The F₁‐ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18‐subunit

Abstract

The F-ATPases (also called the F F -ATPases or ATP synthases) are multi-subunit membrane-bound molecular machines that produce ATP in bacteria and in eukaryotic mitochondria and chloroplasts. The structures and enzymic mechanisms of their F -catalytic domains are highly conserved in all species investigated hitherto. However, there is evidence that the F-ATPases from the group of protozoa known as Euglenozoa have novel features. Therefore, we have isolated pure and active F -ATPase from the euglenozoan parasite, Trypanosoma brucei, and characterized it. All of the usual eukaryotic subunits (α, β, γ, δ, and ε) were present in the enzyme, and, in addition, two unique features were detected. First, each of the three α-subunits in the F -domain has been cleaved by proteolysis in vivo at two sites eight residues apart, producing two assembled fragments. Second, the T. brucei F -ATPase has an additional subunit, called p18, present in three copies per complex. Suppression of expression of p18 affected in vitro growth of both the insect and infectious mammalian forms of T. brucei. It also reduced the levels of monomeric and multimeric F-ATPase complexes and diminished the in vivo hydrolytic activity of the enzyme significantly. These observations imply that p18 plays a role in the assembly of the F domain. These unique features of the F -ATPase extend the list of special characteristics of the F-ATPase from T. brucei, and also, demonstrate that the architecture of the F -ATPase complex is not strictly conserved in eukaryotes.

ATP synthase from Trypanosoma brucei has an elaborated canonical F 1 -domain and conventional catalytic sites

“…The PPR repeat is folded into a helix-turn-helix motif, and PPR proteins usually contain several tandem repeats associated into a superhelix, with a concave groove on one face that serves as a binding surface for RNA ligands. The p18-subunit of the F 1 -ATPase from T. brucei is predicted to be a PPR protein with three PPRs, whereas it was previously thought to have two PPRs (50,55). Although the probability score (49%) is rather low, as reflected in the weak correspondence of the sequences of the three predicted PPRs to the PPR consensus ( Fig.…”

“…Third, and most significantly from a structural standpoint, the T. brucei F 1 -ATPase has an additional p18-subunit bound to each of its three α-subunits (50). The buried surface areas of interaction of the p18-subunits with their partner α E -, α TP -, and α DP -subunits are 2,500, 2,600, and 2,500 Å 2 , respectively.…”

“…First, the α-subunit in T. brucei is cleaved in vivo by proteolysis at two adjacent sites, removing residues 128-135 ( Fig. S2) (50). The cleavage of α-subunits has been noted in other euglenozoan ATP synthases as well (48,(51)(52)(53), although the sites of cleavage have not been characterized precisely.…”

“…However, p18 does not have a site equivalent to the RNAbinding site in PPR10, and other residues required for RNA binding in α-helix-7 of PPR10 have been substituted in H7 of p18. Therefore, there is no evidence suggesting that p18 has any role in binding an RNA molecule, and its role in the T. brucei F 1 -ATPase remains obscure, although its presence is essential for assembly of the enzyme (50). The sequences of p18-subunits, including the PPR repeats, are highly conserved across the euglenozoa, suggesting that the structure and the mode of interaction of the various p18-proteins with their cognate F 1 -ATPases are conserved as well (Fig.…”

Inhibition of F1‐ATPase from Trypanosoma brucei by its regulatory protein inhibitor TbIF1

“…The kinetics of the inhibition of F 1 ‐ATPase by TbIF 1 were characterized by measurement of the rate constants of the inhibitor's binding ( k on ) and dissociation ( k off ), and the dissociation constant of the inhibitor–enzyme complex ( K i ). The kinetic measurements were carried out with F 1 ‐ATPase isolated from the PF of T. brucei . These kinetic data are summarized in Table .…”