Ubiquitylation of BAG-1 Suggests a Novel Regulatory Mechanism during the Sorting of Chaperone Substrates to the Proteasome

Abstract

BAG-1 is a ubiquitin domain protein that links the molecular chaperones Hsc70 and Hsp70 to the proteasome. During proteasomal sorting BAG-1 can cooperate with another co-chaperone, the carboxyl terminus of Hsc70-interacting protein CHIP. CHIP was recently identified as a Hsp70-and Hsp90-associated ubiquitin ligase that labels chaperone-presented proteins with the degradation marker ubiquitin. Here we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP mediates attachment of ubiquitin moieties to BAG-1 in conjunction with ubiquitinconjugating enzymes of the Ubc4/5 family. Ubiquitylation of BAG-1 is strongly stimulated when a ternary Hsp70⅐BAG-1⅐CHIP complex is formed. Complex formation results in the attachment of an atypical polyubiquitin chain to BAG-1, in which the individual ubiquitin moieties are linked through lysine 11. The noncanonical polyubiquitin chain does not induce the degradation of BAG-1, but it stimulates a degradation-independent association of the co-chaperone with the proteasome. Remarkably, this stimulating activity depends on the simultaneous presentation of the integrated ubiquitinlike domain of BAG-1. Our data thus reveal a cooperative recognition of sorting signals at the proteolytic complex. Attachment of polyubiquitin chains to delivery factors may represent a novel mechanism to regulate protein sorting to the proteasome.The control and maintenance of the three-dimensional structure of proteins are prerequisites for cell survival and involve a cooperation of molecular chaperones and energy-dependent proteases (1-4). Molecular chaperones recognize hydrophobic regions exposed on unfolded proteins and stabilize non-native conformations. As a consequence formation of insoluble protein aggregates is prevented, and folding to the native state is promoted. On the other hand, energy-dependent proteases, such as the eukaryotic 26 S proteasome, degrade irreversibly damaged proteins that fail to be folded properly.Selection of proteins for degradation by the proteasome involves ubiquitin conjugation (5-7). A polyubiquitin chain is attached to a protein substrate through the concerted action of a ubiquitin-activating enzyme (E1), 1 a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein isopeptide ligase (E3). In contrast to the presence of only one type of E1 enzyme in the eukaryotic cytosol, E2 and E3 enzymes are recruited from large protein families and mediate a specific recognition of a large repertoire of protein substrates. A polyubiquitin chain generated through the linkage of lysine 48 residues of successive ubiquitin moieties is usually sufficient to target a protein substrate to the proteasome, where finally deubiquitylation, unfolding, and degradation occur.Recent studies shed light onto molecular mechanisms underlying the cooperation of molecular chaperones with the ubiquitin/proteasome system during protein quality control. Two co-chaperones, CHIP and BAG-1, are of central importance in this regard. The CHIP protein was shown to act as a ...

Chaperone-assisted degradation: multiple paths to destruction

“…Degradation is stimulated upon binding of BAG-1 and HSJ-1, respectively, to the chaperone/CHIP complex (Figure 2). BAG-1 facilitates the docking of the complex at the proteasome, because it possesses a ubiquitin-like domain integrated in its primary structure, which is used for proteasome binding (Demand et al, 2001;Alberti et al, 2002). HSJ-1 belongs to the family of J-domain containing substrate-loading factors of Hsc/Hsp70 and in addition displays two ubiquitin interaction motifs that enable the co-chaperone to bind ubiquitylated chaperone clients after their initial encounter with CHIP (Westhoff et al, 2005; Figure 2).…”

CHIP promotes the degradation of mutant SOD1 by reducing its interaction with VCP and S6/S6′ subunits of 26S proteasome

“…Cochaperone Bag-1 interacts with proteasomes through its ubiquitin-like (UBL) domain in an ATP-dependent manner, which enables this protein to act as a 'shuttling factor' between the chaperone and the proteasome complex. Importantly, CHIP-mediated ubiquitylation of Bag-1 further stimulates its association with the proteasome (Alberti et al 2002). By analogy, our finding that CHIP promotes ubiquitylation of mutant SOD1-associated Hsp70 could imply that such a modification facilitates the binding of CHIP/Hsp70/ Bag-1 ternary complex (together with substrates) to the proteasome.…”

“…However, it is unlikely that CHIP is the sole factor in mediating the transfer of ubiquitylated substrates from the chaperone complex to the proteolytic machinery, instead, additional proteins must be present for efficient delivery. Interestingly, CHIP catalyzes ubiquitin conjugation to several other protein functions in the chaperone machinery Á i.e., Hsp70/Hsc70 and Bag-1 Á without influencing their half-lives (Jiang et al 2001;Alberti et al 2002). Cochaperone Bag-1 interacts with proteasomes through its ubiquitin-like (UBL) domain in an ATP-dependent manner, which enables this protein to act as a 'shuttling factor' between the chaperone and the proteasome complex.…”

“…Bag-1, another cochaperone of Hsp70, has also been implicated in the sorting of substrates to proteasomes (Demand et al 2001;Alberti et al 2002). CHIP and Bag-1 can directly interact and possibly function as partners in substrate delivery to proteasomes (Demand et al 2001).…”

Expression profile of Bag 1 in the postmortem brain

“…In addition, Bag 1 contains a domain with heat shock 70 (Hsp70) nucleotide-exchange activity, presumably to assist molecular chaperones with the removal of aberrant proteins from the cytosol (McClellan et al, 2005). It seems, therefore, that Bag 1 alleviates cellular stress by linking chaperones (i.e., Hsp70) with the ubiquitin-proteasome system to facilitate degradation of soluble oligomeric species (Luders et al, 2000;Alberti et al, 2002).…”