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

摘要

The Trypanosoma brucei mitochondrial F(1)-ATPase has been previously isolated and characterized. It is composed of five subunits of molecular weights 55000, 42000, 32000, 22000, and 17000 [1]. We have identified the alpha and beta 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 alpha subunit is unusually small at 42 kDa. The alpha 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 alpha subunit may have an unusually large mitochondrial presequence of 159 amino acids. In contrast, the estimated size of the native beta 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 beta subunit was confirmed by expression in an in vitro and an Escherichia coli expression system. The purified recombinant beta 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 beta subunits of other organisms. The differential binding of this photoaffinity analogue was used to resolve the identities of the alpha and beta subunits of the ATP synthase from T. brucei. These results are in contrast to results previously obtained for a related trypanosomatid Crithidia fasciculata.