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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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Neither deletion of zinc finger like region nor alteration of residues in substrate-binding cleft affects maintenance of mtDNA. A) (Top) Scheme of Mdj1ΔZ with dotted line indicating the deletion of the zinc finger like region (for details see Fig. 3A). (Bottom) Structural model of C module with ZFLR, CTD1 and CTD2 indicated and with residues of the substrate binding cleft red — Ile202, blue — Leu222, yellow — Phe224, green — Ile301, orange — Ile343, magenta — Val345. Arrows indicate residues, which were altered in this study. B) mdj1-Δ [TETr-MDJ1] expressing Mdj1 (wt), Mdj1ΔZ, Mdj1LFI/AAA under the 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 determined as the ratio of the number of red colored versus the total number of colonies on plates. C) Distribution of Mdj1 variants after centrifugation of mitochondrial lysates. Mitochondrial lysates isolated from MDJ1/mdj1-Δ diploid cells expressing Mdj1 (wt) or the Mdj1 variants, Mdj1ΔZ, or Mdj1LFI/AAA 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. Mdj1LFI/AAA GFP fusions were used to allow separation from wt Mdj1 during electrophoresis. D) Cellular localization of Mdj1 variants. Cells expressing GFP fusions of Mdj1ΔZ (∆Z-GFP) or Mdj1LFI/AAA (LFI/AAA-GFP) were analyzed by fluorescence microscopy. Cellular DNA was stained with DAPI. Overlay of the two images (MERGE). Size bars (2 μm) are shown. E) mdj1-Δ cells harboring plasmid-borne copies of wt MDJ1, ΔZ or LFI/AAA variants, as indicated, were plated as 10-fold dilutions on glucose-rich medium (top) or glycerol-rich medium (bottom) and incubated at indicated temperatures for 3 days (glucose) or 4 days (glycerol).
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f0025: Neither deletion of zinc finger like region nor alteration of residues in substrate-binding cleft affects maintenance of mtDNA. A) (Top) Scheme of Mdj1ΔZ with dotted line indicating the deletion of the zinc finger like region (for details see Fig. 3A). (Bottom) Structural model of C module with ZFLR, CTD1 and CTD2 indicated and with residues of the substrate binding cleft red — Ile202, blue — Leu222, yellow — Phe224, green — Ile301, orange — Ile343, magenta — Val345. Arrows indicate residues, which were altered in this study. B) mdj1-Δ [TETr-MDJ1] expressing Mdj1 (wt), Mdj1ΔZ, Mdj1LFI/AAA under the 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 determined as the ratio of the number of red colored versus the total number of colonies on plates. C) Distribution of Mdj1 variants after centrifugation of mitochondrial lysates. Mitochondrial lysates isolated from MDJ1/mdj1-Δ diploid cells expressing Mdj1 (wt) or the Mdj1 variants, Mdj1ΔZ, or Mdj1LFI/AAA 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. Mdj1LFI/AAA GFP fusions were used to allow separation from wt Mdj1 during electrophoresis. D) Cellular localization of Mdj1 variants. Cells expressing GFP fusions of Mdj1ΔZ (∆Z-GFP) or Mdj1LFI/AAA (LFI/AAA-GFP) were analyzed by fluorescence microscopy. Cellular DNA was stained with DAPI. Overlay of the two images (MERGE). Size bars (2 μm) are shown. E) mdj1-Δ cells harboring plasmid-borne copies of wt MDJ1, ΔZ or LFI/AAA variants, as indicated, were plated as 10-fold dilutions on glucose-rich medium (top) or glycerol-rich medium (bottom) and incubated at indicated temperatures for 3 days (glucose) or 4 days (glycerol).

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.


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)

Neither deletion of zinc finger like region nor alteration of residues in substrate-binding cleft affects maintenance of mtDNA. A) (Top) Scheme of Mdj1ΔZ with dotted line indicating the deletion of the zinc finger like region (for details see Fig. 3A). (Bottom) Structural model of C module with ZFLR, CTD1 and CTD2 indicated and with residues of the substrate binding cleft red — Ile202, blue — Leu222, yellow — Phe224, green — Ile301, orange — Ile343, magenta — Val345. Arrows indicate residues, which were altered in this study. B) mdj1-Δ [TETr-MDJ1] expressing Mdj1 (wt), Mdj1ΔZ, Mdj1LFI/AAA under the 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 determined as the ratio of the number of red colored versus the total number of colonies on plates. C) Distribution of Mdj1 variants after centrifugation of mitochondrial lysates. Mitochondrial lysates isolated from MDJ1/mdj1-Δ diploid cells expressing Mdj1 (wt) or the Mdj1 variants, Mdj1ΔZ, or Mdj1LFI/AAA 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. Mdj1LFI/AAA GFP fusions were used to allow separation from wt Mdj1 during electrophoresis. D) Cellular localization of Mdj1 variants. Cells expressing GFP fusions of Mdj1ΔZ (∆Z-GFP) or Mdj1LFI/AAA (LFI/AAA-GFP) were analyzed by fluorescence microscopy. Cellular DNA was stained with DAPI. Overlay of the two images (MERGE). Size bars (2 μm) are shown. E) mdj1-Δ cells harboring plasmid-borne copies of wt MDJ1, ΔZ or LFI/AAA variants, as indicated, were plated as 10-fold dilutions on glucose-rich medium (top) or glycerol-rich medium (bottom) and incubated at indicated temperatures for 3 days (glucose) or 4 days (glycerol).
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f0025: Neither deletion of zinc finger like region nor alteration of residues in substrate-binding cleft affects maintenance of mtDNA. A) (Top) Scheme of Mdj1ΔZ with dotted line indicating the deletion of the zinc finger like region (for details see Fig. 3A). (Bottom) Structural model of C module with ZFLR, CTD1 and CTD2 indicated and with residues of the substrate binding cleft red — Ile202, blue — Leu222, yellow — Phe224, green — Ile301, orange — Ile343, magenta — Val345. Arrows indicate residues, which were altered in this study. B) mdj1-Δ [TETr-MDJ1] expressing Mdj1 (wt), Mdj1ΔZ, Mdj1LFI/AAA under the 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 determined as the ratio of the number of red colored versus the total number of colonies on plates. C) Distribution of Mdj1 variants after centrifugation of mitochondrial lysates. Mitochondrial lysates isolated from MDJ1/mdj1-Δ diploid cells expressing Mdj1 (wt) or the Mdj1 variants, Mdj1ΔZ, or Mdj1LFI/AAA 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. Mdj1LFI/AAA GFP fusions were used to allow separation from wt Mdj1 during electrophoresis. D) Cellular localization of Mdj1 variants. Cells expressing GFP fusions of Mdj1ΔZ (∆Z-GFP) or Mdj1LFI/AAA (LFI/AAA-GFP) were analyzed by fluorescence microscopy. Cellular DNA was stained with DAPI. Overlay of the two images (MERGE). Size bars (2 μm) are shown. E) mdj1-Δ cells harboring plasmid-borne copies of wt MDJ1, ΔZ or LFI/AAA variants, as indicated, were plated as 10-fold dilutions on glucose-rich medium (top) or glycerol-rich medium (bottom) and incubated at indicated temperatures for 3 days (glucose) or 4 days (glycerol).
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.

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