Enigmatic Presence of Mitochondrial Complex I in Trypanosoma brucei Bloodstream Forms

Abstract

The presence of mitochondrial respiratory complex I in the pathogenic bloodstream stages of Trypanosoma brucei has been vigorously debated: increased expression of mitochondrially encoded functional complex I mRNAs is countered by low levels of enzymatic activity that show marginal inhibition by the specific inhibitor rotenone. We now show that epitope-tagged versions of multiple complex I subunits assemble into ␣ and ␤ subcomplexes in the bloodstream stage and that these subcomplexes require the mitochondrial genome for their assembly. Despite the presence of these large (740-and 855-kDa) multisubunit complexes, the electron transport activity of complex I is not essential under experimental conditions since null mutants of two core genes (NUBM and NUKM) showed no growth defect in vitro or in mouse infection. Furthermore, the null mutants showed no decrease in NADH:ubiquinone oxidoreductase activity, suggesting that the observed activity is not contributed by complex I. This work conclusively shows that despite the synthesis and assembly of subunit proteins, the enzymatic function of the largest respiratory complex is neither significant nor important in the bloodstream stage. This situation appears to be in striking contrast to that for the other respiratory complexes in this parasite, where physical presence in a life-cycle stage always indicates functional significance.

Characterization of Two Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-Phosphate Dehydrogenases in Trypanosoma brucei

“…While the presence of several different forms of NAD-G3PDH is relatively common in eukaryotes (15,16), two isoforms of FAD-G3PDH are, as far as we know, unique to trypanosomatids. To date, TAO and other components of the respiratory chain have been subjected to numerous investigations (8,12,14,27,29,(34)(35)(36)(37)(38)(39)(40)(41), while only two studies dealt with the function of mtG3PDH (12,14), and the role of putG3PDH remains totally unknown.…”

“…The respiratory chain of the BF consists only of a trypanosomal alternative terminal oxidase (TAO) and a flavin adenine dinucleotide (FAD)-dependent glycerol-3-phosphate dehydrogenase (FAD-G3PDH) (6). Whereas, together with the other Krebs cycle enzymes, respiratory complex II is missing in the BF (5), complex I is present, although its contribution to the electron flow remains uncertain (7,8).…”

Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

“…In the BS mitochondrion, these cofactors may be incorporated into the nonessential respiratory complexes I and II (12), as well as ferredoxin, glutaredoxin, and lipoate synthase, all of which are expressed at a very low level, however (31,63). Therefore, we propose that in this life cycle stage, the bulk of cofactors is exported outside the mitochondrion, as in these fast-dividing parasites a high demand for them exists in the catalytic subunits of DNA polymerases (25) and other cytosolic and nuclear proteins.…”

“…While cytochrome c-dependent respiratory complexes III and IV are altogether missing from the BS, complex II probably remains active. Moreover, complex I is expressed, yet its enzymatic activity is not significant, and its real function remains elusive (12)(13)(14)(15). Complex V (ATP synthase) is present, but its F1 part rotates in the reverse direction compared to the PS, cleaving ATP and pumping H ϩ out of the mitochondrion in order to create the membrane potential required for protein import (2).…”

Solanesyl Diphosphate Synthase, an Enzyme of the Ubiquinone Synthetic Pathway, Is Required throughout the Life Cycle of Trypanosoma brucei

“…Having a central position in the respiratory chain, UQ receives in a typical cell electrons from complexes I and II and, if present, from alternative NADH dehydrogenase. While both the presence and activity in the T. brucei PCF of complex II and rotenone-insensitive alternative NADH dehydrogenase are undisputed (13,14,21,22), both the composition (23,24) and activity of complex I seem to be highly unusual (25,26). Diminishing the cellular concentration of UQ could then favor an increase of the reduced NADH pool with parallel formation of ubisemiquinone, facilitating the deviation of electrons to oxygen with consequent mitochondrial ROS formation.…”