Functional Characterization Of The Ribosome Associated Zuo1 Ssz1 Chaperone Complex

Ribosome-associated molecular chaperones interact with nascent polypeptide chains, assisting their proper folding as they exit ribosomes. In Saccharomyces cerevisiae, the J-protein co-chaperone Zuo1 promotes interaction between partner Hsp70 Ssb and nascent polypeptides. Interaction of Zuo1 with the ribosome is key, but how it associates remains ill-defined. Zuo1 forms a complex with an atypical Hsp70, Ssz1, whose function is unknown. In this thesis, I report results which better define the interaction between Zuo1 and the ribosome, providing insight into the function of Zuo1. In addition, I report results which indicate direct interaction between Ssz1 and Ssb, suggesting that Ssz1 is a scaffold for Ssb on the ribosome. In Chapter Two, I describe how Zuo1 interacts with the 60S and 40S ribosomal subunits. Based on results of site-specific crosslinking, ribosomal RNA mutagenesis experiments, and computer driven docking using available structural information, I propose a model of Zuo1's interaction with the ribosome: (1) Zuotin Homology Domain of Zuo1 interacts with ribosomal protein eL31 and RNA H24 near the ribosomal tunnel exit; (2) the C-terminus of Zuo1 interacts with the ribosomal RNA H44, which originates at the decoding center. My results provide insight into how the dual interaction of Zuo1 with both ribosomal subunits acts to coordinate protein folding and mRNA decoding. In Chapter Three, results of experiments conducted to elucidate the function of Ssz1 are described. Crosslinking results indicated that Ssz1 interacts with Ssb. To understand the mode of interaction, unbiased computational docking was conducted. These docking results suggested that Ssz1 and Ssb interact via their ATPase domains. I verified this mode of interaction by in vitro crosslinking and mass spectrometry analysis. Residues forming salt bridges mediate the interaction in the docking model. Reverse charge substitutions of these residues in Ssb or Ssz1 destabilized the interaction in vitro and impaired cell growth. The presence of both variants complemented the growth defects caused by a single variant, suggesting that interaction between Ssz1 and Ssb is important in vivo. I hypothesize that Ssz1 positions Ssb on the ribosome, facilitating its interaction with nascent polypeptide.