Figure 5.

Assembly of mtDNA-Encoded Subunits in D2 Mitochondria

(A) Assembly of newly synthesized mtDNA-encoded subunits into OXPHOS complexes was studied by pulse/chase labeling with [³⁵S]methionine/cysteine in whole cells, followed by isolation of mitochondria and analysis by BN-PAGE and phosphorimaging. The CIV holoenzyme was not assembled in D2 mitochondria. The bottom panel shows the steady-state levels of mature complexes IV and I by immunoblotting. Abbreviations are as follows: CIV_I, complex IV subcomplex; CV, complex V.

(B) 2D-PAGE analysis of labeled mtDNA-encoded subunits from D2 revealed a blocked entry of COX1, COX2, and COX3 into the CIV holoenzyme, the stalling of these subunits in various subcomplexes, and an increased turnover of COX2 and COX3. The bottom panel shows the steady-state levels of mature complexes IV and II by immunoblotting. The positions of mtDNA-encoded CIV subunits COX1, COX2, and COX3 are indicated.