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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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Mdj1 localizes to mitochondrial nucleoid. (A,B) In vivo localization of Mdj1. Wild-type (wt) cells expressing Mdj1-GFP, Abf2-GFP or mt-GFP were analyzed by fluorescence microscopy. Images in the two right-most panels were overlaid (MERGE). Size bars (2 μm) are shown. (A) Cellular DNA was stained using DAPI. (B) Mitochondrial network was stained using MitoTracker. (C) Distribution of Mdj1 after centrifugation of mitochondrial lysates. Lysates prepared from isolated mitochondria were subjected to ultracentrifugation through a sucrose gradient. A portion of each fraction was analyzed for mtDNA content by PCR amplification of mtDNA ori5 fragment (mtDNA) and for protein content by immunoblot analysis using antibodies specific for Mdj1, Abf2, Ssc1 and Jac1.
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f0005: Mdj1 localizes to mitochondrial nucleoid. (A,B) In vivo localization of Mdj1. Wild-type (wt) cells expressing Mdj1-GFP, Abf2-GFP or mt-GFP were analyzed by fluorescence microscopy. Images in the two right-most panels were overlaid (MERGE). Size bars (2 μm) are shown. (A) Cellular DNA was stained using DAPI. (B) Mitochondrial network was stained using MitoTracker. (C) Distribution of Mdj1 after centrifugation of mitochondrial lysates. Lysates prepared from isolated mitochondria were subjected to ultracentrifugation through a sucrose gradient. A portion of each fraction was analyzed for mtDNA content by PCR amplification of mtDNA ori5 fragment (mtDNA) and for protein content by immunoblot analysis using antibodies specific for Mdj1, Abf2, Ssc1 and Jac1.

Mentions: Considering that Mdj1 is necessary for maintenance of functional mtDNA and that the human Mdj1 ortholog (DnajA3/Tid1) has been detected associated with the mitochondrial nucleoid complex [9], we decided to determine the localization of Mdj1 within the yeast mitochondrial matrix. We used two approaches, one microscopic, visualizing Mdj1 in living cells and, the second, biochemical, fractionating mitochondrial extracts by sucrose gradient centrifugation. To visualize Mdj1 we constructed a chimeric gene to allow expression of a fusion between Mdj1 and green fluorescent protein (GFP). Mdj1-GFP supported maintenance of mtDNA and rescued the growth defect of mdj1-Δ cells, demonstrating the functionality of the fusion protein (Suppl. Fig. S1). As positive and negative controls, we tested a GFP fusion of Abf2, a major core packaging protein of the mitochondrial nucleoid, and GFP itself, targeted to the mitochondrial matrix by the presence of a fused N-terminal cleavable pre-sequence (mt-GFP), respectively (Fig. 1A). As expected [1], Abf2-GFP was concentrated in distinct, dot-like structures, which co-localized with DAPI stained mtDNA. On the other hand, mt-GFP showed a diffuse pattern throughout mitochondrial tubules [36]. Mdj1-GFP fluorescence showed a very similar pattern to that of Abf2-GFP, concentrated in distinct, dot-like structures, which co-localized with DAPI stained mtDNA (Fig. 1A, B).


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 localizes to mitochondrial nucleoid. (A,B) In vivo localization of Mdj1. Wild-type (wt) cells expressing Mdj1-GFP, Abf2-GFP or mt-GFP were analyzed by fluorescence microscopy. Images in the two right-most panels were overlaid (MERGE). Size bars (2 μm) are shown. (A) Cellular DNA was stained using DAPI. (B) Mitochondrial network was stained using MitoTracker. (C) Distribution of Mdj1 after centrifugation of mitochondrial lysates. Lysates prepared from isolated mitochondria were subjected to ultracentrifugation through a sucrose gradient. A portion of each fraction was analyzed for mtDNA content by PCR amplification of mtDNA ori5 fragment (mtDNA) and for protein content by immunoblot analysis using antibodies specific for Mdj1, Abf2, Ssc1 and Jac1.
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f0005: Mdj1 localizes to mitochondrial nucleoid. (A,B) In vivo localization of Mdj1. Wild-type (wt) cells expressing Mdj1-GFP, Abf2-GFP or mt-GFP were analyzed by fluorescence microscopy. Images in the two right-most panels were overlaid (MERGE). Size bars (2 μm) are shown. (A) Cellular DNA was stained using DAPI. (B) Mitochondrial network was stained using MitoTracker. (C) Distribution of Mdj1 after centrifugation of mitochondrial lysates. Lysates prepared from isolated mitochondria were subjected to ultracentrifugation through a sucrose gradient. A portion of each fraction was analyzed for mtDNA content by PCR amplification of mtDNA ori5 fragment (mtDNA) and for protein content by immunoblot analysis using antibodies specific for Mdj1, Abf2, Ssc1 and Jac1.
Mentions: Considering that Mdj1 is necessary for maintenance of functional mtDNA and that the human Mdj1 ortholog (DnajA3/Tid1) has been detected associated with the mitochondrial nucleoid complex [9], we decided to determine the localization of Mdj1 within the yeast mitochondrial matrix. We used two approaches, one microscopic, visualizing Mdj1 in living cells and, the second, biochemical, fractionating mitochondrial extracts by sucrose gradient centrifugation. To visualize Mdj1 we constructed a chimeric gene to allow expression of a fusion between Mdj1 and green fluorescent protein (GFP). Mdj1-GFP supported maintenance of mtDNA and rescued the growth defect of mdj1-Δ cells, demonstrating the functionality of the fusion protein (Suppl. Fig. S1). As positive and negative controls, we tested a GFP fusion of Abf2, a major core packaging protein of the mitochondrial nucleoid, and GFP itself, targeted to the mitochondrial matrix by the presence of a fused N-terminal cleavable pre-sequence (mt-GFP), respectively (Fig. 1A). As expected [1], Abf2-GFP was concentrated in distinct, dot-like structures, which co-localized with DAPI stained mtDNA. On the other hand, mt-GFP showed a diffuse pattern throughout mitochondrial tubules [36]. Mdj1-GFP fluorescence showed a very similar pattern to that of Abf2-GFP, concentrated in distinct, dot-like structures, which co-localized with DAPI stained mtDNA (Fig. 1A, B).

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