Saccharomyces cerevisiae

The AAA؉ molecular chaperone Hsp104 mediates the extraction of proteins from aggregates by unfolding and threading them through its axial channel in an ATP-driven process. An Hsp104-binding peptide selected from solid phase arrays enhanced the refolding of a firefly luciferase-peptide fusion protein. Analysis of peptide binding using tryptophan fluorescence revealed two distinct binding sites, one in each AAA؉ module of Hsp104. As a further indication of the relevance of peptide binding to the Hsp104 mechanism, we found that it competes with the binding of a model unfolded protein, reduced carboxymethylated ␣-lactalbumin. Inactivation of the pore loops in either AAA؉ module prevented stable peptide and protein binding. However, when the loop in the first AAA؉ was inactivated, stimulation of ATPase turnover in the second AAA؉ module of this mutant was abolished. Drawing on these data, we propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA؉ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA؉ module.Hsp104 is a AAAϩ protein disaggregase that functions in yeast in the resolubilization and reactivation of thermally denatured and aggregated proteins (1, 2). In unstressed cells, Hsp104 is critical to the mitotic stability of the yeast prions [, and [URE3] (3-5). Hsp104 and its bacterial orthologue ClpB are members of the Hsp100/Clp family of proteins (6). Other Hsp100s, such as ClpA, ClpX, and ClpY (HslU), unfold and unidirectionally translocate polypeptides through a central axial channel (7-11). Crystal structures of HslU (12, 13) and cryoelectron microscopic reconstructions of ClpB (14) reveal that the diameter of the axial channel is regulated by flexible loops whose conformation is regulated by the nucleotide status of the nucleotide binding domain of each AAAϩ module. Modification of these loops impairs protein translocation and/or degradation implying that these loops play critical roles in translocation (15-18). Likewise, mutation of the flexible loops of Hsp104 and ClpB results in refolding defects suggesting that all Hsp100s employ a similar unfolding/threading mechanism to process substrates whether they are ultimately degraded or refolded (16,19,20). Despite the growing body of knowledge regarding the unfolding and translocation mechanism of Hsp104, the determinants of the initial stage of the unfolding process, substrate recognition and binding, remain unclear.In other Hsp100s, recognition of specific peptide sequences initiates unfolding and translocation. Protein substrates of ClpXP generally contain recognition signals of roughly 10 -15 residues that can be located either at the N or C termini (21). The SsrA tag, an 11-amino acid peptide (AANDENYALAA) that is appended to the C terminus of polypeptides by the action of transfer-messenger RNA on stalled ribosomes (22), is a particularly well studied example of an Hsp10...

Hsp104 is molecular chaperone in the AAA+ family of ATPases that specializes in the resolubilization and refolding of thermally denatured proteins in yeast. In addition to providing high levels of thermotolerance, Hsp104 plays a pivotal role in the propagation of yeast prions, self-replicating, amyloid-like aggregates that are inherited during mitosis and meiosis. In this review, the structure and function of Hsp104 is discussed, its functional interaction with other molecular chaperones, and a model for disaggregation and refolding is proposed.

Calreticulin is a lectin chaperone of the endoplasmic reticulum that interacts with newly synthesized glycoproteins by binding to Glc 1 Man 9 GlcNAc 2 oligosaccharides as well as to the polypeptide chain. In vitro, the latter interaction potently suppresses the aggregation of various non-glycosylated proteins. Although the lectin-oligosaccharide association is well understood, the polypeptide-based interaction is more controversial because the binding site on calreticulin has not been identified, and its significance in the biogenesis of glycoproteins in cells remains unknown. In this study, we identified the polypeptide binding site responsible for the in vitro aggregation suppression function by mutating four candidate hydrophobic surface patches. Mutations in only one patch, P19K/I21E and Y22K/ F84E, impaired the ability of calreticulin to suppress the thermally induced aggregation of non-glycosylated firefly luciferase. These mutants also failed to bind several hydrophobic peptides that act as substrate mimetics and compete in the luciferase aggregation suppression assay. To assess the relative contributions of the glycan-dependent and -independent interactions in living cells, we expressed lectin-deficient, polypeptide bindingdeficient, and doubly deficient calreticulin constructs in calreticulin-negative cells and monitored the effects on the biogenesis of MHC class I molecules, the solubility of mutant forms of ␣ 1 -antitrypsin, and interactions with newly synthesized glycoproteins. In all cases, we observed a profound impairment in calreticulin function when its lectin site was inactivated. Remarkably, inactivation of the polypeptide binding site had little impact. These findings indicate that the lectin-based mode of client interaction is the predominant contributor to the chaperone functions of calreticulin within the endoplasmic reticulum. Membrane-bound calnexin (Cnx)3 and its soluble paralog calreticulin (Crt) are glycoprotein-specific chaperones of the endoplasmic reticulum (ER). As components of the ER quality control system, they retain glycoprotein folding intermediates within this organelle and assist folding by preventing aggregation and by recruiting folding catalysts such as the thiol oxidoreductase ERp57 and peptidyl-prolyl isomerase cyclophilin B (for reviews, see Refs. 1 and 2). Both chaperones consist of a globular lectin domain and an extended arm or P domain (3-5) with the tip of the arm domain comprising the binding site for ERp57 (6) and cyclophilin B (7). The specificity for glycoproteins resides within their lectin domains, which recognize a monoglucosylated oligosaccharide-processing intermediate of composition Glc 1 Man 9 GlcNAc 2 (8 -10). Cycles of chaperone binding and release are regulated by the availability of the terminal glucose residue on this oligosaccharide with glucose removal catalyzed by glucosidase II and its readdition by UDPglucose:glycoprotein glucosyltransferase I (11). UDP-glucose: glycoprotein glucosyltransferase I acts as the folding sensor in the cycle, only...