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First published online 1 November 2005
doi: 10.1242/jcs.02645

Journal of Cell Science 118, 5357-5367 (2005)
Published by The Company of Biologists 2005
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MIDAS/GPP34, a nuclear gene product, regulates total mitochondrial mass in response to mitochondrial dysfunction

Naomi Nakashima-Kamimura1, Sadamitsu Asoh1, Yoshitomo Ishibashi1, Yuri Mukai1,*, Yujiro Shidara1,2, Hideaki Oda2, Kae Munakata3, Yu-ichi Goto3 and Shigeo Ohta1,{ddagger}

1 Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Kawasaki, Kanagawa, 211-8533, Japan
2 Department of Pathology, Tokyo Women's Medical University, School of Medicine, Shinjuku-ku, Tokyo, 162-8666, Japan
3 Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, 187-8502, Japan



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  		Fig. 1. Enhanced expression of MIDAS/GPP34 in EB8 (mtDNA-free HeLa cells). (A) Comparison of mRNA obtained from Ft2-11 with that from EB8 by differential display. EB8 lacks mtDNA whereas Ft2-11 is derived from EB8 but has wild-type mitochondria. Ten sets of arbitrarily primed PCR products were subjected to 5% PAGE. Three bands indicated by arrows were cloned and sequenced. Bands 1, 2 and 3 corresponded to apurinic/apyrimidinic endonuclease I, DNA ligase III and MIDAS/GPP34, respectively. (B) Northern blots of total RNA extracted from Ft2-11 and EB8 were hybridized with GPP34- and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)-specific probes. (C) GPP34 mRNA levels normalized to GAPDH levels based on the mean values±s.d. for three sets of northern blotting experiments (vertical bars). (D) Western blotting of Ft2-11 and EB8. Whole-cell lysates were separated by 12% SDS-PAGE and transferred onto a PVDF membrane. MIDAS/GPP34 was immunostained with anti-MIDAS antibody. (E) The MIDAS/GPP34 protein normalized against actin. Mean values of three sets of experiments are shown with s.d. (vertical bars).

 


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  		Fig. 2. Expression of MIDAS/GPP34 in SDH++/COX– muscle cells of patients with mitochondrial diseases. Biopsy samples were obtained from the biceps brachii muscle. Activities of SDH and COX were visualized histochemically and the expression of MIDAS/GPP34 was detected with anti-MIDAS antibody. (A-C) Control muscle without mitochondrial disorder. (D-F) Muscle from a patient with CPEO who has a common deletion in mtDNA. (G-I) Muscle from a patient with MELAS who has a point mutation at nucleotide number 3243 in the tRNALeu(UUR) gene. Asterisks indicate SDH++/COX– cells of patients with increased MIDAS/GPP34 expression. Bars, 50 µm.

 


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  		Fig. 3. Localization of MIDAS/GPP34 in mitochondria and the Golgi apparatus. (A-C) HeLa cells in culture were fixed, permeabilized with 5% acetic acid in ethanol and then immunostained with various antibodies. (A) MitoTracker Red (100 nM) was added as a mitochondrial indicator prior to fixation. MIDAS/GPP34 was detected with anti-MIDAS antibody. Arrowhead indicates where MIDAS/GPP34 is more abundant than mitochondria in a perinuclear area. (B) MIDAS/GPP34 and p230 (trans-Golgi) were double-stained immunochemically using different secondary antibodies. Arrowheads indicate where the perinuclear area was co-stained with both antibodies. (C) Hsc70 (cytosol), Tom20 (outer membrane of mitochondria), cytochrome c (cyt. c) (intermembrane space of mitochondria) and p230 (trans-Golgi) were detected with their respective antibodies. (D-F) HeLa cells in culture were fixed with 4% paraformaldehyde and 4% sucrose without treatment for permeabilization and immunostained with the same procedure as in (A-C). (G,H) Localization of Myc-tagged MIDAS. A Myc tag was fused to the N-terminus (G) or C-terminus (H) of MIDAS. Fusion constructs were transfected into HeLa cells and cells were allowed to express protein for 16 hours. Cells were stained with anti-Myc, anti-p230 antibodies or MitoTracker Red with the same procedure as in (A-C). Bars, 20 µm.

