Abstract: Ubiquitin ligase E4B can transfer ubiquitin (UB) that comes from ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) to substrates via U-box motif. Three proteins such as peptidase M20 domain-containing protein 2 (PM20D2), polyadenylate-binding protein 1 (PABPC1), and translational activator of cytochrome C oxidase I (TACO1) ubiquitinated by E4B were verified. Total RNA was extracted from HEK293 cells and reverse-transcribed into cDNA. The cDNA was used as template for obtaining the genes of substrates by PCR, and recombinant plasmids were constructed with genes of substrates and vector, respectively. E. coli system was used to express proteins which were required for the ubiquitination of E4B substrates. Result showed that PM20D2, PABPC1 and TACO1 could be ubiquitinated by E4B.The result indicated that all the three proteins were the substrates of E4B, which would lay a foundation for the further study of the mechanism of ubiquitination in cells.

Key words: ubiquitination, E4B, PM20D2, PABPC1, TACO1