosis. Proteomic and transcriptomic profiling of T cell populations identified proteostasis as a significant factor distinguishing TRM and progenitor-exhausted TIL from terminally-exhausted TIL, including loss of E3 ubiquitin ligases NEURL3, RNF149, and WSB1, with accumulation of unfolded proteins in spite of functional proteasome activity. Enforced expression of these ligases by TIL preserved stem-like TCF1+ populations and improved anti-tumor function, whereas their knockout impaired TIL and altered T cell differentiation in acute infection. Sustained ligase expression rescued accumulation of unfolded proteins in TIL and improved immunotherapy outcome in preclinical models, highlighting the critical role of proteostasis in TIL function and identifying new avenues for advancing cancer immunotherapy.

immunology↗

bioRxiv2026

CRISPR-mediated conditional mutagenesis of Smad1/5/8 reveals BMP/GDF signaling restricts postnatal bone overgrowth

The BMP/GDF branch of TGF-{beta} signaling regulates diverse aspects of skeletal biology, from skeletal development to maintenance and repair. However, the complexity, redundancy, and pleiotropy of BMP/GDF signaling have hamstrung a genetic dissection of its activities in different cell types over time. Here, we tested the feasibility of a three-transgene system using CRISPR/Cas9 to conditionally mutate six target sites, two each in the receptor-mediated Smad1, Smad5, and Smad8 transcriptional effectors of BMP/GDF signaling. Briefly, we used Prx1-cre to activate a conditional Cas9 transgene by recombination in early limb bud mesenchyme; this endonuclease then complexes with gRNAs expressed from a polycistronic tRNA-gRNA array for targeted mutagenesis. Slower than expected accumulation of gRNA-directed mutations in each Smad produced an unexpected postnatal skeletal phenotype. Beginning around one month after birth, all animals developed hyperostosis on the surface of all long limb bones, which progressively worsened with age. This woven bone expansion occurred through proliferation of RUNX2+ osteoprogenitor cells in the cambium layer of the periosteum, producing an abundance of periosteal osteoblasts. Endosteal osteoblasts did not increase in number but increased their mineralizing activity. As a result, the marrow cavities narrowed, and the patella and carpal elements, which have no periosteum, increased internal bone mass without altering shape and size. Thus, while BMP/GDF signaling is known to promote early postnatal bone growth, these data support an additional homeostatic role during late postnatal osteogenesis by regulating both periosteal and endosteal osteoblasts. Although this genetically simple approach requires further optimization to improve efficiency, combining three transgenes produced more than 160 conditionally mutagenized animals with a fully penetrant and reproducible phenotype. This is an advance over traditional cre/lox systems that scale in complexity with the number of target loci, and it highlights the potential to model a wide range of genetically complex traits and disorders.

genetics↗

bioRxiv2025

The functional landscape of the human ubiquitinome

Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. To date, hundreds of thousands of ubiquitination sites (ubi-sites) have been identified, however fewer than 1% have known functional roles. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. To pinpoint critical regulatory events requiring ubiquitination at a precise site, we mapped ubi-site conservation across proteomics data from six non-human species. Perturbation proteomics revealed that highly conserved ubi-sites are more likely to regulate signaling functions rather than proteasomal degradation. To further prioritize site-specific ubiquitination relevant for organismal fitness, we constructed a machine learning-based positional importance score for more than 100,000 ubi-sites, which identifies sites regulating diverse protein functions and rationalizes genetic vulnerabilities. Finally, we employed chemical genomics to validate the functional relevance of high-scoring ubi-sites and leveraged genetic code expansion to demonstrate that ubiquitination of K320 in the RNA-regulator ELAVL1 disrupts RNA binding. Our work reveals systems-level principles of the ubiquitinome and provides a powerful resource for studying site-specific protein ubiquitination.

cell biology↗

bioRxiv2025

RNA triggers chronic stress during neuronal aging

Neurodegenerative diseases are linked with dysregulation of the integrated stress response (ISR), which coordinates cellular homeostasis during and after stress events. Cellular stress can arise from several sources, but there is significant disagreement about which stress might contribute to aging and neurodegeneration. Here, we leverage directed transdifferentiation of human fibroblasts into aged neurons to determine the source of ISR activation. We demonstrate that increased accumulation of cytoplasmic double-stranded RNA (dsRNA) activates the eIF2 kinase PKR, which in turn triggers the ISR in aged neurons and leads to sequestration of dsRNA in stress granules. Aged neurons accumulate endogenous mitochondria-derived dsRNA that directly binds to PKR. This mitochondrial dsRNA leaks through damaged mitochondrial membranes and forms cytoplasmic foci in aged neurons. Finally, we demonstrate that PKR inhibition leads to the cessation of stress, resumption of cellular translation, and restoration of RNA-binding protein expression. Together, our results identify a source of RNA stress that destabilizes aged neurons and may contribute to neurodegeneration.

