All cells synthesize and remove proteins in an ordered fashion, allowing for the regulation of proteins specific to development or signalling. In the eukaryotic cell, the UBIQUITIN (UBQ)-26S proteasome system mediates selective proteolysis of proteins by covalent attachment of UBQ in an E1-E2-E3 enzyme cascade, followed by 26S proteasome-mediated degradation. The UBQ E3 recognizes the specific protein to be degraded. Within the UBQ E3 superfamily, CULLIN (CUL)-RING ligases (CRLs) are significant in plants as they have been implicated in hormonal, developmental and environmental signalling. A primary mechanism for regulation of CRL activity is modification of the CUL subunit by RELATED TO UBIQUITIN (RUB), which occurs by a functionally homologous E1-E2-E3 enzyme cascade. My research has focused on the RUB pathway in the model Arabidopsis thaliana. Firstly, I characterized known RUB-interacting proteins and examined regulatory mechanisms for RUB attachment. Secondly, I used a proteomics screen to identify novel RUB-modified proteins.
Gene duplications are common in angiosperms, and consistent with this, components of the RUB pathway are found in multiples in Arabidopsis . Biochemical and genetic analyses were done to compare the duplicated genes AUXIN RESISTANT1 (AXR1) and AXR1-LIKE1 (AXL1) , encoding homologous RUB E1 subunits. I found that the two proteins have similar biochemical activities in vitro, as both function with E1 C-TERMINAL-RELATED1 (ECR1) in catalyzing RUB1 activation. Similarly, endogenous AXR1 and AXL1 proteins are found in complex with 3HA-RUB1 in mass spectrometric (MS) analyses of protein complexes recovered from Arabidopsis. In contrast, AXR1 and AXL1 differ in ability to correct phenotypic defects associated with an axr1 null mutant, when the respective coding sequences are expressed under the AXR1 5' intergenic region. While both proteins function in the RUB pathway and are biochemically similar in RUB activation, they are not functionally equivalent in vivo.
Additionally, I did a physiological study of axr1 mutants to characterize alterations in ethylene production and response, as it was previously reported that dark-grown axr1-13 hypo-accumulates ethylene. I found that a subgroup of ACS (1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE) genes, encoding a key regulatory protein in ethylene synthesis, is downregulated in axr1. Examination of the response of dark-grown seedlings to inhibition or saturation of ethylene signaling showed that axr1 seedlings have altered ethylene responsiveness.
In Arabidopsis, the only confirmed RUB-modified proteins are the CUL proteins, which have been known RUB substrates for over a decade. In non-plant systems, novel substrates of RUB/Nedd8 modification have been described, including p53 and pVHL (von Hippel Lindau protein). Thus, it is likely that novel RUB substrates await characterization in plants. I screened for putative RUB-modified proteins in Arabidopsis, using a low-stringency threshold for the above-described MS data. In planta assays were subsequently performed to validate candidate RUB-modified proteins, specifically native and denaturing co-immunoprecipitation assays were done with proteins transiently expressed in tobacco. Thus, DNA-DAMAGE BINDING1a (DDB1a), a subunit of the CUL4-type CRLs, was identified as a RUB substrate in Arabidopsis.
|Commitee:||Chan, Simon, Inoue, Kentaro|
|School:||University of California, Davis|
|School Location:||United States -- California|
|Source:||DAI-B 73/07(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Plant biology, Biochemistry|
|Keywords:||Arabidopsis thaliana, Auxin resistant1, Axr1-like1, RUB-associated proteins, RUB-interacting proteins, Related to ubiquitin (rub), Rub pathway|
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