The ribosome-associated chaperone triad from Saccharomyces cerevisiae

Besides NAC, a second ribosome-anchored chaperone system was identified in eukaryotes (see above). In Saccharomyces cerevisiae the Hsp70/40 chaperone pair Ssz and Zuotin (Zuo) assembles into a unique heterodimeric complex termed ribosome-associated complex (RAC) (Gautschi et al, 2001). RAC forms a functional chaperone triad together with another

NAC UBA

N C

N C

N C

NAC

NAC RRK-(X)₂-KK

14 78 136 172

38 103 157

38 103 150

α-NAC (Egd2p) β-NAC (Egd1p)

β‘-NAC (Btt1p)

A B

UBA α

β ^RRK-(X)2-KK

Introduction

genes (SSB1 and SSB2) encode Ssb1p and Ssb2p, respectively, both of which are nearly identical and differ only by four amino acids (collectively referred to as Ssb). The functional cooperation of the members of the yeast chaperone triad became evident from diverse genetic experiments. Yeast strains lacking either one or all three components of the triad show similar pleiotropic phenotypes: sensitivity to high salt concentrations, hypersensitivity to toxic cations including aminoglycosides that increase translational errors, and cold-sensitivity (Gautschi et al, 2001; Jones et al, 2003; Nelson et al, 1992; Yan et al, 1998). Moreover, cells lacking either Ssb or Zuo show additional defects such as hypersensitivity to a broad range of toxic cations (Kim & Craig, 2005). It was thus speculated that the absence of a functional ribosome-associated chaperone system might lead to the accumulation of defective ion transporters in the plasma membrane, causing an increased uptake of cations into the cell.

Figure 11: The chaperone triad from S. cerevisiae. The yeast ribosome-associated chaperone triad consists of Ssb1/2p (Ssb1p is shown in green), Ssz1p (dark blue) and Zuo1p (light blue). Ssb1/2p and Ssz1p belong to the Hsp70 chaperone family and contain an N-terminal nucleotide binding domain (NBD) and a C-terminal substrate-binding domain (SBD). Like canonical Hsp40 cochaperones Zuo1p contains a J-domain required for stimulation of the ATPase activity of its Hsp70 partner. In addition, Zuo1p contains a charged region (+) in its C-terminus. The N-terminus (N) of Zuo is predicted to be unstructured. Ssz1p and Zuo1p form a stable heterodimeric complex (RAC) via unknown interactions.

A direct role of the yeast Ssz/Zuo/Ssb ensemble in folding of newly synthesized proteins has not yet been demonstrated. However, all components of the triad belong to classical chaperone families and associate with ribosomes. In addition, crosslink experiments showed that Ssb contacts short nascent polypeptide chains (Gautschi et al, 2003; Hundley et al, 2002; Pfund et al, 1998)). This suggests its localization close to the ribosomal tunnel exit site.

The finding that the prokaryotic ribosome-associated chaperone TF could partially complement the aminoglycoside sensitivity of triad-deficient yeast cells indicates overlapping functions of the two systems (Rauch et al, 2005). Unlike in the case of NAC, ribosome association of the triad is far less characterized. Only Ssb and Zuo were shown to bind directly to ribosomes (Nelson et al, 1992; Yan et al, 1998). By contrast, Ssz associates with

1 62 102 166 284 363 433

N N J-domain + + + + + + C

N C

1 391 613

NBD SBD

Ssb1p

Zuo1p Ssz1p

1 394 538

N NBD SBD C

RAC

domain with a conserved HPD-motif, which is required to stimulate the ATPase activity of Hsp70 chaperones (Figure 11) (Gautschi et al, 2001; Zhang et al, 1992). However, unlike other Hsp40 co-chaperones, Zuo does not interact with unfolded polypeptides. Instead, Zuo contains an N-terminal Zuotin-homology region, which is critical for its function in vivo (Yan et al, 1998). Another unique feature of Zuo is a positively charged region, which was suggested to be involved in ribosome binding. Although deletion of the charged region resulted in loss of ribosome association (Yan et al, 1998), the mutation of 12 positively charged residues into alanines had no effect (Peisker et al, 2008). A recent study proposed that Zuo interacts with ribosomes at least partly via the ribosomal protein Rpl31p, which is located next to the tunnel exit (Peisker et al, 2008).

Understanding the molecular interactions between Ssz, Zuo and Ssb is of major importance to unravel their functional interplay. Ssb is the only component that contacts nascent polypeptides (Pfund et al, 1998) and the efficiency of this interaction depends on the presence of functional RAC (Gautschi et al, 2002). Zuo was shown to stimulate the ATPase activity of Ssb (Huang et al, 2005). This function of Zuo, however, depends on complex formation with Ssz (Figure 8). Interestingly, Ssz does neither require nucleotide binding, nor hydrolysis for functionality (Huang et al, 2005) and a truncated version of Ssz, lacking the peptide-binding domain, was able to complement the phenotypes of an sszΔ strain (Hundley et al, 2002). These results imply that Ssz carries out a unique function distinct from the one of classical Hsp70 chaperones. It rather seems to modulate the ability of Zuo to act as a cochaperone for Ssb. In summary, the ribosome-associated chaperone triad is special because Ssz and Zuo form a stable complex (RAC), which is required to stimulate the ATPase activity of Ssb.

While ribosome-attached Ssb seems to be confined to fungi (Pfund et al, 2001), homologs of RAC were found in mammals as well. This suggests that the concept of ribosome-anchored Hsp70/40 systems for nascent polypeptides is common in the eukaryotic world (Otto et al, 2005). In accordance with this notion, the human homologs of Ssz (Hsp70L1) and Zuo (Mpp11) can partly substitute for RAC in yeast (Hundley et al, 2005; Otto et al, 2005).

Moreover, the knock-down of mammalian Mpp11 in HeLa cells leads to growth defects similar to those observed in a yeast strain lacking RAC (Jaiswal et al, 2011). Despite the considerable similarities between yeast and mammalian RAC, there are also significant structural and functional differences. Most strikingly, complementation of yeast RAC by mammalian RAC is independent of Ssb. A recent study identified cytosolic Hsp70 as the functional partner for mammalian RAC, which does not bind to ribosomes on its own (Jaiswal

Introduction

associated RAC seems to recruit multifunctional Hsp70s to nascent polypeptide chains (Hundley et al, 2005; Jaiswal et al, 2011). Although it is puzzling why a stable Hsp40/70 complex is required on ribosomes, the conservation of RAC in eukaryotes indicates a significant functional advantage. The characterization of this atypical Hsp70-Hsp40 interaction is therefore particularly important to understand the role of Ssz and its counterparts, and to get new insights how nature evolved chaperone systems to perform specialized tasks.