Disruption of the murine GRIM-19 gene. (A) Schematic presentation of wild-type and targeted GRIM-19 alleles and the gene targeting construct. Four exons are indicated with closed boxes. Arrowhead P1 indicates the primer specific for the wild-type allele, P2 indicates the primer for the GRIM-19⁻ allele, and P3 indicates the primer for both the wild-type and GRIM-19⁻ alleles. Probes 1 and 2 are used for Southern blot analysis. Neo, neomycin resistance cassette; TK, thymidine kinase marker. (B) Southern blot analysis of targeted ES cells. Genomic DNA from individual ES clones were digested with BamHI or KpnI and analyzed by Southern blotting with probe 1 (left panel) or probe 2 (right panel). The targeted clones are shown with the wild-type (12-kb) and mutant (8.8-kb) fragments as marked. (C) PCR genotyping of offspring from intercrossing two GRIM-19^+/− mice. A 370-bp wild-type band and a 491-bp mutant band were amplified by primer pairs P1-P3 and P2-P3, respectively, as indicated in the legend of panel A.

In vitro outgrowth of blastocysts. Intercrossed embryos at E3.5 were collected (day 0) and cultured for 4 days. The blastocysts were photographed at different objective magnifications as indicated. The bottom row of panel B shows semithin sections of the blastocysts stained with toluidine blue. TB, TG, and ICM are indicated by arrows. Scale bar = 50 μm.

Abnormal morphology and cellular distribution of mitochondria in the mutant blastocysts. Ultrathin sections of the blastocysts were examined with a transmission electron microscope. The mitochondria of the normal (A and C) and the mutant (B and D) cells are indicated by arrows. Scale bar = 1 μm.

Mitochondrial complex I assembly and activity assays. (A) Detection of GRIM-19 in complex I by BN-PAGE. Mitochondria from mouse liver were isolated, and 400 μg (lanes 1 and 3) and 600 μg (lanes 2 and 4) of mitochondrial proteins were solubilized and centrifuged. Aliquots of supernatants containing different mitochondrial respiratory complexes were separated by BN-PAGE as described in Materials and Methods and detected by Western blot analysis. Complexes I to V were detected by using mouse monoclonal antibodies against specific subunits of each complex that include the anti-OxPhos complex I 39-kDa subunit, anti-OxPhos complex II 70-kDa subunit, anti-OxPhos complex III FeS subunit, anti-OxPhos complex IV subunit I, and anti-OxPhos complex V inhibitor protein. GRIM-19 was detected in complex I with anti-GRIM-19 antibody (lanes 3 and 4). The positions of the complexes and GRIM-19 (G-19) are indicated by arrows. (B) GRIM-19 is essential for mitochondrial complex I assembly. Blastocysts were isolated and cultured in vitro for 7 days. Twenty-seven blastocysts containing the wild type and GRIM-19^+/− and 27 blastocysts of GRIM-19^−/− were combined and directly solubilized. A total of 20 μg of solubilized proteins was used to analyze the complex assembly as described in panel A. (C) GRIM-19 is essential for mitochondrial complex I activity. Samples from blastocysts were prepared as described in panel B. Solubilized protein (20 μg) was separated by BN-PAGE. In-gel colorimetric reactions for oxidative phosphorylation of complex I and complex II were performed as described in Materials and Methods. (D) Effect of GRIM-19 on the expression of subunits in the other complexes. Cell lysates from wild-type or GRIM-19^−/− blastocysts were separated by SDS-PAGE and subjected to Western blot analysis with antibodies as indicated.

Histological analysis of embryos of wild-type, heterozygous, and homozygous mutants. Wild-type (+/+), GRIM-19^+/−, and GRIM-19^−/− embryos were dissected at 5 μm at E5.5, E6.5, E7.5, and E8.5 as indicated. The sections were stained with hematoxylin and eosin. The objective magnifications are indicated. Scale bar = 100 μm.

Proliferation and apoptosis of the blastocysts. (A) BrdU incorporation during blastocyst outgrowth. E3.5 blastocysts from GRIM-19^+/− intercrossed mice were isolated and cultured for various days. The cells were labeled with BrdU as described in Materials and Methods. TOPRO3 indicates nuclear staining. (B) TUNEL assay was performed as described in Materials and Methods. Control represents the normal blastocysts digested with DNase I prior to the TUNEL assay. The objective magnification is ×10. After assays, the genotypes of the blastocysts were determined by PCR, and representative results from one set of experiments are shown at the bottom of each panel. Scale bar = 50 μm.

Cellular localization of GRIM-19. (A) Mitochondrial localization of GRIM-19 in various cell lines. Various cell types were grown in culture and treated with MitoTracker Red. After fixation, GRIM-19 protein expression and localization were detected by using a specific GRIM-19 mouse primary antibody and a fluorescein isothiocyanate-coupled anti-mouse secondary antibody (top frames). The cells were mounted and examined by using a Bio-Rad MRC 1024 laser scanning confocal microscope. Mitochondria were detected by MitoTracker Red (middle frames). Merged images are shown in the bottom frames. Scale bar = 10 μm. (B) COS-1 (C) and PC12 (P) cells were lysed and fractionated to cytoplasmic (Cyto), mitochondrial (Mito), and nuclear (Nuc) portions as described in Materials and Methods. The expression of GRIM-19 in total cell lysates and different fractions (containing 50 μg of proteins in each) were examined with Western blotting with antibody against GRIM-19. The blots were also probed with anti-poly(ADP-ribose) polymerase as a nuclear marker and anti-COX IV as a mitochondrial marker. (C) Submitochondrial localization of GRIM-19. Mitochondria were isolated from 293T cells and treated with 100 ng of proteinase K per ml for various times in the absence or the presence of 1% Triton X-100. The mitochondrial proteins were separated by SDS-PAGE and subjected to Western blot analysis with antibodies against BCL2, GRIM-19, heat shock protein 60 (HSP 60), and COX IV.