cell biology↗

bioRxiv2024

The Proteostasis Network is a Therapeutic Target in Acute Myeloid Leukemia

Oncogenic growth places great strain and dependence on the proteostasis network. This has made proteostasis pathways attractive therapeutic targets in cancer, but efforts to drug these pathways have yielded disappointing clinical outcomes. One exception is proteasome inhibitors, which are approved for frontline treatment of multiple myeloma. However, proteasome inhibitors are largely ineffective for treatment of other cancers, including acute myeloid leukemia (AML), although reasons for these differences are unknown. Here, we determined that proteasome inhibitors are ineffective in AML due to inability to disrupt proteostasis. In response to proteasome inhibition, AML cells activated HSF1 and autophagy, two key stem cell proteostasis pathways, to prevent unfolded protein accumulation. Inactivation of HSF1 sensitized human AML cells to proteasome inhibition, marked by unfolded protein accumulation, activation of the PERK-mediated integrated stress response, severe reductions in protein synthesis, proliferation and cell survival, and significant slowing of disease progression and extension of survival in vivo. Similarly, combined autophagy and proteasome inhibition suppressed proliferation, synergistically killed AML cells, and significantly reduced AML burden and extended survival in vivo. Furthermore, autophagy and proteasome inhibition preferentially suppressed protein synthesis and induced apoptosis in primary patient AML cells, including AML stem/progenitor cells, without severely affecting normal hematopoietic stem/progenitor cells. Combined autophagy and proteasome inhibition also activated the integrated stress response, but surprisingly this occurred in a PKR-dependent manner. These studies unravel how proteostasis pathways are co-opted to promote AML growth, progression and drug resistance, and reveal that disabling the proteostasis network is a promising strategy to therapeutically target AML.

cancer biology↗

bioRxiv2023

HAPSTR1 localizes HUWE1 to the nucleus to limit stress signaling pathways

HUWE1 is a large, enigmatic HECT domain ubiquitin ligase implicated in the regulation of diverse pathways including DNA repair, apoptosis, and differentiation. How HUWE1 engages its structurally diverse substrates and how HUWE1 activity is regulated are unknown. Using unbiased quantitative proteomics, we identify C16orf72/HAPSTR1 as a dedicated HUWE1 substrate despite HUWE1 targeting substrates in a largely cell type-specific manner. The established physical and genetic interactions between HUWE1 and C16orf72/HAPSTR1 suggest that HAPSTR1 positively regulates HUWE1 function. Here, we show that HAPSTR1 is both a HUWE1 substrate and is required for HUWE1 nuclear localization to facilitate HUWE1 nuclear substrate targeting. HUWE1 or HAPSTR1 loss of function triggers a broad transcriptional stress response. We show that nuclear HUWE1 is both critical for modulating stress signaling pathways, which include p53 and NF-{kappa}B-mediated signaling and required for cell proliferation. Combined, our results define a role for HAPSTR1 in gating critical nuclear HUWE1 functions.

molecular biology↗

bioRxiv2022

An ERAD-independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis

Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER) where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated-degradation (ERAD) removes these clients from the ER membrane to the cytosol in a process known as retrotranslocation. Our recent work demonstrates that rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated integral membrane ERAD substrates. To survey for potential interaction partners of Dfm1, we performed protein-proximity labeling by BioID (proximity-dependent biotin identification) followed by mass spectrometry and identified several interacting proteins known to play a role in the sphingolipid biosynthesis pathway. Specifically, we found that Dfm1 physically interacts with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components and is critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. We demonstrate for the first time that Dfm1 has a role in ER export, a function that is independent of Dfm1s canonical ERAD retrotranslocation function. Specifically, we show that loss of Dfm1 results in the accumulation of phosphorylated Orm2 at the ER, suggesting a novel role for Dfm1 in controlling Orm2 export from the ER and its subsequent degradation by EGAD. Moreover, recruitment of Cdc48 by Dfm1, which is critical for its role in ERAD retrotranslocation, is dispensable for Dfm1s role in ER export. Given that the accumulation of human Orm2 homologs, ORMDLs, are associated with many maladies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.

