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A splicing mutation in the novel mitochondrial protein DNAJC11 causes motor neuron pathology associated with cristae disorganization, and lymphoid abnormalities in mice.

Ioakeimidis F, Ott C, Kozjak-Pavlovic V, Violitzi F, Rinotas V, Makrinou E, Eliopoulos E, Fasseas C, Kollias G, Douni E - PLoS ONE (2014)

Bottom Line: The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog.The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization.Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Agricultural University of Athens, Athens, Greece; Division of Immunology, Biomedical Sciences Research Center "Alexander Fleming", Vari, Greece.

ABSTRACT
Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.

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Mitochondrial and submitochondrial localization of DNAJC11.(A) Representative Western blot analysis on total protein RIPA extracts (T), on cytosolic (C) and mitochondrial (M) fractions of cerebrum and liver tissue from WT mice. The huDnaJC11 transgenic (Tg) samples served as positive controls. Prohibitin served as a mitochondrial specific marker and GAPDH as a cytoplasmic marker. (B) Fluorescence microscopy of HeLa cells transfected with a C-terminally FLAG-tagged huDNAJC11 cDNA of the 63 kDa isoform (green channel) and stained with the mitochondrial specific dye MitoTracker Orange (red channel). Scalebar, 100 µm. (C) Proteinase K protection assay on isolated mitochondria from HeLa cells. 50 µg of mitochondria were either subjected to swelling in the hypotonic buffer (SW, +) or were incubated in the isotonic buffer (SW, -) and then were treated with proteinase K (PK, +) or not (PK, -). A buffer containing 1% TritonX-100 (Tr) was used to solubilize all mitochondrial proteins and render them accessible to proteinase K. Samples were then analyzed by SDS-PAGE and Western blot with the indicated antibodies. Metaxin 1 is an outer membrane protein, mitofilin is an intermembrane space exposed inner membrane protein and Hsp60 is matrix localized. Numbers indicate molecular mass of protein marker in kDa. (D) Sodium carbonate extraction of isolated mitochondria from HeLa cells. Mitochondria were extracted under the two indicated pH conditions and the membranes were collected by ultracentrifugation. Pellet (P) and supernatant (S) were analyzed by SDS-PAGE and Western blot and probed with the indicated antibodies. Tim23 is an integral inner membrane protein and ICDH is a soluble matrix protein. Arrows indicate the 63 kDa isoform and the putative ∼57 kDa isoform. Tim23, Translocase of inner mitochondrial membrane 23 homolog; ICDH, isocitrate dehydrogenase.
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pone-0104237-g006: Mitochondrial and submitochondrial localization of DNAJC11.(A) Representative Western blot analysis on total protein RIPA extracts (T), on cytosolic (C) and mitochondrial (M) fractions of cerebrum and liver tissue from WT mice. The huDnaJC11 transgenic (Tg) samples served as positive controls. Prohibitin served as a mitochondrial specific marker and GAPDH as a cytoplasmic marker. (B) Fluorescence microscopy of HeLa cells transfected with a C-terminally FLAG-tagged huDNAJC11 cDNA of the 63 kDa isoform (green channel) and stained with the mitochondrial specific dye MitoTracker Orange (red channel). Scalebar, 100 µm. (C) Proteinase K protection assay on isolated mitochondria from HeLa cells. 50 µg of mitochondria were either subjected to swelling in the hypotonic buffer (SW, +) or were incubated in the isotonic buffer (SW, -) and then were treated with proteinase K (PK, +) or not (PK, -). A buffer containing 1% TritonX-100 (Tr) was used to solubilize all mitochondrial proteins and render them accessible to proteinase K. Samples were then analyzed by SDS-PAGE and Western blot with the indicated antibodies. Metaxin 1 is an outer membrane protein, mitofilin is an intermembrane space exposed inner membrane protein and Hsp60 is matrix localized. Numbers indicate molecular mass of protein marker in kDa. (D) Sodium carbonate extraction of isolated mitochondria from HeLa cells. Mitochondria were extracted under the two indicated pH conditions and the membranes were collected by ultracentrifugation. Pellet (P) and supernatant (S) were analyzed by SDS-PAGE and Western blot and probed with the indicated antibodies. Tim23 is an integral inner membrane protein and ICDH is a soluble matrix protein. Arrows indicate the 63 kDa isoform and the putative ∼57 kDa isoform. Tim23, Translocase of inner mitochondrial membrane 23 homolog; ICDH, isocitrate dehydrogenase.

Mentions: In order to examine the subcellular localization of muDNAJC11, cerebrum from WT mice was processed to obtain either whole protein extract or cytosolic and mitochondrial fractions that were then subjected to SDS-PAGE and Western blot analysis, using an antibody against human and mouse DNAJC11. Our results showed that DNAJC11 migrated between the range of 60 and 70 kDa as expected (Figure 6A). Specificity of the antibody was also confirmed by increased levels of DNAJC11 in the TghuDnaJC11 mice (Figure 6A). No other isoforms could be verified in this way. DNAJC11 band was present in the mitochondrial but not in the cytosolic fraction and was hardly visible in the whole protein extract (Figure 6A). Similar results were obtained in liver protein extracts.


