Functional Study of Two Putative Multimeric E3 Ligase Subunits: Pollen-Specific SKP1-Like Proteins (LSKs) in Lily and BTB/POZ-MATH Domains Containing Protein1 (AtBPM1) in Arabidopsis

• Main Authors: Liang-Chi Chang, 張良棋
• Other Authors: Guang-Yuh Jauh
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/19136790597000484937

博士 === 國防醫學院 === 生命科學研究所 === 98 === Plants exhibit immovable life and perceive various biotic and abiotic stimulations from surrounding environments during their lifetimes. Quickly and precisely responded to various stimulations is critical for plants growth, development, and survival. Recent studies reveal that proteomic plasticity regulated by ubiquitin-proteasome system (UPS) plays an essential role in all eukaryotic cells. Three enzyme cascade, E1-E2-E3, has been found involving in UPS to selectively tag unwanted protein substrates with ubiquitin and recruit them to 26S proteasome for degradation. The E3 enzymes, protein-ubiquitin ligases, take charge of specific protein substrates recognition and ubiqutination. In this dissertation, the subunits of two putative multimeric E3 ligases, SCF and SCF3, were studied in lily and Arabidopsis, respectively, for their biological functions. In Chapter one, three lily SKP1-like genes, LSK1 to 3, indentified from lily mature pollen and pollen tubes showed pollen specific expression starting at late pollen development stage and sustaining high expression level throughout pollen tube germination. LSKs could functional complement yeast skp1 deletion mutant. Deduced amino acid sequences of LSKs contain putative N-terminal CUL1 protein and C-terminal F-box protein interacting domain. LSKs proteins interact with lily CULLIN1 protein evidenced by yeast two hybrid and in vitro affinity binding assay. Overexpression of full length LSK1:GFP recombinant proteins in lily pollen did not disturb normal pollen tube growth; however, overexpressed LSKs recombinant proteins without the CUL1-interacting domain, LSKsΔ:GFP, significantly retarded pollen tube elongation under both in vitro and in vivo. It suggests that N-terminus deleted LSKs may function as dominant negatively to regulate pollen tube elongation. All these results suggest that LSKs may interact with LLCUL1 protein to form functional SCF complexes, which play a crucial role in regulating pollen tube elongation under both in vitro and in vivo conditions. Next, we characterized a novel Arabidopsis protein called BTB/POZ-MATH domain protein 1 (AtBPM1). Previous yeast two hybrid and in vitro pull-down assays studies showed that AtBPM1 interacted with AtCUL3 to form a putative SCF3 E3 ligase; but its functions and putative protein substrates remained unknown. In Chapter 2, we identified two potential protein substrates of AtBPM1, AtERF4 and AtERF7, by yeast two hybrid screening. In Arabidopsis leaf protoplasts, most of transiently expressed AtBPM1 prominently localized in cytoplasm; however, it translocated from cytosol into nucleus when co-expressed with either AtERF4 or AtERF7. AtERF4 and AtERF7 are members of class II ERF family, contains AP2/ERF domain in N-terminus and EAR repression domain in C-terminus. Domains deletion assays of AtBPM1, AtERF4 and AtERF7 revealed that both BTB/POZ and MATH domains of AtBPM1 and repression domains of AtERF4 and AtERF7 were essential for their interactions. 26S proteasome inhibitor, MG-132, treatment efficiently inhibited AtERF4 and AtERF7 degradation that suggests turnover of AtERF4 and AtERF7 protein are mediated by UPS, but co-overexpression of AtBPM1 with AtERF4 and/or AtERF7 did not dramatically promote AtERF4 and AtERF7 degradation. Interestingly, overexpression of AtBPM1 slightly decreased basal luciferase activity that under control of GAL4 binding sequence and GCC box. It raised the possibility that endogenous AtERF4 and AtERF7 may interact with AtBPM1 to exert their transcriptional repression activity. Based on literatures and above results we speculate that AtBPM1 may have novel function to regulate activities of transcriptional repressors, AtERF4 and AtERF7.