The Multiubiquitin-Chain-Binding Protein Mcb1 Is a Component of the 26S Proteasome in Saccharomyces cerevisiae and Plays a Nonessential, Substrate-Specific Role in Protein Turnover

Abstract

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.

The proteasome: a protein‐destroying machine

“…Similarly, the SUN1 gene (Kominami et al 1997)-also identified as MCB1 (van Nocker et al 1996)-is not essential. This gene, and its human homologue, S5a, encodes a subunit that can bind specifically to proteins conjugated to poly Ub chains, and it therefore functions as a poly Ub-receptor (abbreviated pUb-R) of the 26S proteasome.…”

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

“…On the contrary, recent work has shown that a diverse set of ubiquitin-binding receptors regulate the targeting of ubiquitinated proteins to the proteasome in a substratespecific manner (Wilkinson et al, 2001;Chen and Madura, 2002;Elsasser et al, 2004;Verma et al, 2004; for review, see Elsasser and Finley, 2005;Hicke et al, 2005). The first polyubiquitin-binding receptor identified was Rpn10, a subunit of the 19S regulatory complex that contains a ubiquitin-interacting motif (Deveraux et al, 1994 tional ubiquitin chain recognition mechanisms must exist (van Nocker et al, 1996). A second class of polyubiquitinbinding proteins, referred to as ubiquitin-like/ubiquitin-associated (UbL/UBA) proteins, is exemplified in yeast by the nucleotide excision repair protein Rad23 (Watkins et al, 1993) and the spindle pole duplication factor Dsk2 (Biggins et al, 1996).…”

“…The first polyubiquitin-binding receptor identified was Rpn10, a subunit of the 19S regulatory complex that contains a ubiquitin-interacting motif (Deveraux et al, 1994). However, cells lacking Rpn10 exhibit only mild phenotypes, indicating that addi-tional ubiquitin chain recognition mechanisms must exist (van Nocker et al, 1996). A second class of polyubiquitinbinding proteins, referred to as ubiquitin-like/ubiquitin-associated (UbL/UBA) proteins, is exemplified in yeast by the nucleotide excision repair protein Rad23 (Watkins et al, 1993) and the spindle pole duplication factor Dsk2 (Biggins et al, 1996).…”

The Assembly Pathway of the 19S Regulatory Particle of the Yeast 26S Proteasome

“…The base consists of six AAA-ATPase subunits, regulatory particle triple-A ATPase (Rpt)1p-Rpt6p, and three non-ATPase subunits, regulatory particle non-ATPase (Rpn)1p, Rpn2p, and Rpn13p, whereas the lid is made of nine non-ATPases, Rpn3p, Rpn5p-Rpn9p, Rpn11p, Rpn12p, and Sem1p (Rpn15p) (Glickman et al, 1998b;Leggett et al, 2002;Funakoshi et al, 2004;Sone et al, 2004). Rpn10p, a non-ATPase subunit that binds polyubiquitin (Ub) chains (van Nocker et al, 1996;Saeki et al, 2002;Elsasser et al, 2004), has been suggested to exist in the interface between the base and the lid (Fu et al, 2001).…”