cell biology↗

bioRxiv2022

The 2',3' cyclic phosphatase Angel1 facilitates mRNA degradation during human ribosome-associated quality control

Translation elongation stalls trigger mRNA decay and degradation of the nascent polypeptide via translation-dependent quality control pathways. One such pathway, non-stop mRNA decay (NSD), targets aberrant mRNAs that lack stop codons for example due to premature polyadenylation. Here we identify Angel1, a CCR4 deadenylase homolog whose biochemical activity remains poorly defined, as a rate-limiting factor for NSD in human cells. Angel1 associates with mRNA coding regions and proteins involved in ribosome-associated quality control and mRNA decay, consistent with a factor that monitors translation elongation stalls. Depletion of Angel1 causes stabilization of reporter mRNAs that are targeted for NSD by the absence of stop codons, but not an mRNA targeted for nonsense-mediated decay. A conserved catalytic residue of Angel1 is critical for its function in NSD. Our findings identify Angel1 as a human NSD factor and suggest that Angel1 catalytic activity plays a critical role in the NSD pathway.

molecular biology↗

bioRxiv2021

The OPAQUE1/DISCORIDA2 myosin XI is required for phragmoplast guidance during asymmetric cell division in maize

Formative asymmetric divisions produce cells with different fates and are critical for development. We show the myosin XI protein, OPAQUE1 (O1), is necessary for asymmetric divisions during maize stomatal development. We analyzed stomatal precursor cells prior to and during asymmetric division to determine why o1 mutants have abnormal division planes. Cell polarization and nuclear positioning occur normally in the o1 mutant, and the future site of division is correctly specified. The defect in o1 occurs during late cytokinesis, when the phragmoplast forms the nascent cell plate. Initial phragmoplast guidance in o1 is correct; however, as phragmoplast expansion continues o1 phragmoplasts become misguided. To understand how O1 contributes to phragmoplast guidance, we identified O1-interacting proteins. Maize kinesins related to the Arabidopsis thaliana division site markers PHRAGMOPLAST ORIENTING KINESINs (POKs), which are also required for correct phragmoplast guidance, physically interact with O1. We propose that different myosins are important at multiple steps of phragmoplast expansion, and the O1 actin motor and POK-like microtubule motors work together to ensure correct late-stage phragmoplast guidance.

plant biology↗

bioRxiv2021

iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

Since multiple ribosomes can engage a single mRNA, nonuniform ribosome progression can result in collisions. Ribosome collisions during translation elongation elicit a multifaceted ribosome-associated quality control (RQC) response. Despite advanced mechanistic understanding of translation initiation, a parallel RQC pathway that acts on collided preinitiation complexes has not been described. Here, we show that blocking progression of scanning or elongating ribosomes past the start codon triggers uS3 and uS5 ribosomal ubiquitylation. We demonstrate that conditions that activate the integrated stress response can also induce preinitiation complex collisions. The ubiquitin ligase, RNF10, and the deubiquitylating enzyme, USP10, are the key regulators of uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in an autophagy-independent manner. This study identifies a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on pervasive ribosome collisions during translation initiation to modulate translation activity and capacity.

cell biology↗

bioRxiv2020

Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection

Ribosomes are highly abundant cellular machines that perform the essential task of translating the genetic code into proteins. Cellular translation activity is finely tuned and proteostasis insults, such as those incurred upon viral infection, activate stress signaling pathways that result in translation reprogramming. Viral infection selectively shuts down host mRNA while redistributing ribosomes for selective translation of viral mRNAs. The intricacies of this selective ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome associated quality control (RQC) factor ZNF598, a sensor for collided ribosomes, as a critical factor for vaccinia virus mRNA translation. Collided ribosomes are sensed by ZNF598, which ubiquitylates 40S subunit proteins uS10 and eS10 and thereby initiates RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection in human cells enhances uS10 ubiquitylation indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells which either lack ZNF598 or contain a ubiquitylation deficient version of uS10. Using SILAC-based proteomics and concurrent RNAseq analysis, we determine that host translation of vaccinia virus mRNAs is compromised in cells that lack RQC activity as compared to control cells whereas there was little evidence of differences in host or viral transcription. Additionally, vaccinia virus infection resulted in a loss of cellular RQC activity, suggesting that ribosomes engaged in viral protein production recruit ZNF598 away from its function in host translation. Thus, co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

molecular biology↗

bioRxiv2020

Modular HUWE1 architecture serves as hub for degradation of cell-fate decision factors

