Alternative Editing of Cytochrome C Oxidase III mRNA in Trypanosome Mitochondria Generates Protein Diversity

Abstract

Alternative editing of the cytochrome c oxidase III pre‐mRNA in Trypanosoma brucei (T. brucei) produces a novel mitochondrial membrane protein, alternatively edited protein‐1 (AEP‐1). Immunofluorescence microscopy reveals that this unique protein associates with two distinct regions intracellularly. AEP‐1 is distributed throughout the mitochondria and is also observable as a discrete condensed structure associated with the detergent resistant tripartite attachment complex adjacent to the mitochondrial DNA. Biochemical fractionation and Blue Native PAGE (BNPAGE) analysis has also shown that AEP‐1 associates with the detergent soluble mitochondrial membranes. To further investigate the role of AEP‐1 localized to the mitochondrial membrane, intact mitochondria were isolated using a hypotonic lysis method and the organelles were treated with a tandem nonionic detergent treatment to separate the mitochondrial matrix from the membrane fraction. Separation of this membrane fraction by BNPAGE and Western Blot analysis revealed the association of AEP‐1 with a high molecular weight membrane complex. The AEP‐1 complex was purified further using anion exchange chromatography and the resulting BNPAGE band was subjected to ESI‐ion trap‐tandem mass spectrometry. Sequencing revealed several proteins including the oxoglutarate dehydrogenase (E1) and the dihydrolipoyl succinyltransferase (E2) subunits of the 2‐oxoglutarate dehydrogenase complex (2ODC). The AEP‐1 complex did not retain the same NAD+reductive capacity as the bonafide 2ODC. These data suggest that AEP‐1 associates with proteins that may have bifunctional roles in T. brucei mitochondria.