Introduction
Hsp70s and Hsp40s cooperate in a wide range of cellular processes, including protein translation, translocation across membranes, and folding. Hsp70s bind unfolded or partially unfolded polypeptides in an ATP-regulated cycle (Hartl 1996; Bukau and Horwich 1998; Mayer et al. 2000). Hsp40s stimulate ATP hydrolysis, promoting the ADP-bound form of Hsp70, which has a higher affinity for unfolded protein substrates. In addition, many Hsp40s also bind unfolded polypeptide substrates and are able to prevent their aggregation independently of Hsp70 action. Based on the results of several in vitro studies, the current model of the cycle of Hsp70 and Hsp40 purports that Hsp40 first binds unfolded protein substrates and then transfers them to Hsp70 for folding (Langer et al. 1992; Liberek et al. 1995; Laufen et al. 1999).
Hsp40s are modular proteins. The signature J domain binds the ATPase domain of Hsp70 and is required for stimulating ATP hydrolysis. In class I and II Hsp40s, the J domain is followed by a region rich in glycine and phenylalanine residues (G/F) (Cheetham and Caplan 1998). Class I Hsp40s are distinguished by a cysteine-rich region that has been implicated in substrate binding (Banecki et al. 1996; Szabo et al. 1996). In addition, class I and II Hsp40s contain a poorly conserved COOH terminus that also has been implicated in binding unfolded polypeptides (Lu and Cyr 1998a,Lu and Cyr 1998b).
Multiple Hsp40s exist in both prokaryotic and eukaryotic cells. Here, we focus on two cytosolic Hsp40 chaperones of Saccharomyces cerevisiae, Ydj1 and Sis1 (see Fig. 1) (Caplan and Douglas 1991; Luke et al. 1991). Lack of Ydj1, a class I Hsp40, results in a severe growth phenotype, especially at higher temperatures. Sis1 is an essential type II Hsp40. Some functional overlap exists between these two Hsp40s, as overexpression of SIS1 can partially rescue the growth defect of a ydj1 disruption strain. However, overexpression of YDJ1 does not restore viability to an sis1 disruption strain. Each of these Hsp40s is capable of stimulating the ATPase activity of the abundant essential cytosolic Hsp70, Ssa, and can cooperate with Ssa in refolding denatured luciferase (Lu and Cyr 1998a,Lu and Cyr 1998b).
Both Ydj1 and Sis1 can bind unfolded polypeptides. A proteolytic fragment of Ydj1 encompassing amino acids (a.a.) 179–384, which contains half of the cysteine-rich region and most of the COOH terminus, has been shown to bind denatured substrates in vitro (Lu and Cyr 1998a). The region of Sis1 responsible for binding denatured substrates also maps to the COOH terminus (a.a. 171–352) (see Fig. 1 A), (Lu and Cyr 1998b). The recent crystal structure of this fragment revealed two similar domains, domain I and domain II, as well as a dimerization domain. Domain I contains a large hydrophobic depression and thus is proposed to contain the peptide-binding site (Sha et al. 2000).
Although it has been demonstrated that Ydj1 and Sis1 can bind substrates in vitro, the importance of this function in vivo is not clear. In a ydj1 disruption strain, expression of the first 134 a.a. of Ydj1, containing the J domain, G/F region, and a short spacer region, restores wild-type growth at the optimal growth temperature of 30°C (Johnson and Craig 2000). Likewise, in an sis1 disruption strain, a strain expressing only the J plus G/F region of Sis1 (Sis1-121) grows as well as a wild-type strain at 30°C and exhibits only a mild temperature–sensitive growth phenotype (Yan and Craig 1999). These results suggest that the ability of either Ydj1 or Sis1 to bind substrate is unnecessary. Therefore, we set out to critically test the requirements for Hsp40 function in vivo, using the cytosol of yeast as a test. Using a strain having chromosomal deletions of both YDJ1 and SIS1, we found that the COOH terminus of either Ydj1 or Sis1 must be present for viability and thus, despite limited sequence homology, share overlapping essential functions.