cerevisiae
Together, Zuo and Ssz form the ribosome-associated complex (RAC) (Gautschi et al., 2001). Homologs of RAC are also found in mammals, indicating its conservation in the eukaryotic world (Otto et al., 2005; Hundley et al., 2005). In S. cerevisiae RAC together with the ribosome-attached Hsp70 Ssb forms a functional chaperone triad (Gautschi et al., 2002), while in mammals the cytosolic Hsp70 seems to complete this tripartite system. Ssb is encoded by two genes (ssb1, ssb2), which are more than 99%
identical (hereafter referred to as Ssb).
Figure 1.4.: The ribosome-associated chaperone triad inS. cerevisiaeconsists of Ssz (purple) and Zuotin (orange) that form the ribosome-associated complex (RAC). Only the Hsp40 Zuotin and the Hsp70 Ssb (blue) bind directly to the ribosome. Only Ssb can interact with the nascent polypeptide.
Yeast strains with a deletion of either one or all of its chaperone triad members dis-play three phenotypes: salt sensitivity, cold sensitivity, and hypersensitivity to amino-glycosides such as paromomycin that block ribosomal protein biosynthesis and impair translational fidelity (Gautschi et al., 2001; Yan et al., 1998; Hundley et al., 2002). Al-though the specific function of the ribosome-associated triad in co-translational protein folding is still unknown, it is likely that the triad functions as a chaperone system, since all components are members of classical chaperone families.
In support of the likely role of the triad as a chaperone, the prokaryotic ribosome bound chaperone TF was shown to be able to substitute in vivo the function of the chaperone triad when expressed in a RAC deletion strain ofS. cerevisiae(Rauch et al.,
1.6. The ribosome-associated chaperone triad in S. cerevisiae 2005; Ito, 2005). TF’s ability to rescue these cells is dependent upon its ability to bind
to ribosomes.
The chaperone triad has also been shown to bind to the ribosome. Both, Zuo and Ssb directly bind to the ribosome, whereas Ssz seems to be only indirectly attached via its stable interaction with Zuo (Pfund et al., 1998; Gautschi et al., 2001; Yan et al., 1998). Binding of RAC to the ribosome could be shown in close proximity of Rpl31 at the exit of the polypeptide tunnel in yeast (Peisker et al., 2008). However, stable Zuo ribosome binding is thought to be predominantly achieved via interaction with rRNA (Yan et al., 1998; Peisker et al., 2008). Furthermore, the chaperone triad can be crosslinked to a large variety of nascent chains, indicating its role in de novo protein folding (Pfund et al., 1998; Hundley et al., 2002). In these experiments, only Ssb contacts the nascent chain, but the cross-linking efficiency depends on the presence of RAC.
Recently, Ssb has been linked to the co-translational folding of ribosomal pro-teins and biogenesis factors, indicating a potential client repertoire for the ribosome-associated chaperone triad (Koplin et al., 2010). Furthermore, Ssb is assumed to mod-ulate production and assembly of ribosomal components, thereby adjusting ribosome production and proteins synthesis with the folding capacity of ribosome-associated chaperones. Additionally, Zuo too has been linked to the ribosome maturation (Al-banèse et al., 2010), indicating the involvement of the whole chaperone triad RAC/Ssb in the biogenesis of ribosomes.
Some very interesting features have been found to render the RAC chaperone system quite unique: Namely, Ssz and Zuo do not show a typical Hsp70/40 behavior as demonstrated previously for e.g. the bacterial DnaK/DnaJ chaperone system. Instead Zuo and Ssz form a highly stable complex. It is Ssz (Hsp70) that appears to regulate the activity of Zuotin (Hsp40) and it does so in a manner independent of Ssz’s ATPase activity. Zuotin thus requires the assistance of Ssz to efficiently stimulate the ATPase activity of Ssb (Hsp70) (Pfund et al., 2001; Huang et al., 2005).
1.6.1. Ssz
Although Ssz belongs to the Hsp70 family of chaperones, functionality and structure are changed compared to canonical Hsp70 to cope with its different demands in RAC.
Ssz’s ATPase binding domain is similar to this of canonical Hsp70s and was shown to
bind ATP. However, the Ssz-ATP complex is rather unstable and no ATPase activity could be detected so far (Huang et al., 2005; Conz et al., 2007). A non ATP binding mutant version of Ssz (Ssz-LKA) fully complements the ssz∆ phenotype suggesting that ATP binding is not a prerequisite for function (Conz et al., 2007).
The substrate binding domain (SBD) of Ssz is shortened compared to canonical Hsp70s, missing the lid structure which closes the binding pocket through a salt bridge and two hydrogen bonds. Substrate binding within in the SBD could not be shown so far (Huang et al., 2005). Interestingly, Ssz lacking its peptide-binding domain fully complements ssz∆ mutants, but fails to stably interact with Zuo, indicating that a tight interaction between Ssz and Zuo is not strictly required in vivo (Huang et al., 2005; Conz et al., 2007). However, the combination of the C-terminal deletion and the inability of the N-terminal domain to bind ATP results in a loss of function phenotype in vivo (Conz et al., 2007).
Figure 1.5.: Schematic model of the domain structure of Ssz~58 kDa, 538 aa
1.6.2. Zuo
Zuo, as a type III J-protein, holds a highly conserved J-domain, but differs in its domain organization compared to other Hsp40 members. At the N-terminus a so called Zuotin-homology-region is a characteristic of all Zuo homologs, however, so far no functionality could be assigned. At the C-terminal region Zuo contains an internal, highly charged region localized between aa 284 - 360, which was found to be important for Zuo’s interaction with the ribosome as well as for its ability to interact with nucleic acids (Yan et al., 1998). A mutant version of Zuo lacking aa 282-331 was still able to form a complex with Ssz, but the complex was not bound to the ribosome (Peisker et al., 2008). However, a severe mutation, replacing a total of 15 aa to alanines in the region aa 296 - 305, showed only minor effects on Zuo-ribosome interaction (Peisker et al., 2008). Instead a chimera mutant, containing the J-domain from the cytosolic Hsp40 protein Ydj1 and a part of the charged region spanning aa 306-363, could be
1.7. Analysis of Protein Dynamics