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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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Related in: MedlinePlus

Mdj1 depletion results in loss of functional mtDNA. Doxycycline was added to (Dox +) or, as a control, omitted from a culture (Dox −) of mdj1-Δ [TETr-MDJ1] cells. At the indicated generations, aliquots of cells were collected and subject to one of three types of analysis: A) plated on either glycerol (upper panel)- or glucose (lower panel)-based media, B) pelleted prior to preparation of cellular extracts, which were subjected to immunoblot analysis or D) used to start subcultures for preparation of mtDNA, which was subjected to restriction endonuclease testing. A) Representative plates from samples harvested at the indicated generations are shown. Red colony color on glucose-based media is an indicator of respiratory competence; white colony color is an indicator of respiratory incompetence. B) Immunoblot analysis of extracts prepared from cells at the indicated number of generations after doxycycline addition, using antibodies specific for Mdj1 and, as a loading control, porin. Also included is an extract of wt cells (wt), as a control to indicate the level of expression of Mdj1. C) Plot of the data obtained. Left Y axis (open circles): the relative amount of Mdj1 obtained by quantitative analysis of results shown in (B), setting the level present prior to the addition of doxycycline at 100%. Right Y axis (closed squares): percentage of cells able to respire, calculated as the ratio of red colonies to total number of colonies (red and white) on glucose containing media. X axis: number of generations of growth in the presence of doxycycline. D) One red colony from a control culture, and three white colonies from a doxycycline treated culture from a 20 generation time point were picked and used to start liquid cultures in glucose-based media. After 12 h at 30 °C total cellular DNA was isolated from each culture. mtDNA was prepared and subjected to digestion with EcoRV, separated by agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light. MW, molecular weight DNA standard Mass Ruler™ DNA Ladder (Fermentas).
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f0010: Mdj1 depletion results in loss of functional mtDNA. Doxycycline was added to (Dox +) or, as a control, omitted from a culture (Dox −) of mdj1-Δ [TETr-MDJ1] cells. At the indicated generations, aliquots of cells were collected and subject to one of three types of analysis: A) plated on either glycerol (upper panel)- or glucose (lower panel)-based media, B) pelleted prior to preparation of cellular extracts, which were subjected to immunoblot analysis or D) used to start subcultures for preparation of mtDNA, which was subjected to restriction endonuclease testing. A) Representative plates from samples harvested at the indicated generations are shown. Red colony color on glucose-based media is an indicator of respiratory competence; white colony color is an indicator of respiratory incompetence. B) Immunoblot analysis of extracts prepared from cells at the indicated number of generations after doxycycline addition, using antibodies specific for Mdj1 and, as a loading control, porin. Also included is an extract of wt cells (wt), as a control to indicate the level of expression of Mdj1. C) Plot of the data obtained. Left Y axis (open circles): the relative amount of Mdj1 obtained by quantitative analysis of results shown in (B), setting the level present prior to the addition of doxycycline at 100%. Right Y axis (closed squares): percentage of cells able to respire, calculated as the ratio of red colonies to total number of colonies (red and white) on glucose containing media. X axis: number of generations of growth in the presence of doxycycline. D) One red colony from a control culture, and three white colonies from a doxycycline treated culture from a 20 generation time point were picked and used to start liquid cultures in glucose-based media. After 12 h at 30 °C total cellular DNA was isolated from each culture. mtDNA was prepared and subjected to digestion with EcoRV, separated by agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light. MW, molecular weight DNA standard Mass Ruler™ DNA Ladder (Fermentas).

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).


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)

Mdj1 depletion results in loss of functional mtDNA. Doxycycline was added to (Dox +) or, as a control, omitted from a culture (Dox −) of mdj1-Δ [TETr-MDJ1] cells. At the indicated generations, aliquots of cells were collected and subject to one of three types of analysis: A) plated on either glycerol (upper panel)- or glucose (lower panel)-based media, B) pelleted prior to preparation of cellular extracts, which were subjected to immunoblot analysis or D) used to start subcultures for preparation of mtDNA, which was subjected to restriction endonuclease testing. A) Representative plates from samples harvested at the indicated generations are shown. Red colony color on glucose-based media is an indicator of respiratory competence; white colony color is an indicator of respiratory incompetence. B) Immunoblot analysis of extracts prepared from cells at the indicated number of generations after doxycycline addition, using antibodies specific for Mdj1 and, as a loading control, porin. Also included is an extract of wt cells (wt), as a control to indicate the level of expression of Mdj1. C) Plot of the data obtained. Left Y axis (open circles): the relative amount of Mdj1 obtained by quantitative analysis of results shown in (B), setting the level present prior to the addition of doxycycline at 100%. Right Y axis (closed squares): percentage of cells able to respire, calculated as the ratio of red colonies to total number of colonies (red and white) on glucose containing media. X axis: number of generations of growth in the presence of doxycycline. D) One red colony from a control culture, and three white colonies from a doxycycline treated culture from a 20 generation time point were picked and used to start liquid cultures in glucose-based media. After 12 h at 30 °C total cellular DNA was isolated from each culture. mtDNA was prepared and subjected to digestion with EcoRV, separated by agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light. MW, molecular weight DNA standard Mass Ruler™ DNA Ladder (Fermentas).
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f0010: Mdj1 depletion results in loss of functional mtDNA. Doxycycline was added to (Dox +) or, as a control, omitted from a culture (Dox −) of mdj1-Δ [TETr-MDJ1] cells. At the indicated generations, aliquots of cells were collected and subject to one of three types of analysis: A) plated on either glycerol (upper panel)- or glucose (lower panel)-based media, B) pelleted prior to preparation of cellular extracts, which were subjected to immunoblot analysis or D) used to start subcultures for preparation of mtDNA, which was subjected to restriction endonuclease testing. A) Representative plates from samples harvested at the indicated generations are shown. Red colony color on glucose-based media is an indicator of respiratory competence; white colony color is an indicator of respiratory incompetence. B) Immunoblot analysis of extracts prepared from cells at the indicated number of generations after doxycycline addition, using antibodies specific for Mdj1 and, as a loading control, porin. Also included is an extract of wt cells (wt), as a control to indicate the level of expression of Mdj1. C) Plot of the data obtained. Left Y axis (open circles): the relative amount of Mdj1 obtained by quantitative analysis of results shown in (B), setting the level present prior to the addition of doxycycline at 100%. Right Y axis (closed squares): percentage of cells able to respire, calculated as the ratio of red colonies to total number of colonies (red and white) on glucose containing media. X axis: number of generations of growth in the presence of doxycycline. D) One red colony from a control culture, and three white colonies from a doxycycline treated culture from a 20 generation time point were picked and used to start liquid cultures in glucose-based media. After 12 h at 30 °C total cellular DNA was isolated from each culture. mtDNA was prepared and subjected to digestion with EcoRV, separated by agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light. MW, molecular weight DNA standard Mass Ruler™ DNA Ladder (Fermentas).
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).

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