The glycine-rich regions of Hsp40 J-type proteins, which frequently have been considered simply linker regions between the J domain and the unfolded protein-binding domain, are critical for the protein's function. Herein, we report that the sequences in this region are important for the specific function of the Sis1/Hdj1 Hsp40s (class II), and distinguish them from the Ydj1/Hdj2 Hsp40s (class I). Class I and II J-type proteins often function with Hsp70s as soluble proteins within the same cellular compartment. Under such circumstances a single Hsp70 may function with more than one Hsp40, thus being a part of a complex network of chaperones. An example of such cross-function is the interaction of both Sis1 and Ydj1 with the Ssa class of Hsp70s.
The results presented above demonstrate that residues in the G/F region of Sis1 play important roles in Sis1-specific functions required for cell viability, as well as maintenance of [RNQ+
]. Consistent with our hypothesis based on comparison of the G/F regions of Sis1 and Ydj1, a short sequence of amino acids found in Sis1, but not Ydj1, is required for both viability and prion maintenance. Therefore, this “insertion” in the G/F region of Sis1 is important for Sis1-specific function. However, this result does not mean that the conserved sequences in the G/F region do not play an important function in both Ydj1 and Sis1. The G/F region of Sis1 likely plays two roles, one generic and held in common with that of the G/F region of class I Hsp40s such as Ydj1, and the second specific for Sis1-type Hsp40s. This idea is consistent with the fact that overexpression of Sis1 can rescue growth defects of a ydj1
strain (Caplan and Douglas, 1991
), but overexpression of Ydj1 cannot compensate for the absence of Sis1 (Luke et al., 1991
Our observations demonstrate that Ydj1 does not complement Sis1's function in [RNQ+
] maintenance. Nevertheless, it can interact with the prion form of Rnq1. Recently, it was reported that overexpression of Ydj1 can cure some variants of [RNQ+
] (also known as [PIN+
]) (Bradley et al., 2002
), suggesting that Ydj1 antagonizes Sis1 in prion maintenance.
The sufficiency of the J domain and G/F region (Sis1-121) to support viability, as well as to maintain the prion form of Rnq1, facilitated dissection of the requirements for certain sequences in this truncated protein. The results of the analysis of full-length protein were more complex, revealing a redundancy in function within the glycine-rich region. The G/M portion of the glycine-rich region also plays a partially overlapping role with the G/F region. The single amino acid alterations in the G/F region that resulted in a nonfunctional Sis1-121 protein resulted in only mild defects when in the full-length protein. Similarly, deletion of only the G/M region had a modest effect on prion maintenance. However, in combination with the single amino alterations (N108I or D110G), the G/M deletion had dramatic effects on prion maintenance. Therefore, both sections of the glycine-rich region play a role in prion maintenance. Actually, the initial division of extended glycine-rich region into G/F and G/M segments was quite arbitrary. In fact, these two glycine-rich segments likely function together.
What does the glycine-rich region of Sis1 do? In vitro experiments indicate that deletion of the G/F region reduces the ability of an Hsp40 to enhance the interaction of an Hsp70 with a target protein (Wall et al., 1995
) (Lopez, Johnson, and Craig, unpublished data). Because Sis1, but not Ydj1, is required for maintenance of the Rnq1 prion, and the sequences responsible are within the glycine-rich region, we propose that Sis1 type molecular chaperones uniquely have the ability to facilitate the productive interaction of Hsp70 with particular substrates, including Rnq1. More generally, the glycine-rich regions of an Hsp40 may determine the specificity of interaction of an Hsp70 with substrate proteins. This idea is an expansion of the hypothesis of Rapoport and coworkers developed from the results of experiments in which the presence of only the J domain greatly enhanced the affinity of an Hsp70 for a variety of protein/peptide substrates (Misselwitz et al., 1998
). According to our expanded hypothesis, the J domain is required for this enhanced interaction with substrates, but the G/F region has evolved to determine the specificity of such interactions. In the case analyzed in our laboratory, the G/F region of the Sis1 family of Hsp40 can specifically enhance the productive interaction of Rnq1 with Hsp70.
However, it should be pointed out that glycine-rich regions are not required for function of all J-type proteins because members of class III, by definition, have no obvious region rich in glycine residues. Several members of that class have been found to function with Hsp70s at particular sites within organelles and some are important for targeting the Hsp70 to the site of function. Sec63 is localized to the ER membrane with a J domain located in the lumen targets Kar2/BiP to the translocation pore (Lyman and Schekman, 1995
). Auxilin targets Hsp70 of mammalian cells to clathrin-coated vesicles where it functions in vesicle uncoating (Ungewickell et al., 1995
). Zuo1 functions with Ssb Hsp70 on yeast ribosomes as a chaperone for nascent chains (Yan et al., 1998
). G/F regions that may function to modulate the affinity of Hsp70 for particular substrates may not be as critical in cases where the Hsp70 is tethered to the location of the polypeptide substrate, thus dramatically increasing the local concentration. On the other hand, it is also possible that sequences, not recognizable to us because they are not rich in glycine residues, play a similar role.
An important goal of this study was to assess whether the role of Sis1 in prion maintenance was functionally different from its role in other cellular functions. This question was raised by the observation that Sis1 lacking the G/F region resulted in complete loss of the prion, but had little effect on cell viability. However, the analysis of mutants reported herein suggests that the sequence requirements are very similar, but that prion maintenance is particularly sensitive to slight alterations in Sis1 function. Therefore, we propose that biochemically Sis1 functions in prion maintenance as it does in other cellular processes, although its exact role in prion maintenance remains to be elucidated.
The ability of the Sis1 homologs from both Drosophila and humans to not only carry out the essential function of Sis1 but also maintain the Rnq1 prion was surprising. Neither of these Hsp40s have any apparent sequence similarity with Sis1 in the glycine-rich regions, other than the general preponderance of glycine and phenylalanine residues that are also found in yeast Ydj1 and human Hdj2. Therefore, divergent sequences are able to perform the function carried out by amino acids 101–113 of Sis1. This divergence suggests that a certain structural conformation that can be manifested through a variety of interactions is required for class II Hsp40-specific function, rather than a requirement for specific amino acid contacts between the G/F region and other domains. Current research to determine whether the G/F sequences affect the structure of the adjacent J domain or directly interact with Hsp70 and alter its function is in progress.