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: Two features within the region encompassing CTD1 and CTD2 have been defined structurally and functionally, the zinc finger-like region (ZFLR) and the peptide binding cleft (Fig. 5A) [20–22]. To assess their importance in maintenance of mtDNA, we constructed two types of MDJ1 mutations: (i) a complete deletion of the ZFLR (Mdj1∆Z) and (ii) alterations of hydrophobic amino acids in the peptide-binding cleft (Mdj1LFI/AAA) (Fig. 5A). The respiratory competence of Mdj1∆Z and Mdj1LFI/AAA cells remained constant after depletion of wt Mdj1 (Fig. 5B), indicating that neither the zinc finger-like region nor the peptide cleft is critical for maintenance of mtDNA. Both sucrose gradient analysis of mitochondrial extracts and microscopic evaluation indicated that these variants were nucleoid-associated (Fig. 5C, D). We also tested the growth phenotype of a strain having mdj1∆Z or mdj1LFI/AAA as the only copy of the MDJ1 gene (Fig. 5E). Both mdj1∆Z and mdj1LFI/AAA grew on glycerol based medium, indicating mtDNA function. However, they grew more slowly at 37 °C on rich glucose-based media, with mdj1LFI/AAA barely able to form colonies (Fig. 5E). This temperature sensitive growth is consistent with client protein binding being needed for growth at borderline temperatures, perhaps in general protein folding. However, our data provides no evidence that either of the two well-defined features of the region encompassing CTD1 and CTD2 is necessary for mtDNA maintenance.
Affiliation: Department of Molecular and Cellular Biology, University of Gdansk, Gdansk, Poland.