The RPN10 D11 residue critical for the 26S proteasome structural stability and the RPN13 F67 residue critical for RPN2 binding are important in vivo.

RESEARCH INTEREST: Function and Mechanism Studies for Critical Components of the Ubiquitin/26S Proteasome System

The ubiquitin/26S proteasome system (UPS) represents a key regulatory mechanism for nearly all aspects of plant growth and development. Illustration of functional roles and associated mechanisms for the critical UPS components will greatly increase our understanding of various aspects of plant biology and ultimately provide potential manipulation sites for crop improvement.

I.Functional roles and mechanisms of ubiquitylated substrate recognition pathways.

Ubiquitylated protein substrates are recognized by distinct pathways mediated by multiple evolutionarily conserved ubiquitin receptors. By biochemical characterization, we demonstrated clearly divergence of major recognition pathways across species, suggesting their functional and mechanistic divergence. To dissect the complexity and functional roles of the distinct substrate recognition pathways in Arabidopsis, we further characterized major ubiquitin receptors, including the proteasome subunits RPN10 and RPN13 and the UBL and UBA domain-containing factors such as RAD23, DSK2, DDI1, and NUB1, using protein-protein interaction and genetic approaches. Our studies show that the major ubiquitin receptors involved in targeting ubiquitylated proteins for proteasome-mediated proteolysis are functionally redundant in Arabidopsis. Interestingly, in addition to playing a redundant role in substrate recognition, the Arabidopsis proteasome subunit RPN10 maintains the structural integrity of the proteasome, which is critical for vegetative and reproductive growth.

II.Biochemical and functional analyses of a novel Arabidopsis deubiquitylation enzyme family (OTU).

Distinct phylogeny, biochemical properties, and mutant phenotypes suggest the twelve Arabidopsis OTU deubiquitinases participate in different plant functions. One member OTU5 is involved in activation of major flowering repressors FLC, MAF4, and MAF5 through histone modification to suppress flowering. Interestingly, the otu5 mutant plants phenocopy mutants for various subunits of SWR1 complex involved in H2A.Z deposition. The OTU5 proteins, nucleus-localized and chromatin-associated, were found on FLC and MAF4-5 and associated in a substantial amount with protein complex(es) that are distinct from the SWR1 complex. Reduced abundance but not molecular weight of the OTU5 complex(es) was observed in the absence of a SWR1 subunit ARP6. Moreover, synthetic phenotypes and H2A.Z CHIP reveal that OTU5-mediated activation of flowering repressors is likely distinct from the H2A.Z deposition-mediated pathway.