As stated above, a single Hsp70 protein can interact with multiple members of the Hsp40 family to form unique Hsp70-Hsp40 couples that have unique functions. In the case of type III Hsp40s, specification of Hsp70 function occurs through the binding of specific clients via their unique PPDs (Cyr et al 1994
; Cheetham and Caplan 1998
). However, because type I and type II Hsp40s appear to have overlapping substrate specificity, how they specify Hsp70 is not clear.
To investigate this question, investigators have used yeast as a model system and examined the functional relationships between Ydj1 and Sis1 that occur with the cytosolic Hsp70 Ssa1–4 and Hsp70 Ssb1–2 proteins. Biochemical studies suggest that Ydj1 and Sis1 interact with Hsp70 Ssa proteins but not with members of the Hsp70 Ssb protein family (Cyr et al 1992
; Cyr and Douglas 1994
; Cyr 1995
). Genetic studies indicate that Ydj1 and Sis1 have specific functional properties that enable them to direct Hsp70 Ssa proteins to facilitate different cellular processes. For example, the overexpression of Sis1 can complement the slow-growth phenotype of ydj1Δ strains, but Ydj1 cannot complement the lethal phenotype of sis1Δ strains (Caplan and Douglas 1991
; Luke et al 1991
). In addition, the cellular functions of Ydj1 and Sis1 are different. Ydj1 and its human homolog Hdj2 function on the cytoplasmic face of the endoplasmic reticulum to promote membrane protein folding and protect cells from stress (Caplan et al 1992b
; Meacham et al 1999b
). Ydj1 is known to be required for proper folding of the insulinase-like protease Axl1 (Meacham et al 1999a
) and regulation of cyclin 3 phophporylation and ubiquitination (Yaglom et al 1996
). In addition, Ydj1 is more efficient than Sis1 in maintaining hormone receptors in ligand-binding competent conformations (Fliss et al 1999
). In contrast, Sis1 is found in association with translating ribosomes and is required to facilitate the assembly of translation initiation complexes (Zhong and Arndt 1993
; Horton et al 2001
). Sis1, but not Ydj1, is required for the maintenance of the prion [RNQ+] (Sondheimer et al 2001
; Lopez et al 2003
Examination of the domain structures of Ydj1 and Sis1 reveals 2 structural differences. First, the glycine-and phenylalanine-rich region (G/F) of Ydj1 and Sis1 are different, with that of Sis1 containing a 10-residue-long insert (Lopez et al 2003
). Second, as mentioned above, the protein modules located in the middle of Ydj1 and Sis1 are different (). Thus, it is plausible that either the G/F domain or the chaperone modules of Ydj1 and Sis1 serve to specify their in vivo functions.
In tests of the latter hypothesis, chimeric forms of Ydj1 and Sis1 were constructed in which the chaperone modules were swapped to form YSY and SYS (Fan et al 2004
). Purified SYS and YSY were found to exhibit protein-folding activity and substrate specificity that mimicked that of Ydj1 and Sis1, respectively (Fan et al 2004
). In in vivo studies YSY exhibited a gain of function and, unlike Ydj1, could complement the lethal phenotype of sis1Δ and promote the propagation of the yeast prion [RNQ1+]. SYS exhibited a loss of function and was unable to maintain [RNQ1+]. These in vitro and in vivo data suggest that the chaperone modules of Ydj1 and Sis1 are exchangeable and that they help specify Hsp70s cellular functions (Fan et al 2004
To determine whether the G/F regions of type I and type II Hsp40s help specify Hsp70 functions, the Craig group has carried out a number of complementation studies with Hsp40 fragments (Yan and Craig 1999
). In these studies, which were conducted with a sis1Δ strain, the G/F region of Sis1, but not that of Ydj1, was shown to be important for suppression of the inviability caused by the loss of Sis1 function (Yan and Craig 1999
). In addition, Sis1Δ G/F was demonstrated to be defective at [RNQ1+] maintenance (Sondheimer et al 2001
). The G/F region of Sis1 contains a 10-residue insert GHAFSNEDAF that corresponds to amino acids 102–112 that are not present in the G/F region of Ydj1 (Lopez et al 2003
). When the G/M region is deleted from the Sis1 chaperone module (see ), residues N108I and D110G in the G/F region become important for Sis1 in vivo function (Lopez et al 2003
). Thus, the Sis1 G/F rich region is clearly important for its in vivo function, and it plays an important role in modulating the conformation of at least some substrates.
How the G/F domain functions to help specify Hsp40 action is not established, and a direct interaction between the G/F domain and a substrate protein has not been demonstrated. However, the G/F region is enriched in hydrophobic residues and lies adjacent to regions in Hsp40s that are involved in substrate binding. Thus, it is possible that the G/F region operates as a component of the Hsp40 PPD. On the other hand, the G/F region may have evolved to mediate interactions between Hsp40 and Hsp70 that are important for the conformational maturation of different substrate proteins. Nonetheless, it is clear that the G/F domain and chaperone modules of type I and type II Hsp40 both act to specify Hsp70 cellular functions.