HECT ubiquitin ligases play essential roles in metazoan development and physiology. The HECT ligase HUWE1 is central to the cellular stress response by mediating degradation of key death or survival factors including Mcl1, p53, DDIT4, and Myc. As a step toward understanding regulation of HUWE1 engagement with its diverse substrates, we present here the cryo-EM structure of HUWE1, offering a first complete molecular picture of a HECT ubiquitin ligase. The ~4400 amino acid residue polypeptide forms an alpha solenoid-shaped assembly with a central pore decorated with protein interaction modules. This modularity enables HUWE1 to target a wide range of substrates for destruction. The locations of human mutations associated with severe neurodevelopmental disorders link functions of this essential enzyme with its three-dimensional organization.

molecular biology↗

bioRxiv2020

Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-protein Interactions

Throughout their cellular lifetime, RNA transcripts are bound to proteins, playing crucial roles in RNA metabolism, trafficking, and function. Despite the importance of these interactions, identifying the proteins that interact with an RNA of interest in mammalian cells represents a major challenge in RNA biology. Leveraging the ability to site-specifically and covalently label an RNA of interest using E. Coli tRNA guanine transglycosylase and an unnatural nucleobase substrate, we establish the identification of RNA-protein interactions and the selective enrichment of cellular RNA in mammalian systems. We demonstrate the utility of this approach through the identification of known binding partners of 7SK snRNA via mass spectrometry. Through a minimal 4-nucleotide mutation of the long noncoding RNA HOTAIR, enzymatic biotinylation enables identification putative HOTAIR binding partners in MCF7 breast cancer cells that suggest new potential pathways for oncogenic function. Furthermore, using RNA sequencing and qPCR, we establish that an engineered enzyme variant achieves high levels of labeling selectivity against the human transcriptome allowing for 145-fold enrichment of cellular RNA directly from mammalian cell lysates. The flexibility and breadth of this approach suggests that this system could be routinely applied to the functional characterization of RNA, greatly expanding the toolbox available for studying mammalian RNA biology.

biochemistry↗

bioRxiv2019

A newly defined cullin-RING ubiquitin ligase promotes thermotolerance as part of the Intracellular Pathogen Response in C. elegans

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode C. elegans called the Intracellular Pathogen Response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other ubiquitin ligase components. Here we use co-immunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4 and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that co-immunoprecipitate with CUL-6 and find that FBXA-158 promotes thermotolerance. In summary, we have defined the three core components and an F-box adaptor of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.\n\nSignificance StatementIntracellular pathogen infection in the nematode Caenorhabditis elegans induces a robust transcriptional response as the host copes with infection. This response program includes several ubiquitin ligase components that are predicted to function in protein quality control. In this study, we show that these infection-induced ubiquitin ligase components form a protein complex that promotes increased tolerance of acute heat stress, an indicator of improved protein homeostasis capacity. These findings show that maintaining protein homeostasis may be a critical component of a multifaceted approach allowing the host to deal with stress caused by intracellular infection.

biochemistry↗

bioRxiv2018

Active protein neddylation or ubiquitylation is dispensable for stress granule dynamics

Many protein homeostasis stressors induce the formation of membraneless cytoplasmic stress granules (SGs) that contain large assemblies of repressed mRNAs and associated RNA binding proteins. Similar stressors have been shown to globally alter the function of the ubiquitin proteasome system (UPS) resulting in the accumulation of ubiquitylated proteins. Previous studies have demonstrated that ubiquitin and specific UPS components co-localize with SGs and that reducing the abundance or activity of ubiquitin pathway proteins can inhibit SG formation. These studies suggest that SG dynamics and composition may be regulated by ubiquitylation of SG resident proteins. Using ubiquitin-specific proteomic approaches, we demonstrate that many proteins, including some SG proteins are dynamically ubiquitylated upon SG-inducing sodium arsenite treatment. We utilized potent and selective inhibitors of the ubiquitin activating enzyme (UAE) or the NEDD8 activating enzyme (NAE) to directly test if active protein ubiquitylation or neddylation was required for SG dynamics. Using ubiquitin-site specific proteomics, we establish that UAE inhibition results in the rapid loss of nearly all protein ubiquitylation regardless of ubiquitin chain type. Addition of UAE or NAE inhibitors to cells did not alter arsenite-induced SG formation or dissolution. While we confirmed that ubiquitin co-localizes with both sodium arsenite and thapsigargin-induced SGs, antibodies that recognize all forms of ubiquitin more strongly co-localize with SGs compared to antibodies that preferentially recognize polyubiquitin or specific polyubiquitin-linkages. Interestingly, ubiquitin itself co-localizes with SGs in a UAE independent manner suggesting that the ubiquitin present within SGs is likely unconjugated ubiquitin. Our findings clearly demonstrate that active protein ubiquitylation or neddylation is not required for SG dynamics. These results suggest that ubiquitin-binding SG proteins may recruit free ubiquitin into SGs to modulate SG protein interactions.

