Abstract
Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked polyubiquitin chains. The UBA domains bind Lys48-linked polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.
Highlights
The conserved protein Ub1 becomes covalently linked through its C terminus (Gly76) to lysine residues of substrates destined for degradation by the 26 S proteasome, a 2.5 MDa assembly consisting of a central cylindrical 20 S core complex and two distally positioned 19 S regulatory complexes [1]
Rad23 Inhibits Ub-dependent Proteolysis in a Reticulocyte Fraction II in a ubiquitin-associated domain(s) (UBA) Domain-dependent Manner—To assess the effects of Rad23 on Ub-dependent proteolysis we first used Ub-depleted reticulocyte extract in which the model substrate bovine lactalbumin is recognized by the N-end rule E3/E2 complex (E3␣/E2–14K, equivalent to yeast Ubr1/ Ubc2), conjugated to Lys48-linked polyUb chains and degraded by proteasomes [5, 32, 36, 39]
Effects of Rad23 on Proteasome-catalyzed Proteolysis—UbLUBA proteins can play a positive role in proteasome-catalyzed degradation [19, 22, 23], but their mechanism of action remains poorly defined
Summary
Ubiquitin; Gal, -galactosidase; DHFR, dihydrofolate reductase; DUB, deubiquitinating enzyme; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme, E3, ubiquitin-protein ligase; polyUb, polyubiquitin (refers to a branched, isopeptide-linked ubiquitin chain); UBA, ubiquitin-associated domain; Ubal, ubiquitin aldehyde; UbL, ubiquitin-like domain; GST, glutathione S-transferase; FPLC, fast protein liquid chromatography. A Lys48-linked chain of at least four Ubs in length is an autonomous signal that affords high affinity for purified proteasomes in vitro (Kd of ϳ50 nM, Ref. 7), recent studies have suggested that factors extrinsic to the 19 S complex might assist in targeting some polyubiquitinated substrates to proteasomes. Leading candidates for such a role are members of the UbL-UBA protein family, including Rad23/Rhp and Dsk2/Dph. We describe new properties of UBA domains that have a high likelihood to be relevant for the biological function(s) of these domains
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