Subunit 9 of the mitochondrial ATP synthase of Trypanosoma brucei is nuclearly encoded and developmentally regulated

Abstract

We have previously shown that the mitochondrial ATP synthase is developmentally regulated through the life cycle of Trypanosoma brucei. The mechanism of this regulation is as yet unknown. We are currently examining regulation of expression of several key subunits of the ATP synthase to investigate this mechanism. In the work presented here, we have cloned, sequenced, and confirmed the identity of the ATPase subunit 9 homologue from T. brucei. The ATPase subunit 9 gene that we have identified from T. brucei has between 40 and 600% identity with subunit 9 from a variety of organisms. This gene possesses a putative mitochondrial import sequence at the N terminus of the encoded protein sequence. The protein expressed from this gene by in vitro transcription/translation comigrates with native protein isolated from inner mitochondrial membrane vesicles from T. brucei. We have shown that the cDNA identifies a copy of this gene in the nuclear genome, but does not identify a similar gene in kinetoplast DNA (kDNA) prepared from T. brucei. This gene does not show homology to any published sequence data from maxicircle DNA or edited maxicircle derived sequences. Steady state transcripts of a single size have been identified by Northern analysis and demonstrate significant developmental regulation through the T. brucei life cycle. Northern analysis and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results show that the transcript is 10-14-fold higher in procyclic form than in early and late bloodstream forms.