cell biology↗

bioRxiv2018

USP21 and OTUD3 Antagonize Regulatory Ribosomal Ubiquitylation and Ribosome-Associated Quality Control

Defects within mRNAs or nascent chains that halt ribosomal progression can trigger ribosome-associated quality control (RQC) pathways that facilitate mRNA and nascent polypeptide destruction as well as ribosome recycling. Failure to remove defective mRNAs or nascent chains can lead to the accumulation of cytotoxic protein aggregates and proteotoxic stress. We previously established that the E3 ligase ZNF598 catalyzes regulatory ribosomal ubiquitylation of specific 40S ribosomal proteins required for downstream RQC events. Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and facilitate ribosomal deubiquitylation following RQC activation. Overexpression of either USP21 or OTUD3 enhances readthrough of stall-inducing sequences as compared to knock-in cells lacking individual RRub sites suggesting that combinatorial ubiquitylation of RPS10 (eS10) and RPS20 (uS10) is required for optimal resolution of RQC events and that deubiquitylating enzymes can limit RQC activation.

molecular biology↗

bioRxiv2017

Proximity labeling reveals an extensive steady-state stress granule interactome and insights to neurodegeneration

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form in response to proteotoxic stress. Although SGs are distinct from aggregates observed in neurodegenerative disorders, they share protein components. We used APEX-mediated proximity labeling combined with quantitative mass spectrometry and high-throughput imaging to identify >100 previously unknown SG proteins in human cells, about 10% of which localize to SGs in a cell type- or stress type-dependent manner. Supporting a link between SG proteins and neurodegeneration, we demonstrate aberrant SG composition and subcellular distribution in iPSC-derived motor neurons from ALS patients, and identify several known and previously unidentified SG proteins that modify toxicity of mutant FUS and TDP-43 overexpression in Drosophila. We show that even in an unstressed steady-state, SG proteins form a densely-connected protein interaction network (PIN) and propose a model in which existing RNPs coalesce rapidly into microscopically visible granules that can act as gateways to pathological protein aggregation.\n\nHighlights O_LIAPEX proximity labeling of dynamic RNP granules identifies over 100 novel SG proteins\nC_LIO_LISG proteins form a densely-connected protein interaction network in unstressed cells\nC_LIO_LISystematic immunofluorescence analysis reveals stress- and cell type-specific SG composition\nC_LIO_LIALS motor neurons contain SGs with distinct content and subcellular distribution\nC_LI

cell biology↗

bioRxiv2017

Decoys reveal the genetic and biochemical roles of redundant plant E3 ubiquitin ligases

The ubiquitin proteasome system (UPS) is the main cellular route for protein degradation in plants and is important for a wide range of biological processes including daily and seasonal timing. The UPS relies on the action of E3 ubiquitin ligases to specifically recognize substrate proteins and facilitate their ubiquitylation. In plants, there are three major challenges that inhibit studies of E3 ligase function: 1) rampant genetic redundancy, 2) labile interactions between an E3 ligase and its cognate substrates, and 3) a lack of tools for rapid validation of bona fide substrates. To overcome these 3 challenges, we have developed a decoy method that allows for rapid genetic analysis of E3 ligases, in vivo identification of substrates using immunoprecipitation followed by mass spectrometry, and reconstitution of the ubiquitylation reaction in mammalian cells to rapidly validate potential substrates. We employ the strategy to study the plant F-box proteins, ZTL, LKP2, and FKF1 revealing differential genetic impacts on circadian clock period and seasonal flowering. We identify a group of circadian clock transcriptional regulators that interact with ZTL, LKP2, and FKF1 in vivo providing a host of potential substrates that have not been seen previously. We then validate one substrate of ZTL, the plant circadian clock transcription factor CHE, and show that ZTL mediates CHE ubiquitylation and that the levels of the CHE protein cycle in daily timecourses. This work further untangles the complicated genetic roles of this family of E3 ligases and suggests that ZTL is a master regulator of a diverse set of critical clock transcription factors. Furthermore, the method that is validated here can be tool employed widely to overcome traditional challenges in studying redundant plant E3 ubiquitin ligases.

plant biology↗