Hayashi, K.-I. | Search bioRxiv

bioRxiv2024

Combination of AID2 and BromoTag expands the utility of degron-based protein knockdowns

Acute protein knockdown is a powerful approach to dissecting protein function in dynamic cellular processes. We previously reported an improved auxin-inducible degron system, AID2, but recently noted that its ability to induce degradation of some essential replication factors, such as ORC1 and CDC6, was not enough to induce lethality. Here, we present combinational degron technologies to control two proteins and enhance target depletion. For this purpose, we initially compared PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrated control of cohesin and condensin with AID2 and BromoTag, respectively. We developed a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrated that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we found that co-depletion of ORC1 and CDC6 by the double-degron system completely suppressed DNA replication, and the cells entered mitosis with single-chromatid chromosomes, indicating DNA replication was uncoupled from the cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses.

cell biology↗

bioRxiv2019

A fuzzy encounter complex precedes formation of the fully-engaged TIR1-Aux/IAA auxin co-receptor system

The plant hormone auxin regulates almost every aspect of plant development via the TIR1/AFB-auxin-Aux/IAA auxin co-receptor complex. Within this ternary complex, auxin acts as a molecular glue to promote the binding of Aux/IAA transcriptional repressor proteins to SCFTIR1/AFB ubiquitin-ligase complexes, thereby catalysing their ubiquitin-mediated proteolysis. A conspicuous feature of the crystal structure of the complex is a rare cis W-P bond within the Aux/IAA degron motif. To study receptor complex assembly, we have used NMR to determine the solution structure of the amino-terminal half of the Aux/IAA protein AXR3/IAA17, including the degron, both in isolation and in complex with TIR1 and auxin. We show that this region of AXR3 is intrinsically-disordered with only limited elements of structure and yet the critical degron W-P bond occurs with an unusually high (1:1) ratio of cis to trans isomers. We show that assembly of the co-receptor complex involves both auxin-dependent and -independent interaction events in which the disorder of the Aux/IAA is retained. Further, using the synthetic auxin molecule cvxIAA and by analysing specific Aux/IAA conformers, we show that a subset of auxin-dependent binding events occur away from the base of the canonical auxin binding pocket in TIR1. Our results reveal the existence of a fuzzy, topologically-distinct ternary encounter complex and thus that auxin perception is not limited to sequential, independent binding of auxin and then Aux/IAA to TIR1.

plant biology↗