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Molecular Mechanism of the Ubiquitin Conjugating Enzyme E2‐25K to Synthesize the Polyubiquitin Chains
Author(s) -
Shin Dong Yeon,
Park Eun Sil,
Yoo Yung Joon
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.842.2
Subject(s) - ubiquitin , thioester , deubiquitinating enzyme , chemistry , mechanism (biology) , microbiology and biotechnology , biochemistry , enzyme , biology , gene , philosophy , epistemology
Ubiquitin(Ub) is involved in various cellular functions through the ubiquitination of many proteins. The assembly of polyUb chains with different topology determines the consequence of ubiquitinated proteins but the detailed mechanism for the synthesis of polyUb chains is largely unknown. In this study, we tried to elucidate the molecular mechanism of the K48‐polyUb synthesis. E2‐25K contains a C‐terminal Ub‐associated (UBA) domain and has a catalytic activity of E3‐independent ubiquitination. The most plausible mechanism of Ub chain synthesis is the aminolysis‐based transfer between a donor Ub on the active site cysteine(C92) and an acceptor Ub(s) interacting with UBA domain. However, UBA‐deleted E2‐25K mutant was able to synthesize polyUb although the catalytic activity was reduced. This results indicated that E2‐25K core domain alone could recruit the acceptor Ub(s) to synthesize the polyUb chains, facilitated by the UBA domain. In addition, we found that the polyUb chains were linked onto C92 of E2‐25K through the thioester bond using the nonreduced/reduced two‐dimensional electrophoresis, suggesting that protein substrates could be ubiquitinated en bloc by preassembled polyUb chains from E2‐25K in addition to conventional one‐by‐one ubiquitination. (Supported by a grant for the Study of Ubiquitome Functions from the KMEST and the Systems Biology Infrastructure Establishment Grant by GIST)