Distinct activation of an E2 XXXX -conjugating enzyme by its cognate E3 ligases.

@entity49654 @entity53563 @entity47502 @entity375 @entity8290
A significant portion of @entity8290 (Ub)-dependent cellular protein quality control takes place at the endoplasmic reticulum (ER) in a process termed "ER-associated degradation" (ERAD). @entity375 ERAD employs two integral ER membrane E3 Ub ligases: @entity53563 (also termed @entity53563 ) and @entity47502 , which recognize a distinct set of substrates. However, both E3s bind to and activate a common E2-conjugating enzyme, @entity49654 . Here we describe a novel feature of the ERAD system that entails differential activation of @entity49654 by its cognate E3s. We found that residues within helix a2 of @entity49654 that interact with donor Ub were essential for polyUb conjugation. Mutagenesis of these residues inhibited the in vitro activity of @entity49654 by preventing the conjugation of donor Ub to the acceptor. Unexpectedly, Ub chain formation by mutant @entity49654 was restored selectively by the @entity53563 RING domain but not by the @entity47502 RING domain. In agreement with the in vitro data, @entity49654 a2 helix mutations selectively impaired the in vivo degradation of @entity47502 substrates but had no apparent effect on the degradation of @entity53563 substrates. To our knowledge, this is the first example of distinct activation requirements of a single E2 by two E3s. We propose a model in which the RING domain activates Ub transfer by stabilizing a transition state determined by noncovalent interactions between the a2 helix of @entity49654 and Ub and that this transition state may be stabilized further by some E3 ligases, such as @entity53563 , through additional interactions outside the RING domain.
