Research Interest: Function and mechanism studies for critical components of the ubiquitin/26S proteasome system.
The ubiquitin/26S proteasome system (UPS) is intimately integrated into numerous cellular processes and confers critical and multiple regulatory mechanisms for nearly all aspects of plant growth and development. Many challenges in the field are remained to be resolved such as: a. identification of new substrates; b. revelation of conjugation signals for substrates; c. conjugation chemistry; d. assembly mechanisms of ubiquitin chains; e. specificity determinants of substrates; f. regulation by deubiquitylation enzymes (DUBs); g. perception/targeting mechanisms of conjugates; h. processing of substrate by 26S proteasome. Undertaking some of these challenges, our primary efforts include: a. structural/functional analyses of 26S proteasome (Fig. 1); b. biochemical characterization of critical UPS components including major ubiquitin receptors (Fig. 2-3), members of a novel DUB family OTU (Fig. 4) and two small RING E3 families; c. functional assessment by reverse genetic approaches for above-mentioned critical UPS components. Illustration of all mechanistic details for the ubiquitin/26S proteasome system will greatly increase our understanding of various aspects of plant biology and ultimately provide potential manipulation sites for crop improvement.
I.Heterologous replacement and overexpression of various 26S proteasome RPN subunits affect proteolysis in Saccharomyces cerevisiae.
A nearly complete set of yeast deletion strains for various 26S proteasome subunits was established. Complementation of various RPN subunits using corresponding Arabidopsis orthologs generated mutants with clear proteolytic defects for misfolded proteins, in vivo artificial substrates and native cell cycle regulators (Fig.1). These mutants were sensitive to various stresses and found to be defective in G2-M cell cycle. Overexpression of various endogenous RPN subunits also caused mild proteolytic defects. This set of yeast mutants is valuable for structural/functional analyses of the 26S proteasome and can be employed by proteomic approaches for global searches of substrates and regulatory factors of the 26S proteasome (manuscript in preparation).
Figure 1. Yeast strains with various RPN subunits replaced with Arabidopsis orthologs showed proteolytic defects. (A) Growth sensitivity to various stresses for RPN-replacement strains; (B) Mutant strains showed reduced degradation of the artificial substrate Arg-ß-Gal; (C) Mutant strains showed G2-M cell cycle delay.
II.The major Arabidopsis ubiquitin receptors have distinct ubiquitin chain binding and proteasomal receiving properties.
To illustrate ubiquitin perception mechanisms and functions, we characterized major Arabidopsis ubiquitin receptors (Fig.2-3) and compared with yeast and human homologs. The proteasome base subunit Rpn10 and Ubl-Uba adaptor families Rad23 and Dsk2 showed a strong and similar binding preference for longer K48-linked ubiquitin chains as compared to K63-chains. Two other Ubl-Uba factors, the Ddi1 has weak and similar affinities for both K48- and K63-chains whereas the Nub1 has only a weak affinity for K63-chains. Divergence on chain binding properties and interacting motifs were observed with human and yeast homologs. Proteasomal receiving of Rad23 and Dsk2 homologs from Arabidopsis and human are likely mediated by Rpn10; in contrast, yeast Rad23 and Dsk2 are mediated by Rpn1 and Rpn10, respectively. The Ddi1 homologs from different examined species and Arabidopsis Nub1 did not interact with any of tested proteasome subunits including Rpn10. Our studies reveal the biochemical differences of major Arabidopsis ubiquitin receptors indicating their potential functional/mechanistic differentiation. In addition, structural and mechanistic divergences of major ubiquitin receptors among different species are apparent (manuscript in preparation).
Figure 2. Ubiquitin chain binding profiles of major Arabidopsis ubiquitin receptors.
Chain binding profiles of Rad23 members, Ddi1 (A) and Nub1 (B) were examined by GST-pull down using K48- or K63-linked ubiquitin chains (2-7-mer). Chain binding profiles were also examined by K48- or K63-linked tetra ubiquitin (C). Human Rpn10 homolog S5a was compared.
Figure 3. Proteasome receiving of Rad23 members, Dsk2 and Ddi1 is mediated by base subunit Rpn10 in Arabidopsis. Rad23 and Dsk2 are recognized by UIM3 and UIM1 of Rpn10, respectively.
III.Members of a novel Arabidopsis deubiquitination enzyme family OTU have distinct substrate specificities.
DUBs potentially regulate ubiquitination with substrate-specificity by removing ubiquitin units from conjugated substrates. We have identified a novel OTU domain-containing deubiquitination enzyme family. To explore potential functional differences, we expressed, purified and assayed for all family members, except OTU8, which likely is a pseudogene (Fig.4). Enzyme activities, kinetics and optimal pH were determined by using K48- or K63-linked ubiquitin tetramer as a substrate. OTU1 showed strong preference for K48-linked chain whereas OTU3, 4, 7 and 10 have a clear preference for K63-chain. In contrast, OTU2 and 9 have relatively weak activities and similar preference against both substrates. There was no activity observed with OTU5, 6, 11 and 12 with examined substrates. A broad optimal pH ranges were observed in general (manuscript in preparation).
Figure 4. Different chain type preference for members of a novel Arabidopsis DUB family OTU. K48- or K63-linked tetra ubiquitin chains were used as substrates. OTU1, 2, 3, 4iib, 5ii, 6, 7, 9, 11, and 12 were expressed with a HIS tag. Due to insolubility, OTU4iia and 10 were expressed as GST fusions (OTU1 also and served as a control).
IV.Functional assessment of critical UPS components by phenotypical analyses of Arabidopsis T-DNA insertion mutants.
To determine functional roles, we are establishing and characterizing Arabidopsis T-DNA insertion lines for various UPS components including the major ubiquitin receptors, OTUs and two small RING E3 families. Preliminary experiments observed different phenotypes which include UV-sensitivity, enhanced root growth and early flowering for insertion in genes encoding Rad23, Nub1 and a RING-type E3, respectively.
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Yang, P§., Fu, H.§*, Walker, J. Papa, C. M. Smalle, J. Ju, Y. M. and Vierstra, R. D*. (2004). Purification of the Arabidopsis 26S proteasome: biochemical and molecular analyses revealed the presence of multiple isoforms. J. Biol. Chem. 279: 6401-6413. § equal contribution; *corresponding authors
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