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: To begin to narrow down the region of Mdj1 important for nucleoid localization and mtDNA maintenance, we first tested two deletion variants: (i) Mdj1∆C, lacking the C-terminal domains, CTD1and CTD2, but maintaining the putative dimerization domain and (ii) Mdj1∆D, lacking the C-terminal 81 amino acids of this dimerization domain (Fig. 4A). Both were expressed at levels similar to normal Mdj1 levels (Fig. 4C). Cells expressing the ΔC allele lost respiratory competence with kinetics similar to that observed for Mdj1H89Q (compare Fig. 4B with Fig. 3B, Suppl. Fig. S4). Cells expressing Mdj1∆D also showed a decrease in respiratory competence. However, this loss was significantly slower than that of cells expressing either Mdj1H89Q or Mdj1∆C, as 70% of the cells were competent 20 generations after the addition of drug (Fig. 4B, Suppl. Fig. S4). This observation is consistent with the putative dimerization domain, being important, but not critical for Mdj1's role in the maintenance of mtDNA. Next, we tested the nucleoid association of these two variants. Mdj1∆D, but not Mdj1∆C, was associated with the nucleoid, as judged both by sucrose gradient centrifugation and microscopic observations of GFP fusions (Fig. 4D, E). Thus, the presence of CTD1 and CTD2 domains, but not the dimerization domain, is critical for both maintenance of functional mtDNA and mitochondrial nucleoid association.
Affiliation: Department of Molecular and Cellular Biology, University of Gdansk, Gdansk, Poland.