 


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  		Fig. 4. Localization of MIDAS/GPP34 in the mitochondrial intermembrane space. (A) Fractionation of organelles of HeLa cells in 7-35% (w/v) preformed density gradients. The distribution of MIDAS was detected by western blotting. Syntaxin6 was used for a Golgi marker. Tom20 and Hsp60 were used for mitochondrial markers. (B) Mitoplasts were obtained from the mitochondrial fraction (fraction 15 in A) by osmotic disruption of the outer membrane. The mitochondria (M; lanes 1-3) and the mitoplasts (MP; lanes 4-6) were treated with proteinase K (PK; 10 µg/ml or 50 µg/ml). Mitochondrial sub-fractions were monitored by western blotting with antibodies directed against Tom20 (an outer membrane protein; most of which is exposed outside mitochondria), Tom40 (an outer membrane protein), cytochrome c (cyt c) (an intermembrane space protein), Cox4 (an inner membrane protein) and Hsp60 (a matrix space protein).

 


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  		Fig. 5. The increased or decreased mass of intact mitochondria related to MIDAS concentration. (A) CMV-CTL7 and Si-CTL1 are stable control transfectants derived from HeLa cells. Although CMV-MIDAS3 and CMV-MIDAS9 are stable MIDAS transfectants under the control of the CMV promoter. Si-MIDAS5 and Si-MIDAS11 were transfectants expressing siRNA of MIDAS constitutively. Total cell lysate was extracted from each transfectant cell line and subjected to western blotting with anti-MIDAS and anti-actin antibodies. (B) Control (CMV-CTL7 and Si-CTL1), MIDAS transfectants (CMV-MIDAS3) and siRNA MIDAS transfectants (Si-MIDAS11) were stained with MitoTracker Red and visualized by confocal scanning laser microscopy. (C) Electron micrographs (x8000) of mitochondria in the transfectants. Bar, 20 µm (B); 1 µm (C).

 


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  		Fig. 6. Changes in mitochondrial mass with MIDAS. (A) Flow cytometric profiles of the fluorescence of dyes specific to mitochondria in CMV-CTL7 (white) and CMV-MIDAS3 (gray). Living transfectants were stained with MitoTracker Green (left) or MitoTracker Red (right) and analyzed with a flow cytometer. (B) Flow cytometric analysis performed with Si-CTL1 (white) and Si-MIDAS11 (gray) cells as described in A. (C) Fluorescence intensity (MitoTracker Green and MitoTracker Red) as well as forward scatter (FS) of HeLa cells and transfectants as quantified by flow cytometry. Values are the mean±s.d. (D) The mitochondria and nucleus in control transfectants (CMV-CTL7 and Si-CTL1), MIDAS transfectants (CMV-MIDAS3) and siRNA MIDAS transfectants (Si-MIDAS11) were stained with MitoTracker Red and SYTO 16 (green), respectively and scanned by confocal laser microscopy in each 0.4 µm section. Then the side view of a three-dimensional image was reconstructed with Fluoview software. (E) Areas of the nucleus and mitochondria were measured for each section. (F) The total mass of mitochondria in the transfectants was calculated based on the values in E and normalized to that of nucleus. Data represent the mean±s.d. of three sets of experiments. Bars, 20 µm.

 


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  		Fig. 7. Effects of MIDAS on amounts of cardiolipin (a mitochondria-specific lipid), mitochondrial DNA, RNA and proteins. (A) Total lipids were extracted from control transfectants (CMV-CTL7 and Si-CTL1), MIDAS transfectants (CMV-MIDAS3) and siRNA MIDAS transfectants (Si-MIDAS11) and fractionated by HPLC as described in the Materials and Methods. The elution of phospholipids was monitored at 205 nm. CL, cardiolipin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine. (B) The amount of cardiolipin in the transfectants was quantified based on the values in A and normalized to that of phosphatidylcholine. Mean values for three sets of experiments are shown with the s.d. (C) The amount of DNA in HeLa cells and transfectants was analyzed by Southern blotting. To detect mtDNA we used the COX II region in mtDNA as a probe. The nDNA (nuclear DNA) was a loading control and the 18S ribosomal DNA region was used as a probe. The expression of mtDNA and nDNA was analyzed by northern blotting. Blots of total RNA extracted from HeLa and transfectants were hybridized with COX II- and GAPDH -specific probes. Proteins were analyzed by western blotting. SDH30 and SDH70 are the nucleus-encoded 30 kDa and 70 kDa subunit of SDH, respectively. COX I and COX II are the mitochondrial DNA-encoded subunits.

 

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© The Company of Biologists Ltd 2005