Abstract
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.
Highlights
J-domain proteins (JDPs) are obligate co-chaperones of Hsp70-based molecular chaperone systems [1, 2]
Consistent with previously published data [11, 12], SANT domains are present in Zuotin from animal and protists, but not fungal, species (Fig 1B)
Most of the fungal 4-helix bundle (4HB) segment align with animal and protist sequences, two disparities were noted: (i) the plug sequence does not align with the rest of the 4HB; (ii) in a monophyletic subgroup of Basidiomycota species, the 4HB region is truncated, with only 9–16 residues present (Fig 1B, S1 Fig)
Summary
J-domain proteins (JDPs) are obligate co-chaperones of Hsp70-based molecular chaperone systems [1, 2]. Such chaperone machineries are ubiquitous, functioning in diverse biological processes [3,4,5]. Zuotin is the JDP of the eukaryotic ribosome-associated Hsp system, which facilitates folding of nascent polypeptide chains [6, 7]. Plants and protists, but not fungi, Zuotin orthologs have a C-terminal, ~200 residue, segment containing SANT domains [11, 12]—domains typically involved in protein-protein interactions [13]. Changes that occurred in Zuotin upon SANT domain loss is one focus of this report
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