JCS02645 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1.  Schematic diagram for the primary structures of MIDAS/GPP34 and GPP34R.  Amino acid residues conserved among five species (human, mouse, D. melanogaster, C. elegans and S. cerevisiae) are indicated by black lines, whereas those found in only four or three of the five species are indicated by dark gray or light gray lines, respectively. Human and mouse MIDAS/GPP34s have an isoform named GPP34R.  Positions of amino acid residues that were identical between MIDAS/GPP34 and GPP34R are indicated by black dots.  MIDAS/GPP34 and GPP34R have a common acidic region (indicated with blue dots) near the N-terminus whereas a leucine-zipper motif (indicated with red dots) was found only in MIDAS/GPP34.

• Supplemental Figure 2 -

  Fig. S2.  Expression of MIDAS/GPP34 and its isoform GPP34R in HeLa derivative cells. (A) Northern blotting for MIDAS/GPP34 and GPP34R in EB8 (the mtDNA-less HeLa cell) and Ft2-11 (EB8 with wild-type mtDNA). Total RNA (1, 2 or 4 mg) was hybridized with PCR products specific to MIDAS/GPP34 or GPP34R.  The primers used were 5¢-AAGCAGCGCCTCATCAAGA-3¢ and 5¢-TGTCCATGCGGTGAGGGT-3¢ to amplify MIDAS/GPP34; and 5¢-CCAAACATGGATAGAGCTACTCACTG-3¢ and 5¢-TTCTTTGCGATGCGCTCTC-3¢ were used for GPP34R. F and E indicate cell lines Ft2-11 and EB8, respectively. (B) Semi-quantification of mRNA of MIDAS/GPP34 and GPP34R isolated from HeLa cells by the Taq Man probe method. Poly(A⁺) RNA isolated from HeLa cells was reverse transcribed to cDNA.  To estimate starting amount of cDNA, increases in the fluorescence intensity from the TaqMan probe were monitored at each PCR cycle with an automatic sequence detection system (Prism 7700 system; Applied Biosystems) as described in Nomiyama et al., 2002. Starting amounts of MIDAS/GPP34 (red circles) and GPP34R (green circles) were semi-quantified as assessed by the threshold cycle, where the PCR products reach a fixed amount. A plasmid containing the MIDAS/GPP34 cDNA (black circles) and the GPP34R (gray circles) was used as a standard sample. Three independent results were indicated by dots in a chart (red circles, MIDAS/GPP34 and green circles, GPP34R). The relative value of GPP34R to MIDAS/GPP34 was estimated as 1/55. The primers used were 5¢-AAGCAGCGCCTCATCAAGA-3¢ and 5¢-TGTCCATGCGGTGAGGGT-3¢ for amplifying MIDAS/GPP34; and 5¢-CCAAACATGGATAGAGCTACTCACTG-3¢ and 5¢-TTCTTTGCGATGCGCTCTC-3¢ for amplifying GPP34R.  The nucleotide sequences of the TaqMan probes for MIDAS/GPP34 and GPP34R were 5¢-AGTACAGGAAGCCGTTCTTGACAAATGGG-3¢ and 5¢-TGGAACCCCTTCAAATTACAGTACCAGCTGA-3¢, respectively. There were no detectable PCR fragments from a cross-reaction between MIDAS/GPP34 and GPP34R.

• Supplemental Figure 3 -

  Fig. S3.  The effect of an acetate/ethanol pretreatment in cultured cells on the detection of mitochondrial proteins. SDH70 (succinate dehydrogenase the 70 subunit) and Hsp60 (Heat shock protein 60) are located in the inner membrane and matrix, respectively. HeLa cells in culture were fixed with 4% paraformaldehyde and pretreated with 5% acetic acid in ethanol (acetate/ethanol treatment +) for permeabilization pretreatment, or cells were fixed with 4% paraformaldehyde and 4% sucrose with no acetate/ethanol pretreatment (acetate/ethanol treatment –), then immunostained with antibody against SDH70 (Molecular Probes) or Hsp60 (purchased from MBL). The mitochondrial proteins were detected only when the membranes were leaky with the acetate/ethanol treatment. Bar, 20 mm.

• Supplemental Figure 4 -

  Fig. S4.  Images of mitochondria in transfectants overexpressing MIDAS or suppressing the MIDAS expression.  Control- (CMV-CTL7 and Si-CTL1), MIDAS-transfectants (CMV-MIDAS3 and CMV-MIDAS9) and siRNA MIDAS-transfectants (Si-MIDAS5 and Si-MIDAS11) were stained with 10 nM of a membrane-potential-dependent fluorescent dye TMRM (tetra-methyl-rhodamine methyl ester; purchased from Molecular Probes) specific to mitochondria for 10 minutes at 37°C and visualized by confocal laser-scanning microscopy. The intensity of fluorescence depended upon the expression of MIDAS. Bar, 100 mm.

• Supplemental Figure 5 -

  Fig. S5.  The effect of MIDAS on the amount of mitochondria-specific phospholipids.  Concentration of cardiolipin was analyzed with a fluorometer using NAO (nonyl acridine orange; purchased from Molecular Probes), a dye that binds to cardiolipin, as previously described (Oubrahim et al., 2001). In brief, the cells were incubated with 1 mM NAO for 30 minutes and then lysed with SDS and the fluorescence intensity of NAO was measured with a Spectra Fluor (TECAN), with normalization for total protein concentration. Control (CMV-CTL7 and Si-CTL1), MIDAS transfectants (CMV-MIDAS3 and CMV-MIDAS9) and siRNA MIDAS transfectants (Si-MIDAS5 and Si-MIDAS11) were used. Mean values of three sets of experiments are shown with s.d. (vertical bars). The amounts of cardiolipin increased depending upon the expression of MIDAS.

• Supplemental Figure 6 -

  Fig. S6.  There was no influence of overexpression of MIDAS on cytoskeletal structure.  Control (CMV-CTL7) and MIDAS transfectants (CMV-MIDAS3) were co-stained with anti-b-tubulin (purchased from Sigma) and anti-MIDAS antibodies and visualized by confocal laser-scanning microscopy. Mitochondria gathered around the nucleus in transfectants expressing MIDAS and were independent of the cytoskeletal structure. Bar, 20 mm.