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Nucleoid localization of Hsp40 Mdj1 is important for its function in maintenance of mitochondrial DNA.

Ciesielski GL, Plotka M, Manicki M, Schilke BA, Dutkiewicz R, Sahi C, Marszalek J, Craig EA - Biochim. Biophys. Acta (2013)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Gdansk, Gdansk, Poland.

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CTD1/2 domains of Mdj1 are critical for maintenance of mtDNA. A) Scheme of domain composition of Mdj1 deletion variants as in Fig. 3A. Internal deletion of Mdj1ΔC is indicated by dotted line. B) mdj1-Δ [TETr-MDJ1] expressing Mdj1 (wt), Mdj1ΔC (∆C) or Mdj1ΔD (∆D) under control of the MDJ1 promoter was grown in the presence of doxycycline. At the indicated number of generations after doxycycline addition, aliquots were collected and plated on glucose-based media. The percentage of respiring cells was taken as the ratio of the number of red colored versus the total number of colonies. C) Mitochondrial lysates were prepared from cells harvested 10 generations after doxycycline addition and subjected to immunoblot analysis using antibodies specific for Mdj1 and, as loading control, porin. As a quantity reference 0.2 pmol of purified wt Mdj1 (wt), Mdj1ΔC (∆C) or Mdj1ΔD (∆D), was run on the same gel (protein), as the polyclonal Mdj1 antibody does not recognize Mdj1, Mdj1ΔC and Mdj1ΔD with the same efficiency. Slower migration of the purified ΔC and ΔD variants is due to the presence of 6 His tag at the C-terminus. Red star indicates unspecific bands. D) Distribution of Mdj1 variants after centrifugation of mitochondrial lysates. Mitochondrial lysates isolated from MDJ1/mdj1-Δ diploid cells expressing wt Mdj1 (wt) or the variants, Mdj1ΔC (∆C) or Mdj1ΔD (∆D) were subjected to ultracentrifugation through a sucrose gradient. Each fraction was analyzed for protein content using immunoblot analysis with antibodies specific for Mdj1 or, as a control, Abf2. E) Cellular localization of Mdj1 variants. Cells expressing GFP fused to Mdj1ΔC (∆C) or Mdj1ΔD (∆D) were analyzed by fluorescence microscopy. Cellular DNA was stained with DAPI. Overlay of the two images (MERGE). Size bars (2 μm) are shown.
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f0020: CTD1/2 domains of Mdj1 are critical for maintenance of mtDNA. A) Scheme of domain composition of Mdj1 deletion variants as in Fig. 3A. Internal deletion of Mdj1ΔC is indicated by dotted line. B) mdj1-Δ [TETr-MDJ1] expressing Mdj1 (wt), Mdj1ΔC (∆C) or Mdj1ΔD (∆D) under control of the MDJ1 promoter was grown in the presence of doxycycline. At the indicated number of generations after doxycycline addition, aliquots were collected and plated on glucose-based media. The percentage of respiring cells was taken as the ratio of the number of red colored versus the total number of colonies. C) Mitochondrial lysates were prepared from cells harvested 10 generations after doxycycline addition and subjected to immunoblot analysis using antibodies specific for Mdj1 and, as loading control, porin. As a quantity reference 0.2 pmol of purified wt Mdj1 (wt), Mdj1ΔC (∆C) or Mdj1ΔD (∆D), was run on the same gel (protein), as the polyclonal Mdj1 antibody does not recognize Mdj1, Mdj1ΔC and Mdj1ΔD with the same efficiency. Slower migration of the purified ΔC and ΔD variants is due to the presence of 6 His tag at the C-terminus. Red star indicates unspecific bands. D) Distribution of Mdj1 variants after centrifugation of mitochondrial lysates. Mitochondrial lysates isolated from MDJ1/mdj1-Δ diploid cells expressing wt Mdj1 (wt) or the variants, Mdj1ΔC (∆C) or Mdj1ΔD (∆D) were subjected to ultracentrifugation through a sucrose gradient. Each fraction was analyzed for protein content using immunoblot analysis with antibodies specific for Mdj1 or, as a control, Abf2. E) Cellular localization of Mdj1 variants. Cells expressing GFP fused to Mdj1ΔC (∆C) or Mdj1ΔD (∆D) were analyzed by fluorescence microscopy. Cellular DNA was stained with DAPI. Overlay of the two images (MERGE). Size bars (2 μm) are shown.

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.


