The ubiquitin domain protein BAG-1 and the CHIP ubiquitin ligase can cooperate to shift the activity of the Hsc/Hsp70 chaperone system from protein folding to degradation. The chaperone cofactors thus act as key regulators to influence protein quality control.

The BAG-1 protein modulates the chaperone activity of Hsc70 and Hsp70 in the mammalian cytosol and nucleus. Remarkably, BAG-1 possesses a ubiquitin-like domain at its amino terminus, suggesting a link to the ubiquitin/proteasome system. Here we show that BAG-1 is indeed associated with the 26 S proteasome in HeLa cells. Binding of the chaperone cofactor to the proteolytic complex is regulated by ATP hydrolysis and is not mediated by Hsc70 and Hsp70. The presented findings reveal a role of BAG-1 as a physical link between the Hsc70/Hsp70 chaperone system and the proteasome. In fact, targeting of BAG-1 to the proteasome promotes an association of the chaperones with the proteolytic complex in vitro and in vivo. A regulatory function of the chaperone cofactor at the interface between protein folding and protein degradation is thus indicated.

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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 ...

In the mammalian cytosol and nucleus the activity of the molecular chaperone Hsc70 is regulated by chaperone cofactors that modulate ATP binding and hydrolysis by Hsc70. Among such cofactors is the anti-apoptotic protein BAG-1. Remarkably, BAG-1 is expressed as multiple isoforms, which are distinguished by their amino termini. We investigated whether distinct isoforms differ with respect to their Hsc70-regulating activity. By comparing the mainly cytosolic isoforms BAG-1M and BAG-1S, opposite effects of the two isoforms were observed in chaperone-assisted folding reactions. Whereas BAG-1M was found to inhibit the Hsc70-mediated refolding of nonnative polypeptide substrates, the BAG-1S isoform stimulated Hsc70 chaperone activity. The opposite effects are not due to differences in the regulation of the ATPase activity of Hsc70 by the two isoforms. Both isoforms stimulated ATP hydrolysis by Hsc70 in an Hsp40-dependent manner through an acceleration of ADP-ATP exchange. Our results reveal that the different amino termini of the distinct BAG-1 isoforms determine the outcome of an Hsc70-mediated folding event, most likely by transiently interacting with the polypeptide substrate. Employing isoforms of a cofactor with different substrate binding properties appears to provide the means to influence the chaperone function of Hsc70 in addition to modulating its ATPase cycle.

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