Cloning and characterization of the subunits comprising the catalytic core of the Trypanosoma brucei mitochondrial ATP synthase

Abstract

The Trypanosoma brucei mitochondrial F 1-ATPase has been previously isolated and characterized. It is composed of five subunits of molecular weights 55 000, 42 000, 32 000, 22 000, and 17 000 [1]. We have identified the α and β subunits of the T. brucei F 1-ATPase by N-terminal sequence determination together with analysis of cDNA and genomic clones. The genes for both subunits are homologous to the same subunits from other organisms. They contain the Walker A and B boxes of homology and a putative mitochondrial import sequence. The isolated T. brucei α subunit is unusually small at 42 kDa. The α cDNA clone encodes a protein of predicted size 59 kDa with a mitochondrial import presequence at the N-terminus. The predicted size was confirmed by expression of a 59 kDa protein from the cDNA clone in vitro. These results suggest that the α subunit may have an unusually large mitochondrial presequence of 159 amino acids. In contrast, the estimated size of the native β subunit (55 kDa) correlates well with the size predicted from the cDNA clone, 57 kDa, from which a 21 amino acid presequence has been removed in vivo. The size of the β subunit was confirmed by expression in an in vitro and an Escherichia coli expression system. The purified recombinant β subunit, like the native F 1-ATPase, can be labeled by the photoaffinity nucleotide analogue 8-azido ATP. Binding of the 8-azido ATP probe is best competed by the natural substrate ATP, and is significantly reduced by pretreatment with the inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazide as has been shown with β subunits of other organisms. The differential binding of this photoaffinity analogue was used to resolve the identities of the α and β subunits of the ATP synthase from T. brucei. These results are in contrast to results previously obtained for a related trypanosomatid Crithidia fasciculata.