The J-domain protein Zuotin is a multi-domain eukaryotic Hsp70 co-chaperone. Though it is primarily ribosome-associated, positioned at the exit of the 60S subunit tunnel where it promotes folding of nascent polypeptide chains, Zuotin also has off-ribosome functions. Domains of Zuotin needed for 60S association and interaction with Hsp70 are conserved in eukaryotes. However, whether the 4-helix bundle (4HB) domain is conserved remains an open question. We undertook evolutionary and structural approaches to clarify this issue. We found that the 4HB segment of human Zuotin also forms a bundle of 4 helices. The positive charge of Helix I, which in Saccharomyces cerevisiae is responsible for interaction with the 40S subunit, is particularly conserved. However, the C-termini of fungal and human 4HBs are not similar. In fungi the C-terminal segment forms a plug that folds back into the bundle; in S. cerevisiae it plays an important role in bundle stability and, off the ribosome, in transcriptional activation. In human, C-terminal helix IV of the 4HB is extended, protruding from the bundle. This extension serves as a linker to the regulatory SANT domains, which are present in animals, plants and protists, but not fungi. Further analysis of Zuotin sequences revealed that the plug likely arose as a result of genomic rearrangement upon SANT domain loss early in the fungal lineage. In the lineage leading to S. cerevisiae, the 4HB was subjected to positive selection with the plug becoming increasingly hydrophobic. Eventually, these hydrophobic plug residues were coopted for a novel regulatory function-activation of a recently emerged transcription factor, PDR1. Our data suggests that Zuotin evolved off-ribosome functions twice-once involving SANT domains, then later in fungi, after SANT domain loss, by coopting the hydrophobic plug. Zuotin serves as an example of complex intertwining of molecular chaperone function and cell regulation.

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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.

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Shrestha OK, Sharma R, Tomiczek B, Lee W, Tonelli M, Cornilescu G, Stolarska M, Nierzwicki L, Czub J, Markley JL, ... Show all

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