Unfolding of the C-Terminal Domain of the J-Protein Zuo1 Releases Autoinhibition and Activates Pdr1-Dependent Transcription

Abstract

The C-terminal 69 residues of the J-protein Zuo1 are sufficient to activate Pdr1, a transcription factor involved in both pleiotropic drug resistance (PDR) and growth control. Little is understood about the pathway of activation by this primarily ribosome-associated Hsp40 co-chaperone. Here we report that only the C-terminal 13 residues of Zuo1 are required for activation of Pdr1, with hydrophobic residues being critical for activity. Two-hybrid interaction experiments suggest that the interaction between this 13-residue Zuo1 peptide and Pdr1 is direct, analogous to the activation of Pdr1 by xenobiotics. However, simply dissociation of Zuo1 from the ribosome is not sufficient for induction of Pdr1 transcriptional activity, as the C-terminal 86 residues of Zuo1 fold into an autoinhibitory left-handed four-helix bundle. Hydrophobic residues critical for interaction with Pdr1 are sequestered within the structure of this C-terminal domain (CTD), necessitating unfolding for activation. Thus, although expression of the CTD does not result in activation, alterations that destabilize the structure cause induction of PDR. These destabilizing alterations also result in dissociation of the full-length protein from the ribosome. Thus, our results are consistent with an activation pathway in which unfolding of Zuo1’s C-terminal helical bundle domain results in ribosome dissociation followed by activation of Pdr1 via a direct interaction.

Dual interaction of the Hsp70 J-protein cochaperone Zuotin with the 40S and 60S ribosomal subunits

“…It is also interesting to note that Zuo1 and its orthologs have diverse regulatory functions off the ribosome, consistent with evidence that its interaction with the ribosome is marginally stable and likely dynamic 9,27 . For example, S. cerevisiae Zuo1 activates Pdr1, a transcription factor implicated in quorum sensing 27,49 .…”

“…For example, S. cerevisiae Zuo1 activates Pdr1, a transcription factor implicated in quorum sensing 27,49 . Human DnaJC2 functions as a chromatin modulator, affecting development and DNA repair 50,51 .…”

“…1a). Based on its position on the 40S subunit, the C-terminal 4-helix bundle (residues 348–433) 25,27 of Zuo1 has been proposed to interact with expansion segment 12 (ES12), an extension of helix 44 (H44) of 18S rRNA 26 . A long internal alpha helix, called the middle domain (MD), is proposed to span the subunits.…”

The Expanding Landscape of Moonlighting Proteins in Yeasts

“…Zuotin has also been found to be associated with ribosomes by centrifugation through sucrose density gradients (58) and to coimmunoprecipitate with Pdr13 (59). Pdr13, also called Ssz1, is a chaperone that enhances the activity of the transcription factor Pdr1, which is involved in pleiotropic drug resistance (PDR) (60,61). These observations suggest that Zuo1 may play different functional roles, and this is supported by the fact that the protein is found in different cellular compartments and that diverse zuo1 mutants do not present the same phenotype, as described below.…”

How Do J-Proteins Get Hsp70 to Do So Many Different Things?

“…4A), discussed above, has been linked to cellular regulatory pathways associated with growth control in yeast and metazoans. In S. cerevisiae , the 13 residues at the C terminus are sufficient for activation of the Pdr1 transcription factor, which has been linked to quorum sensing [62, 63]. In metazoans, Zuotin (Fig.…”