A splicing mutation in the novel mitochondrial protein DNAJC11 causes motor neuron pathology associated with cristae disorganization, and lymphoid abnormalities in mice.

Ioakeimidis F, Ott C, Kozjak-Pavlovic V, Violitzi F, Rinotas V, Makrinou E, Eliopoulos E, Fasseas C, Kollias G, Douni E - PLoS ONE (2014)

Mitochondrial and submitochondrial localization of DNAJC11.(A) Representative Western blot analysis on total protein RIPA extracts (T), on cytosolic (C) and mitochondrial (M) fractions of cerebrum and liver tissue from WT mice. The huDnaJC11 transgenic (Tg) samples served as positive controls. Prohibitin served as a mitochondrial specific marker and GAPDH as a cytoplasmic marker. (B) Fluorescence microscopy of HeLa cells transfected with a C-terminally FLAG-tagged huDNAJC11 cDNA of the 63 kDa isoform (green channel) and stained with the mitochondrial specific dye MitoTracker Orange (red channel). Scalebar, 100 µm. (C) Proteinase K protection assay on isolated mitochondria from HeLa cells. 50 µg of mitochondria were either subjected to swelling in the hypotonic buffer (SW, +) or were incubated in the isotonic buffer (SW, -) and then were treated with proteinase K (PK, +) or not (PK, -). A buffer containing 1% TritonX-100 (Tr) was used to solubilize all mitochondrial proteins and render them accessible to proteinase K. Samples were then analyzed by SDS-PAGE and Western blot with the indicated antibodies. Metaxin 1 is an outer membrane protein, mitofilin is an intermembrane space exposed inner membrane protein and Hsp60 is matrix localized. Numbers indicate molecular mass of protein marker in kDa. (D) Sodium carbonate extraction of isolated mitochondria from HeLa cells. Mitochondria were extracted under the two indicated pH conditions and the membranes were collected by ultracentrifugation. Pellet (P) and supernatant (S) were analyzed by SDS-PAGE and Western blot and probed with the indicated antibodies. Tim23 is an integral inner membrane protein and ICDH is a soluble matrix protein. Arrows indicate the 63 kDa isoform and the putative ∼57 kDa isoform. Tim23, Translocase of inner mitochondrial membrane 23 homolog; ICDH, isocitrate dehydrogenase.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128653&req=5

pone-0104237-g006: Mitochondrial and submitochondrial localization of DNAJC11.(A) Representative Western blot analysis on total protein RIPA extracts (T), on cytosolic (C) and mitochondrial (M) fractions of cerebrum and liver tissue from WT mice. The huDnaJC11 transgenic (Tg) samples served as positive controls. Prohibitin served as a mitochondrial specific marker and GAPDH as a cytoplasmic marker. (B) Fluorescence microscopy of HeLa cells transfected with a C-terminally FLAG-tagged huDNAJC11 cDNA of the 63 kDa isoform (green channel) and stained with the mitochondrial specific dye MitoTracker Orange (red channel). Scalebar, 100 µm. (C) Proteinase K protection assay on isolated mitochondria from HeLa cells. 50 µg of mitochondria were either subjected to swelling in the hypotonic buffer (SW, +) or were incubated in the isotonic buffer (SW, -) and then were treated with proteinase K (PK, +) or not (PK, -). A buffer containing 1% TritonX-100 (Tr) was used to solubilize all mitochondrial proteins and render them accessible to proteinase K. Samples were then analyzed by SDS-PAGE and Western blot with the indicated antibodies. Metaxin 1 is an outer membrane protein, mitofilin is an intermembrane space exposed inner membrane protein and Hsp60 is matrix localized. Numbers indicate molecular mass of protein marker in kDa. (D) Sodium carbonate extraction of isolated mitochondria from HeLa cells. Mitochondria were extracted under the two indicated pH conditions and the membranes were collected by ultracentrifugation. Pellet (P) and supernatant (S) were analyzed by SDS-PAGE and Western blot and probed with the indicated antibodies. Tim23 is an integral inner membrane protein and ICDH is a soluble matrix protein. Arrows indicate the 63 kDa isoform and the putative ∼57 kDa isoform. Tim23, Translocase of inner mitochondrial membrane 23 homolog; ICDH, isocitrate dehydrogenase.
Mentions: In order to examine the subcellular localization of muDNAJC11, cerebrum from WT mice was processed to obtain either whole protein extract or cytosolic and mitochondrial fractions that were then subjected to SDS-PAGE and Western blot analysis, using an antibody against human and mouse DNAJC11. Our results showed that DNAJC11 migrated between the range of 60 and 70 kDa as expected (Figure 6A). Specificity of the antibody was also confirmed by increased levels of DNAJC11 in the TghuDnaJC11 mice (Figure 6A). No other isoforms could be verified in this way. DNAJC11 band was present in the mitochondrial but not in the cytosolic fraction and was hardly visible in the whole protein extract (Figure 6A). Similar results were obtained in liver protein extracts.

Bottom Line: The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog.The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization.Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Agricultural University of Athens, Athens, Greece; Division of Immunology, Biomedical Sciences Research Center "Alexander Fleming", Vari, Greece.

ABSTRACT
Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.

Show MeSH
Related in: MedlinePlus