Pam16 and Pam18 were repurposed during Trypanosoma brucei evolution to regulate the replication of mitochondrial DNA.

Protein import and genome replication are essential processes for mitochondrial biogenesis and propagation. The J-domain proteins Pam16 and Pam18 regulate the presequence translocase of the mitochondrial inner membrane. In the protozoan Trypanosoma brucei, their counterparts are TbPam16 and TbPam18, which are essential for the procyclic form (PCF) of the parasite, though not involved in mitochondrial protein import. Here, we show that during evolution, the 2 proteins have been repurposed to regulate the replication of maxicircles within the intricate kDNA network, the most complex mitochondrial genome known. TbPam18 and TbPam16 have inactive J-domains suggesting a function independent of heat shock proteins. However, their single transmembrane domain is essential for function. Pulldown of TbPam16 identifies a putative client protein, termed MaRF11, the depletion of which causes the selective loss of maxicircles, akin to the effects observed for TbPam18 and TbPam16. Moreover, depletion of the mitochondrial proteasome results in increased levels of MaRF11. Thus, we have discovered a protein complex comprising TbPam18, TbPam16, and MaRF11, that controls maxicircle replication. We propose a working model in which the matrix protein MaRF11 functions downstream of the 2 integral inner membrane proteins TbPam18 and TbPam16. Moreover, we suggest that the levels of MaRF11 are controlled by the mitochondrial proteasome.

Vestiges of the Bacterial Signal Recognition Particle-Based Protein Targeting in Mitochondria.

The main bacterial pathway for inserting proteins into the plasma membrane relies on the signal recognition particle (SRP), composed of the Ffh protein and an associated RNA component, and the SRP-docking protein FtsY. Eukaryotes use an equivalent system of archaeal origin to deliver proteins into the endoplasmic reticulum, whereas a bacteria-derived SRP and FtsY function in the plastid. Here we report on the presence of homologs of the bacterial Ffh and FtsY proteins in various unrelated plastid-lacking unicellular eukaryotes, namely Heterolobosea, Alveida, Goniomonas, and Hemimastigophora. The monophyly of novel eukaryotic Ffh and FtsY groups, predicted mitochondrial localization experimentally confirmed for Naegleria gruberi, and a strong alphaproteobacterial affinity of the Ffh group, collectively suggest that they constitute parts of an ancestral mitochondrial signal peptide-based protein-targeting system inherited from the last eukaryotic common ancestor, but lost from the majority of extant eukaryotes. The ability of putative signal peptides, predicted in a subset of mitochondrial-encoded N. gruberi proteins, to target a reporter fluorescent protein into the endoplasmic reticulum of Trypanosoma brucei, likely through their interaction with the cytosolic SRP, provided further support for this notion. We also illustrate that known mitochondrial ribosome-interacting proteins implicated in membrane protein targeting in opisthokonts (Mba1, Mdm38, and Mrx15) are broadly conserved in eukaryotes and nonredundant with the mitochondrial SRP system. Finally, we identified a novel mitochondrial protein (MAP67) present in diverse eukaryotes and related to the signal peptide-binding domain of Ffh, which may well be a hitherto unrecognized component of the mitochondrial membrane protein-targeting machinery.

Large-Scale Phylogenetic Analysis of Trypanosomatid Adenylate Cyclases Reveals Associations with Extracellular Lifestyle and Host-Pathogen Interplay.

Receptor adenylate cyclases (RACs) on the surface of trypanosomatids are important players in the host-parasite interface. They detect still unidentified environmental signals that affect the parasites' responses to host immune challenge, coordination of social motility, and regulation of cell division. A lesser known class of oxygen-sensing adenylate cyclases (OACs) related to RACs has been lost in trypanosomes and expanded mostly in Leishmania species and related insect-dwelling trypanosomatids. In this work, we have undertaken a large-scale phylogenetic analysis of both classes of adenylate cyclases (ACs) in trypanosomatids and the free-living Bodo saltans. We observe that the expanded RAC repertoire in trypanosomatids with a two-host life cycle is not only associated with an extracellular lifestyle within the vertebrate host, but also with a complex path through the insect vector involving several life cycle stages. In Trypanosoma brucei, RACs are split into two major clades, which significantly differ in their expression profiles in the mammalian host and the insect vector. RACs of the closely related Trypanosoma congolense are intermingled within these two clades, supporting early RAC diversification. Subspecies of T. brucei that have lost the capacity to infect insects exhibit high numbers of pseudogenized RACs, suggesting many of these proteins have become redundant upon the acquisition of a single-host life cycle. OACs appear to be an innovation occurring after the expansion of RACs in trypanosomatids. Endosymbiont-harboring trypanosomatids exhibit a diversification of OACs, whereas these proteins are pseudogenized in Leishmania subgenus Viannia. This analysis sheds light on how ACs have evolved to allow diverse trypanosomatids to occupy multifarious niches and assume various lifestyles.

ZapE/Afg1 interacts with Oxa1 and its depletion causes a multifaceted phenotype.

ZapE/Afg1 is a component of the inner cell membrane of some eubacteria and the inner mitochondrial membrane of eukaryotes. This protein is involved in FtsZ-dependent division of eubacteria. In the yeast and human mitochondrion, ZapE/Afg1 likely interacts with Oxa1 and facilitates the degradation of mitochondrion-encoded subunits of respiratory complexes. Furthermore, the depletion of ZapE increases resistance to apoptosis, decreases oxidative stress tolerance, and impacts mitochondrial protein homeostasis. It remains unclear whether ZapE is a multifunctional protein, or whether some of the described effects are just secondary phenotypes. Here, we have analyzed the functions of ZapE in Trypanosoma brucei, a parasitic protist, and an important model organism. Using a newly developed proximity-dependent biotinylation approach (BioID2), we have identified the inner mitochondrial membrane insertase Oxa1 among three putative interacting partners of ZapE, which is present in two paralogs. RNAi-mediated depletion of both ZapE paralogs likely affected the function of respiratory complexes I and IV. Consistently, we show that the distribution of mitochondrial ZapE is restricted only to organisms with Oxa1, respiratory complexes, and a mitochondrial genome. We propose that the evolutionarily conserved interaction of ZapE with Oxa1, which is required for proper insertion of many inner mitochondrial membrane proteins, is behind the multifaceted phenotype caused by the ablation of ZapE.