Probing for primary functions of prohibitin in Trypanosoma brucei Jir ˇí Ty ´c ˇ a , Drahomíra Faktorová a , Eva Kriegová a , Milan Jirku ˚ a , Zuzana Vávrová a , Dmitri A. Maslov b, * , Julius Lukeš a, * a Biology Centre, Institute of Parasitology, Czech Academy of Sciences, and Faculty of Natural Sciences, University of South Bohemia, C ˇ eské Bude ˇjovice (Budweis), Czech Republic b Department of Biology, University of California, Riverside, CA 92521, USA article info Article history: Received 12 March 2009 Received in revised form 9 July 2009 Accepted 10 July 2009 Keywords: Prohibitin Trypanosoma Mitochondrion Morphology Mitochondrial translation abstract Prohibitins (PHBs) 1 and 2 are small conserved proteins implicated in a number of functions in the mito- chondrion, as well as in the nucleus of eukaryotic cells. The current understanding of PHB functions comes from studies of model organisms such as yeast, worm and mouse, but considerable debate remains with regard to the primary functions of these ubiquitous proteins. We exploit the tractable reverse genet- ics of Trypanosoma brucei, the causative agent of African sleeping sickness, in order to specifically analyse the function of PHB in this highly divergent eukaryote. Using inducible RNA interference (RNAi) we show that PHB1 is essential in T. brucei, where it is confined to the cell’s single mitochondrion forming a high molecular weight complex. PHB1 and PHB2 appear to be indispensible for mitochondrial translation. Their ablation leads to a decrease in mitochondrial membrane potential, however no effect on the level of reactive oxygen species was observed. Flagellates lacking either PHB1 or both PHB1 and PHB2 exhibit significant morphological changes of their organelle, most notably its inflation. Even long after the loss of the PHB proteins, mtDNA was unaltered and mitochondrial cristae remained present, albeit displaced to the periphery of the mitochondrion, which is in contrast to other eukaryotes. Ó 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Prohibitins (PHBs) comprise evolutionary conserved proteins ubiquitously present in eukaryotes, where they were proposed to function in multiple capacities. They consist primarily of PHB1 and a closely related PHB2, sometimes called prohibitone. As sug- gested by the name, PHBs were originally associated with regula- tion of cell-cycle proliferation (McClung et al., 1989), although this function was later challenged (Manjeshwar et al., 2003). Since the time of their original description, PHBs have been localised to several cellular compartments such as the mitochondrion, nucleus and cell membrane, and have been implicated in an ever increasing number of cellular processes, such as apoptosis, transcriptional control, cell signalling, senescence and mitochondrial biogenesis (for recent reviews see Mishra et al., 2005; Merkwirth and Langer, 2009). In mitochondria of Saccharomyces cerevisiae, Caenorhabditis ele- gans and humans, the PHB proteins are assembled into a large ring- like inner membrane protein complex, the existence of which is supported by mutual dependence of PHB1 and PHB2 (Tatsuta et al., 2005). Both proteins co-immunoprecipitate (Coates et al., 2001) and the elimination of one PHB leads to the disappearance of the other (Berger and Yaffe, 1998; Artal-Sanz et al., 2003; Mer- kwirth et al., 2008). The PHB complex was proposed to be critical for proper folding and stabilization of mitochondrial-encoded sub- units of respiratory complexes (Nijtmans et al., 2000; Bourges et al., 2004) and its depletion is followed by apoptosis and frag- mentation of mitochondria (Kasashima et al., 2006). However, growing evidence from several organisms favours the association of PHB function primarily with mitochondrial mor- phology, corroborating the early observations from C. elegans (Artal-Sanz et al., 2003). The PHB proteins appear to be critical for organellar integrity (Sun et al., 2008), in particular in the mor- phogenesis of mitochondrial cristae and for the proper function of another mitochondrial protein, OPA1, with which they interact (Merkwirth et al., 2008). Putative interactions of PHBs with the in- ner membrane F 1 F 0 -ATP synthase-specific chaperones (Osman et al., 2007) can be interpreted as a potential scaffolding function of the PHB complex (Merkwirth and Langer, 2009). Furthermore, in human mitochondria, PHB1 was recently implicated in the maintenance of mtDNA within the mitochondrial nucleoids, as well as in regulation of its copy number within the organelle (Kasashima et al., 2008). A more extensive list of putative functions is associated with the extramitochondrial PHBs. Their anti-proliferative function, 0020-7519/$36.00 Ó 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2009.07.008 * Corresponding authors. Address: Biology Centre, Branišovská 31, 37005 C ˇ eské Bude ˇjovice, Czech Republic. Tel.: +420 38 7775416; fax: +420 38 5310388. E-mail addresses: maslov@ucr.edu (D.A. Maslov), jula@paru.cas.cz (J. Lukeš). International Journal for Parasitology 40 (2010) 73–83 Contents lists available at ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara