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.

• Keywords
• Ffh, FtsY, LECA, evolution, mitochondrion, protein targeting, protists, signal recognition particle,
• MeSH
• Bacterial Proteins genetics   MeSH
• Biological Evolution * MeSH
• Genome, Mitochondrial * MeSH
• Naegleria genetics   MeSH
• Escherichia coli Proteins genetics   MeSH
• Receptors, Cytoplasmic and Nuclear genetics   MeSH
• Sequence Homology, Nucleic Acid MeSH
• Signal Recognition Particle genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Ffh protein, E coli MeSH Browser
• FtsY protein, Bacteria MeSH Browser
• Escherichia coli Proteins MeSH
• Receptors, Cytoplasmic and Nuclear MeSH
• Signal Recognition Particle MeSH

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.

• Keywords
• Kinetoplastida, adenylate cyclase, oxygen, phylogenetics, receptor, trypanosomatid,
• MeSH
• Adenylyl Cyclases genetics   MeSH
• Gene Duplication MeSH
• Phylogeny * MeSH
• Genome, Protozoan MeSH
• Host-Pathogen Interactions genetics   MeSH
• Evolution, Molecular * MeSH
• Trypanosomatina enzymology   genetics   MeSH
• Up-Regulation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylyl Cyclases MeSH

Dynamins and dynamin-like proteins (DLPs) belong to a family of large GTPases involved in membrane remodelling events. These include both fusion and fission processes with different dynamin proteins often having a specialised function within the same organism. Trypanosoma brucei is thought to have only one multifunctional DLP (TbDLP). While this was initially reported to function in mitochondrial division only, an additional role in endocytosis and cytokinesis was later also proposed. Since there are two copies of TbDLP present in the trypanosome genome, we investigated potential functional differences between these two paralogs by re-expressing either protein in a TbDLP RNAi background. These paralogs, called TbDLP1 and TbDLP2, are almost identical bar a few amino acid substitutions. Our results, based on cell lines carrying tagged and RNAi-resistant versions of each protein, show that overexpression of TbDLP1 alone is able to rescue the observed endocytosis and growth defects in the mammalian bloodstream form (BSF) of the parasite. While TbDLP2 shows no rescue in our experiments in BSF, this might also be due to lower expression levels of the protein in this life stage. In contrast, both TbDLP proteins apparently play more complementary roles in the insect procyclic form (PCF) since neither TbDLP1 nor TbDLP2 alone can fully restore wildtype growth and morphology in TbDLP-depleted parasites.

• MeSH
• Cell Line MeSH
• Dynamins chemistry   genetics   metabolism   MeSH
• Protozoan Proteins chemistry   genetics   metabolism   MeSH
• RNA Interference MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Trypanosoma brucei brucei metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Dynamins MeSH
• Protozoan Proteins MeSH

The kinetoplast (k), the uniquely packaged mitochondrial DNA of trypanosomatid protists is formed by a catenated network of minicircles and maxicircles that divide and segregate once each cell cycle. Although many proteins involved in kDNA replication and segregation are now known, several key steps in the replication mechanism remain uncharacterized at the molecular level, one of which is the nabelschnur or umbilicus, a prominent structure which in the mammalian parasite Trypanosoma brucei connects the daughter kDNA networks prior to their segregation. Here we characterize an M17 family leucyl aminopeptidase metalloprotease, termed TbLAP1, which specifically localizes to the kDNA disk and the nabelschur and represents the first described protein found in this structure. We show that TbLAP1 is required for correct segregation of kDNA, with knockdown resulting in delayed cytokinesis and ectopic expression leading to kDNA loss and decreased cell proliferation. We propose that TbLAP1 is required for efficient kDNA division and specifically participates in the separation of daughter kDNA networks.

• MeSH
• Cell Cycle physiology   MeSH
• DNA, Kinetoplast genetics   MeSH
• Leucyl Aminopeptidase genetics   metabolism   MeSH
• DNA, Mitochondrial genetics   MeSH
• Mitochondria metabolism   ultrastructure   MeSH
• DNA, Protozoan genetics   MeSH
• Protozoan Proteins metabolism   MeSH
• DNA Replication physiology   MeSH
• Trypanosoma brucei brucei genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Kinetoplast MeSH
• Leucyl Aminopeptidase MeSH
• DNA, Mitochondrial MeSH
• DNA, Protozoan MeSH
• Protozoan Proteins MeSH

There are a variety of complex metabolic processes ongoing simultaneously in the single, large mitochondrion of Trypanosoma brucei. Understanding the organellar environment and dynamics of mitochondrial proteins requires quantitative measurement in vivo. In this study, we have validated a method for immobilizing both procyclic stage (PS) and bloodstream stage (BS) T. brucei brucei with a high level of cell viability over several hours and verified its suitability for undertaking fluorescence recovery after photobleaching (FRAP), with mitochondrion-targeted yellow fluorescent protein (YFP). Next, we used this method for comparative analysis of the translational diffusion of mitochondrial RNA-binding protein 1 (MRP1) in the BS and in T. b. evansi. The latter flagellate is like petite mutant Saccharomyces cerevisiae because it lacks organelle-encoded nucleic acids. FRAP measurement of YFP-tagged MRP1 in both cell lines illuminated from a new perspective how the absence or presence of RNA affects proteins involved in mitochondrial RNA metabolism. This work represents the first attempt to examine this process in live trypanosomes.

• MeSH
• Mitochondrial Proteins genetics   MeSH
• Mutation MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• RNA Interference MeSH
• RNA, Mitochondrial MeSH
• RNA genetics   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Trypanosoma brucei brucei genetics   MeSH
• Cell Survival genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• gBP21 protein, Trypanosoma brucei MeSH Browser
• Mitochondrial Proteins MeSH
• RNA-Binding Proteins MeSH
• Protozoan Proteins MeSH
• RNA, Mitochondrial MeSH
• RNA MeSH

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

• MeSH
• Active Transport, Cell Nucleus MeSH
• Cell Nucleus metabolism   MeSH
• Ferredoxins metabolism   MeSH
• Frataxin MeSH
• Nuclear Localization Signals MeSH
• Carbon-Sulfur Lyases chemistry   genetics   metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria metabolism   MeSH
• Molecular Sequence Data MeSH
• Protein Multimerization MeSH
• Nuclear Matrix-Associated Proteins chemistry   genetics   metabolism   MeSH
• Iron-Binding Proteins metabolism   MeSH
• Protozoan Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Trypanosoma brucei brucei enzymology   genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• cysteine desulfurase MeSH Browser
• Ferredoxins MeSH
• Nuclear Localization Signals MeSH
• Carbon-Sulfur Lyases MeSH
• Mitochondrial Proteins MeSH
• Nuclear Matrix-Associated Proteins MeSH
• Iron-Binding Proteins MeSH
• Protozoan Proteins MeSH