Wiley et al. 10.1073/pnas.0701078104. |
Fig. 6. MitoNEET mRNA and protein are widely expressed. (A) RT-PCR analysis of mitoNEET from mouse tissues. (B) Anti-mitoNEET IHC staining in mouse tissues. Shown are preimmune serum (PI) (image 1) and anti-mitoNEET staining (images 2-6). Cells staining red are positive for endogenous mitoNEET. EC, Erythrocytes.
Fig. 7. Protein expression levels in wild-type and mitoNEET-null mice. Protein (50 mg) from cardiac tissue homogenates and purified mitochondria from mitoNEET (+/+) and (−/−) mice were separated by SDS/PAGE and analyzed by immunoblotting (IB) with antibodies to mitochondrial proteins. Antibodies to NDUFB6, complex III FeS protein, and complex II 30-kDa protein were from MitoSciences (Eugene, OR). Calreticulin was used to assess mitochondrial purity (Calbiochem, La Jolla, CA).
SI Materials and Methods
Construction of Bacterial Expression Plasmids.
The portion of the human mitoNEET cDNA corresponding to amino acids 27-108 was amplified by PCR and cloned into the pET28b-MBP-His/TEV vector (a gift from P. Ghosh, University of California at San Diego, La Jolla). The resulting protein has an MBP tag, two 6X His tags, and a TEV protease cleavage site, all amino-terminal to the mitoNEET sequence. In addition, the same portion of human mitoNEET (amino acids 27-108) was also PCR-amplified and cloned into the pGEX-6P-2 vector (GE Healthcare Bio-Sciences, Piscataway, NJ) to generate a GST-fusion protein.Expression and Purification of MitoNEET Fusion Proteins in Escherichia coli.
Expression of MBP-His-mitoNEET27-108 in BL21-CodonPlus-RIL cells (Stratagene, La Jolla, CA) was induced with 0.4 mM isopropyl-1-thio-d-galactopyranoside at 37°C for 3.5 h. The bacteria were grown in 2XYT that was supplemented with 1 mM FeCl3 1 h before induction with isopropyl-1-thio-d-galactopyranoside. Fusion proteins were purified using nickel-agarose (Qiagen, Valencia, CA) according to the manufacturer's protocols. Purified fusion proteins were dialyzed against 50 mM Tris, pH 8.0, 14 mM b-mercaptoethanol, and 50 mM NaCl, and stored at 4° C until metal analysis.Expression of GST-mitoNEET27-108 protein in BL21-CodonPlus-RIL cells (Stratagene) was induced with 0.4 mM isopropyl-1-thio-d-galactopyranoside for 18 h at room temperature. Recombinant protein was purified using glutathione-coupled agarose and eluted with reduced glutathione following the manufacturer's protocols (Sigma, St. Louis, MO).
Cell Culture and Immunocytochemistry.
COS-7 cells were grown under standard conditions (American Type Culture Collection, Manassas, VA). Cells transiently expressing mitoNEET family fusion proteins were plated on glass coverslips, loaded with MitoTracker Red CMXRos (Invitrogen, San Diego, CA) following the manufacturer's protocol, fixed, and immunostained (for V5-tagged proteins) as described (1). Cells were visualized with a ´100 objective using a Leica (Nussloch, Germany) DMR microscope with an attached Hamamatsu (Hamamatsu City, Japan) camera. Images were pseudocolored and overlays compiled with Openlab imaging software (Improvision, Lexington, MA).Preparation of mitoNEET Antisera.
Purified GST-mitoNEET27-108 protein (see SI Methods for expression details) was dialyzed against PBS with 1 mM DTT and used to generate a rabbit polyclonal antibody (Cocalico Biologicals, Reamstown, PA). To remove the undesired anti-GST reactive antibodies, the serum was initially passed through an agarose column bound to purified GST protein. This was followed by protein A affinity purification of the IgG population from the flow-through using the Montage kit (Millipore, Bedford, MA). The resulting antibody was tested for the ability to specifically immunostain mitoNEET protein.Purification and Subfractionation of Rat Liver Mitochondria.
Mitochondria were histodenz purified from fresh rat livers as described previously (2). Mitochondrial subfractions [outer membranes (OMM), mitoplasts, and intermembrane space fractions] were obtained using a swell-shrink procedure and purified by sucrose gradient ultracentrifugation (2). Submitochondrial particles and the soluble fraction were generated by sonication and ultracentrifugation (2). Proteins (14 mg) from each fraction were separated by SDS/PAGE and immunoblotted with antibodies to mitoNEET and to mitochondrial marker proteins of known localization.SDS/PAGE and Immunoblotting.
Proteins were separated by SDS/PAGE using NOVEX 12% precast gels (Invitrogen), transferred to PVDF membranes, and analyzed by immunoblotting. Antibodies recognizing the following proteins were used for mitochondrial markers: voltage-dependent anion channel and second mitochondrial activator of caspases (EMD Biosciences, San Diego, CA); mitoNEET (gift from J. Colca, Pfizer, St. Louis, MO); protein tyrosine phosphatase localized to the mitochondrion 1 (2); cytochrome c and BCL2 (BD PharMingen, San Diego, CA); and mitochondrial heat shock protein of 70 kDa (Stressgen Bioreagents, Victoria, Canada).1. Copp J, Wiley S, Ward MW, van der Geer P (2005) Am J Physiol 288:C403-C415.
2. Pagliarini DJ, Wiley SE, Kimple ME, Dixon JR, Kelly P, Worby CA, Casey PJ, Dixon JE (2005) Mol Cell 19:197-207.