A HECT domain E3 enzyme assembles novel polyubiquitin chains

Although polyubiquitin chains linked through Lys(29) of ubiquitin have been implicated in the targeting of certain substrates to proteasomes, the sign aling properties of these chains are poorly understood. We previously descr ibed a ubiquitin-protein isopeptide ligase (E3) from erythroid cells that a ssembles polyubiquitin chains through either Lys(29) Or LyS(48) of ubiquiti n (Mastrandrea, L. D., You, J., Niles, E. G., and Pickart, C. M. (1999) J. Biol. Chem. 274, 27299-27306). Here we describe the purification of this E3 based on its affinity for a linear fusion of ubiquitin to the ubiquitin co njugating enzyme UbcH5A. Among five major polypeptides in the affinity colu mn eluate, the activity of interest was assigned to the product of a previo usly cloned human cDNA known as KIAA10 (Nomura, N., Miyajima, N., Sazuka, T ., Tanaka, A., Kawarabayasi, Y., Sato, S., Nagase, T., Seki, N., Ishikawa, K., and Tabata, S. (1994) DNA Res. 1, 27-35). The KIAA10 protein is a membe r of the HECT ((h) under bar omologous to (E) under bar6-AP (c) under bar a rboxyl (t) under bar erminus) domain family of E3s. These E3s share a conse rved C-terminal (HECT) domain that functions in the catalysis of ubiquitina tion, while their divergent N-terminal domains function in cognate substrat e binding (Huibregtse, J. M., Scheffner, M., Beaudenon, S., and Howley, P. M. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2563-2567). Recombinant KIAA10 catalyzed the assembly of both Lys(29)- and Lys(48)-linked polyubiquitin ch ains. Surprisingly, the C-terminal 428 residues of KIAA10 were both necessa ry and sufficient for this activity, suggesting that the ability to assembl e polyubiquitin chains may be a general property of HECT domains. The N-ter minal domain of KIAA10 interacted in vitro with purified 26 S proteasomes a nd with the isolated S2/Rpn1 subunit of the proteasome's 19 S regulatory co mplex, suggesting that the N-terminal domains of HECT E3s may function in p roteasome binding as well as substrate binding.