Downregulation of Mitochondrial Porin Inhibits Cell Growth and Alters Respiratory Phenotype in Trypanosoma brucei

Porin is the most abundant outer membrane (OM) protein of mitochondria. It forms the aqueous channel on the mitochondrial OM and mediates major metabolite flux between mitochondria and cytosol. Mitochondrial porin in Trypanosoma brucei, a unicellular parasitic protozoan and the causative agent of African trypanosomiasis, possesses a β-barrel structure similar to the bacterial OM porin OmpA. T. brucei porin (TbPorin) is present as a monomer as well as an oligomer on the mitochondrial OM, and its expression is developmentally regulated. In spite of its distinct structure, the TbPorin function is similar to those of other eukaryotic porins. TbPorin RNA interference (RNAi) reduced cell growth in both procyclic and bloodstream forms. The depletion of TbPorin decreased ATP production by inhibiting metabolite flux through the OM. Additionally, the level of trypanosome alternative oxidase (TAO) decreased, whereas the levels of cytochrome-dependent respiratory complexes III and IV increased in TbPorin-depleted mitochondria. Furthermore, the depletion of TbPorin reduced cellular respiration via TAO, which is not coupled with oxidative phosphorylation, but increased the capacity for cyanide-sensitive respiration. Together, these data reveal that TbPorin knockdown reduced the mitochondrial ATP level, which in turn increased the capacity of the cytochrome-dependent respiratory pathway (CP), in an attempt to compensate for the mitochondrial energy crisis. However, a simultaneous decrease in the substrate-level phosphorylation due to TbPorin RNAi caused growth inhibition in the procyclic form. We also found that the expressions of TAO and CP proteins are coordinately regulated in T. brucei according to mitochondrial energy demand.Trypanosoma brucei belongs to a group of parasitic protozoa that possess a single tubular mitochondrion with a concatenated structure of mitochondrial DNA known as kinetoplast (30). T. brucei is the infectious agent of the disease African trypanosomiasis, which is spread from one mammal to another by the bite of the tsetse fly (53). During transmission from the insect vector to the mammalian host and vice versa, the parasite undergoes various developmental stages accompanied by dramatic changes in mitochondrial activities (15). The bloodstream form that grows in mammalian blood uses glucose as its energy source and suppresses many mitochondrial activities. The bloodstream-form mitochondria lack cytochromes; thus, respiration in this form is solely dependent on the cytochrome-independent trypanosome alternative oxidase (TAO) (15). In contrast, the procyclic form that lives in the insect''s midgut possesses a developed mitochondrion with a full complement of the cytochrome-dependent respiratory system and a reduced level of TAO. The procyclic-form mitochondria produce ATP by both oxidative and substrate-level phosphorylations (7). On the other hand, the bloodstream-form mitochondria do not produce ATP but hydrolyze ATP to maintain the inner membrane (IM) potential (10, 33, 39, 48). Many of the mitochondrial IM- and matrix-localized proteins in T. brucei are well characterized (11, 29, 34, 43, 45). However, the mitochondrial outer membrane (OM) proteins in this group of parasitic protozoa have been poorly explored.Mitochondrial porin, which is also known as the voltage-dependent anion-selective channel (VDAC), is the most abundant protein in the OM (17, 28). The sizes and the secondary structures of this protein are very similar among different organisms. The VDAC possesses a N-terminal α-helical domain, and the rest of the protein consists of a number of amphiphilic β-strands, which form a barrel-like structure that integrates into the lipid bilayer (16, 17, 28). Recently, the three-dimensional structure of the human VDAC has been elucidated by nuclear magnetic resonance spectroscopy and X-ray crystallography, which showed a β-barrel architecture composed of 19 β-strands and the N-terminal α-helix located horizontally midway in the pore (5). Saccharomyces cerevisiae and Neurospora crassa VDACs also possess 16 to 19 β-strands, similar to the mammalian VDAC (17).The VDAC exists as different isomeric forms in different species (16, 19). In yeasts, there are two forms: VDAC1 and VDAC2. Only VDAC1 has the channel activity and is abundantly expressed (22, 23). Animals have three isoforms: VDAC1 to VDAC3. These isoforms showed more than 80% sequence homology among themselves. However, their expression levels and tissue specificities are different (16). Plants also have multiple isoforms of the VDAC with various expression levels under different pathological conditions (19). The VDAC plays a crucial role in regulated transport of ADP, ATP, Ca²⁺, and other metabolites in and out of mitochondria (17, 28, 41). Two ATP-binding sites found at the N- and C-terminal regions in the VDAC are critical for its function (54). Downregulation of VDAC expression disrupts mitochondrial energy production (22, 25). In contrast, overexpression of the VDAC in metazoa induces apoptosis, which can be blocked by compounds that inhibit its channel activity (1, 47).The OM of gram-negative bacteria also consists of various types of porins (24, 32, 40). Based on their structures and functions, they are divided into five groups. OmpA belongs to the small β-barrel integral membrane protein family, which is composed of eight β-strands. It is highly abundant and ubiquitous among most gram-negative bacteria (21). Other types of porins include general porin OmpF, which consists of 16 β-strands; substrate-specific porins, such as LamB or maltoporin, which contains 18 β-strands; receptor-type porin FhuA, the largest β-barrel, with 22 β-strands; and phospholipase A or OMPLA, an integral membrane enzyme containing 12 β-strands (21, 24, 32, 40). The OmpA plays important roles in bacterial conjugation, adhesion, invasion, and immune evasion and also acts as the receptor for several bacteriophages through its surface-exposed loops (44).Here, we show that the T. brucei mitochondrial porin (TbPorin) possesses a predicted β-barrel structure that has fewer β-strands than other mitochondrial porins but is similar to bacterial OmpA. TbPorin is crucial for mitochondrial energy production via both oxidative and substrate-level phosphorylations. The depletion of TbPorin reduced cell growth of the procyclic form as well as the bloodstream form. Furthermore, it reveals that depletion of mitochondrial ATP level by downregulation of porin alters the electron flow via TAO and the cytochrome-dependent pathway (CP) as well as the levels of proteins in these pathways.