Nucleoid localization of Hsp40 Mdj1 is important for its function in maintenance of mitochondrial DNA.
Bottom Line: Underscoring the importance of Hsp70 chaperone activity in the maintenance of mtDNA, an Mdj1 variant having an alteration in the Hsp70-interacting J-domain does not maintain mtDNA.We found that Mdj1 has DNA binding activity and that variants retaining DNA-binding activity also retained nucleoid association.Together, our results are consistent with a model in which Mdj1, tethered to the nucleoid via DNA binding, thus driving a high local concentration of the Hsp70 machinery, is important for faithful DNA maintenance and propagation.
Affiliation: Department of Molecular and Cellular Biology, University of Gdansk, Gdansk, Poland.Show MeSH
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Mentions: The strict requirement of Mdj1 for mtDNA maintenance [12–14], coupled with its substantial nucleoid association, prompted us to begin a more thorough analysis of Mdj1 function in mtDNA maintenance. In the past, the rigid requirement of Mdj1 has been a detriment to analyze Mdj1's role. Therefore, we developed a strain to allow monitoring mtDNA function as Mdj1 is being depleted. The strain we constructed, mdj1-Δ [TETr-MDJ1], has MDJ1 under the control of the TETr promoter, which allows repression of Mdj1 synthesis upon addition of the drug doxycycline [27,30]. Two methods were used to assess respiratory competence, an indicator of functional mtDNA: (i) the ability of cells to form colonies on media having a nonfermentable carbon source such as glycerol and (ii) colony color on glucose-based media, as respiratory competent cells having a mutation in the ADE2 gene form red colonies, while respiratory incompetent cells form small white, so-called “petite”, colonies (Fig. 2A).
Affiliation: Department of Molecular and Cellular Biology, University of Gdansk, Gdansk, Poland.