Ubiquitin C‐terminal hydrolases 1 and 2 affect shoot architecture in Arabidopsis

Abstract

SummaryUbiquitin C-terminal hydrolases (UCHs) are a subset of de-ubiquitinating proteases that release covalently linked ubiquitin (Ub), and as such play essential roles in recycling Ub and reversing the action of Ub conjugation. We show here that two related Arabidopsis UCHs, UCH1, and UCH2, are important for shoot development. The UCH1 and 2 genes are ubiquitously expressed, with the corresponding proteins present in both the cytoplasm and nucleus. Unlike their animal and fungal counterparts, we found no evidence that the Arabidopsis UCH1 and 2 proteins stably associate with the 26S proteasome. Altering the levels of UCH1 and 2 has substantial effects on Arabidopsis shoot development, especially with respect to inflorescence architecture, with over-expression and double mutants enhancing and suppressing the outgrowth of cauline branches, respectively. Neither UCH1-over-expressing nor uch1-1 uch2-1 plants have detectably altered sensitivity to cytokinins or auxins individually, but exhibit an altered sensitivity to the ratio of the two hormones. UCH1-over-expressing plants show dramatically enhanced phenotypes when combined with auxin-insensitive mutants axr1-3 and axr2-1, suggesting that one or more aspects of auxin signaling are affected by this enzyme pair. Previous studies revealed that the ubiquitination and degradation of the AUX/IAA family of repressors is a key step in auxin signaling. Here, we show that turnover of a reporter fused to a representative AUX/IAA protein AXR3 is faster in the uch1-1 uch2-1 double mutant but slower in the UCH1 over-expression backgrounds. Taken together, our results indicate that de-ubiquitination helps to modify plant shoot architecture, possibly via its ability to directly or indirectly protect upstream target proteins involved in auxin/ cytokinin signaling from Ub-mediated degradation.

Evidence that the Arabidopsis Ubiquitin C-terminal Hydrolases 1 and 2 associate with the 26S proteasome and the TREX-2 complex

“…We did not find any interaction between the full length UCH1/UCH2 and RPNs, nor their C-terminal extension alone in Y2H, which is consistent with Yang et al 5 It is interesting that the Arabidopsis UCH1/UCH2 C-terminal extensions alone are not sufficient for interacting with 26S proteasome subunits, while the yeast and animal counterparts do interact. Apparently additional amino acid residues from the ubiquitin hydrolase activity domain are required.…”

“…3,4 Thus it is expected that the same physical association exists in Arabidopsis, but the experimental evidence to support this has been lacking. 5 Our interaction data presented here provide strong as a physical link between the two complexes. 14 Interestingly, the truncated versions, UCH1 124-334 and UCH2 123-330 , interacted with several additional components of the 26S proteasome lid complex, RPN3s, RPN12s and AtDSS1(V) (Fig.…”

“…3 In Arabidopsis, three genes encoding UCHs, designated UCH1 to UCH3, have been identified. 5 The C-terminus of human UCH37 and yeast Uch2 orthologs have been reported to interact with the RPN12 and RPN10 subunits of the 26S proteasome, respectively. 1,6 Since the Arabidopsis UCH1 and UCH2 have a similar C-terminal extension, Yang et al (2007) attempted but failed to detect a direct association of UCH1/UCH2 with the 26S proteasome, either by analyzing isolated 26S proteasome components or by yeast two hybrid (Y2H) assays.…”

“…1,6 Since the Arabidopsis UCH1 and UCH2 have a similar C-terminal extension, Yang et al (2007) attempted but failed to detect a direct association of UCH1/UCH2 with the 26S proteasome, either by analyzing isolated 26S proteasome components or by yeast two hybrid (Y2H) assays. 5 The transcription-export complex 2 (TREX-2) is an important component of mRNA export machinery, which targets genes that are being transcribed [7][8][9] and facilitates the export of mature mRNAs. The TREX-2 complex was first identified and characterized in yeast.…”

DENEDDYLASE1 Deconjugates NEDD8 from Non-Cullin Protein Substrates in Arabidopsis thaliana

“…Based on sequence similarities, we and others have recently identified four proteins as candidates for NEDD8 processing enzymes from Arabidopsis: a DEN1 homologous protein belonging to the C48 peptidase family (Colby et al, 2006;Mergner and Schwechheimer, 2014) as well as three C12 family peptidases UCH1 (UBIQUITIN CARBOXYL-TERMINAL HYDROLASE1), UCH2, and UCH3 (Yang et al, 2007). Here, we examine the DEN1 homologous protein as well as den1 mutants from Arabidopsis.…”