Nucleoid localization of Hsp40 Mdj1 is important for its function in maintenance of mitochondrial DNA.

Ciesielski GL, Plotka M, Manicki M, Schilke BA, Dutkiewicz R, Sahi C, Marszalek J, Craig EA - Biochim. Biophys. Acta (2013)

CTD1/2 domains of Mdj1 are critical for maintenance of mtDNA. A) Scheme of domain composition of Mdj1 deletion variants as in Fig. 3A. Internal deletion of Mdj1ΔC is indicated by dotted line. B) mdj1-Δ [TETr-MDJ1] expressing Mdj1 (wt), Mdj1ΔC (∆C) or Mdj1ΔD (∆D) under control of the MDJ1 promoter was grown in the presence of doxycycline. At the indicated number of generations after doxycycline addition, aliquots were collected and plated on glucose-based media. The percentage of respiring cells was taken as the ratio of the number of red colored versus the total number of colonies. C) Mitochondrial lysates were prepared from cells harvested 10 generations after doxycycline addition and subjected to immunoblot analysis using antibodies specific for Mdj1 and, as loading control, porin. As a quantity reference 0.2 pmol of purified wt Mdj1 (wt), Mdj1ΔC (∆C) or Mdj1ΔD (∆D), was run on the same gel (protein), as the polyclonal Mdj1 antibody does not recognize Mdj1, Mdj1ΔC and Mdj1ΔD with the same efficiency. Slower migration of the purified ΔC and ΔD variants is due to the presence of 6 His tag at the C-terminus. Red star indicates unspecific bands. D) Distribution of Mdj1 variants after centrifugation of mitochondrial lysates. Mitochondrial lysates isolated from MDJ1/mdj1-Δ diploid cells expressing wt Mdj1 (wt) or the variants, Mdj1ΔC (∆C) or Mdj1ΔD (∆D) were subjected to ultracentrifugation through a sucrose gradient. Each fraction was analyzed for protein content using immunoblot analysis with antibodies specific for Mdj1 or, as a control, Abf2. E) Cellular localization of Mdj1 variants. Cells expressing GFP fused to Mdj1ΔC (∆C) or Mdj1ΔD (∆D) were analyzed by fluorescence microscopy. Cellular DNA was stained with DAPI. Overlay of the two images (MERGE). Size bars (2 μm) are shown.
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f0020: CTD1/2 domains of Mdj1 are critical for maintenance of mtDNA. A) Scheme of domain composition of Mdj1 deletion variants as in Fig. 3A. Internal deletion of Mdj1ΔC is indicated by dotted line. B) mdj1-Δ [TETr-MDJ1] expressing Mdj1 (wt), Mdj1ΔC (∆C) or Mdj1ΔD (∆D) under control of the MDJ1 promoter was grown in the presence of doxycycline. At the indicated number of generations after doxycycline addition, aliquots were collected and plated on glucose-based media. The percentage of respiring cells was taken as the ratio of the number of red colored versus the total number of colonies. C) Mitochondrial lysates were prepared from cells harvested 10 generations after doxycycline addition and subjected to immunoblot analysis using antibodies specific for Mdj1 and, as loading control, porin. As a quantity reference 0.2 pmol of purified wt Mdj1 (wt), Mdj1ΔC (∆C) or Mdj1ΔD (∆D), was run on the same gel (protein), as the polyclonal Mdj1 antibody does not recognize Mdj1, Mdj1ΔC and Mdj1ΔD with the same efficiency. Slower migration of the purified ΔC and ΔD variants is due to the presence of 6 His tag at the C-terminus. Red star indicates unspecific bands. D) Distribution of Mdj1 variants after centrifugation of mitochondrial lysates. Mitochondrial lysates isolated from MDJ1/mdj1-Δ diploid cells expressing wt Mdj1 (wt) or the variants, Mdj1ΔC (∆C) or Mdj1ΔD (∆D) were subjected to ultracentrifugation through a sucrose gradient. Each fraction was analyzed for protein content using immunoblot analysis with antibodies specific for Mdj1 or, as a control, Abf2. E) Cellular localization of Mdj1 variants. Cells expressing GFP fused to Mdj1ΔC (∆C) or Mdj1ΔD (∆D) were analyzed by fluorescence microscopy. Cellular DNA was stained with DAPI. Overlay of the two images (MERGE). Size bars (2 μm) are shown.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Gdansk, Gdansk, Poland.

Show MeSH
Related in: MedlinePlus