Preliminary Study on the Function of Ubiquitin Ligase of CrgA Protein from Blakeslea trispora
CrgA has been identified as a negative regulator of the carotenoid biosynthesis in Blakeslea trispora and some other zygomycetes.The presence of the ring finger domains suggest that CrgA may function as an ubiquitin ligase,a key enzyme in the ubiquitination regulation system.In order to test this hypothesis,one ubiquitin activating enzyme(BtE1)and 18 putative ubiquitin-conjugation enzymes(UBC)from B.trispora were isolated and identified.Bioinformatics analysis indicated that each of the 18 UBC proteins contained a conserved domain of UBC,indicating that all of them belong to ubiquitin binding enzymes.Phylogenetic relationship analysis showed that 18 UBC proteins belong to 7 protein subfamilies,homologous with those of Saccharomyces cerevisiae.6 candidate UBC proteins were screened based on phylogenetic analysis,and BtEl,6 BtUBC proteins,BtCrgA and BtWC-lb were obtained by heterologous expression and affinity purification,which were ubiquitinated in vitro.BtCrgA can perform ubiquitin modification function on itself with the assistance of BtE1 and BtUBC D.
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