Bloodstream forms of Trypanosoma hrueei were found to maintain a significant membrane potential across their mitochondrial inner membrane (dym) in addition to a plasma membrane potential (d yp). Significantly, the dy, was selectively abolished by low concentrations of specific inhibitors of the FIFn-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondria1 membrane potential is generated and maintained exclusively by the electrogenic translocation of H +, catalysed by the mitochondrial FIFo-ATPase at the expense of ATP rather than by the mitochondrial electrontransport chain present in T. brucei. Consequently, bloodstream forms of T . brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F,F,-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh,P+) and was shown to have a value of approximately 150mV. When the value for the dym is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondria1 proton-motive force serves a role in the directional transport of ions and rnctabolites across thc promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclearencoded protein into the promjtochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. hrucei.

Proteome analysis by conventional approaches is biased against hydrophobic membrane proteins, many of which are also of low abundance. We have isolated plasma membrane sheets from bloodstream forms of Trypanosoma brucei by subcellular fractionation, and then applied a battery of complementary protein separation and identification techniques to identify a large number of proteins in this fraction. The results of these analyses have been combined to generate a subproteome for the pellicular plasma membrane of bloodstream forms of T. brucei as well as a separate subproteome for the pellicular cytoskeleton. In parallel, we have used in silico approaches to predict the relative abundance of proteins potentially expressed by bloodstream form trypanosomes, and to identify likely polytopic membrane proteins, providing quality control for the experimentally defined plasma membrane subproteome. We show that the application of multiple high-resolution proteomic techniques to an enriched organelle fraction is a valuable approach for the characterisation of relatively intractable membrane proteomes. We present here the most complete analysis of a protozoan plasma membrane proteome to date and show the presence of a large number of integral membrane proteins, including 11 nucleoside/nucleobase transporters, 15 ion pumps and channels and a large number of adenylate cyclases hitherto listed as putative proteins.

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Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA (ϳ1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP). Analysis of the amino acid sequence predicted the presence of a typical NH 2 -terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor. A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense. BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass (ϳ50 -70 kDa) significantly higher than predicted from the amino acid sequence (ϳ26 kDa). Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface. Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction. These data suggested that BARP may be sequestered in lipid rafts.

The distribution of 86Rb+ and the radiolabelled lipophilic cation [3H]methyltriphenylphosphonium (MePh3P+) was used to investigate the membrane potentials that exist in bloodstream forms of Trypanosoma brucei. Even after correction for binding to cellular constituents, the accumulation of MePh3P+ was approximately tenfold greater than the accumulation of Rb+ under resting conditions. The addition of low concentrations of carbonylcyanide p‐trifluoromethoxyphenylhydrazone or valinomycin reduced the accumulation of MePh3P+ tenfold without perturbing the accumulation of Rb+. Although selective permeabilization of the plasma membrane abolished the accumulation of Rb+ and caused a substantial decrease in the accumulation of MePh3P+, a significant carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone‐sensitive accumulation of MePh3P+ persisted under these conditions. These data were consistent with the presence of at least two distinct membrane potentials (Ψ) in bloodstream forms of T. brucei; a potential across the plasma membrane (Ψp) and an additional Ψ, generated by the electrogenic movement of H+, across the membrane of an intracellular organelle that possesses no electrical permeability to Rb+ or K+.