Sequential Duplications of an Ancient Member of the DnaJ-Family Expanded the Functional Chaperone Network in the Eukaryotic Cytosol

Abstract

Across eukaryotes, Hsp70-based chaperone machineries display an underlying unity in their sequence, structure, and biochemical mechanism of action, while working in a myriad of cellular processes. In good part, this extraordinary functional versatility is derived from the ability of a single Hsp70 to interact with an array of J-protein cochaperones to form a functional chaperone network. Among J-proteins, the DnaJ-type is the most prevalent, being present in all three kingdoms and in several different compartments of eukaryotic cells. However, because these ancient DnaJ-type proteins diverged at the base of the eukaryotic phylogeny, little is understood about the evolutionary basis of their diversification and thus the functional expansion of the chaperone network. Here, we report results of evolutionary and experimental analyses of two more recent members of the cytosolic DnaJ family of Saccharomyces cerevisiae, Xdj1 and Apj1, which emerged by sequential duplications of the ancient YDJ1 in Ascomycota. Sequence comparison and molecular modeling revealed that both Xdj1 and Apj1 maintained a domain organization similar to that of multifunctional Ydj1. However, despite these similarities, both Xdj1 and Apj1 evolved highly specialized functions. Xdj1 plays a unique role in the translocation of proteins from the cytosol into mitochondria. Apj1's specialized role is related to degradation of sumolyated proteins. Together these data provide the first clear example of cochaperone duplicates that evolved specialized functions, allowing expansion of the chaperone functional network, while maintaining the overall structural organization of their parental gene.

Roles of Intramolecular and Intermolecular Interactions in Functional Regulation of the Hsp70 J-protein Co-Chaperone Sis1

“…Within 45 min 100% of luciferase was reactivated with Hsp70, but only 19% with Hsp70 ΔEEVD . To test whether Ydj1 was unique in being able to function efficiently with Hsp70 ΔEEVD we also tested Xdj1, a paralog of Ydj1 arising from a gene duplication during the fungal lineage [20]. Xdj1 was also able to promote luciferase refolding with Hsp70 ΔEEVD .…”

“…Ssa1 ΔEEVD and variant J protein plasmids were constructed using the QuikChange Site-Directed mutagenesis kit from Stratagene (La Jolla, CA). Hsp104 (pMal His Tev Hsp104) was purified in a manner similar to that described for Sis1 and Sse1 (Smt3 (SUMO)-His 6 fusion protein) was purified in a manner similar to that described for Apj1 in Sahi et al [20]. …”

“…Single-turnover ATPase assays of Ssa1 were performed as described in Sahi et al [20]. 32 P-α-ATP-Ssa1 complexes were incubated with a 2-fold molar excess of J proteins at 24°C.…”

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

“…Ydj1 is also required for Ste6* turnover but only when a second Hsp40, Hlj1, is also deleted (16). Redundancy for Hsp40 co-chaperones is common, making it challenging to determine all the co-chaperones required for turnover of a particular substrate (54,55).…”

Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1

“…Besides Djp1, two more J proteins of (Makanae et al 2013). While toxicity of Xdj1 which has been attributed to mitochondrial import defects requires a functional J domain (Sahi et al 2013), the molecular basis of Caj1 toxicity is not understood. Moderate over expression of Djp1 resulted into growth defects at 30°C (Fig.…”