Analysis of diverse eukaryotes suggests the existence of an ancestral mitochondrial apparatus derived from the bacterial type II secretion system

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion

Data from: A single Tim translocase in the mitosomes of Giardia intestinalis illustrates convergence of protein import machines in anaerobic eukaryotes

Mitochondria have evolved diverse forms across eukaryotic diversity in adaptation to anaerobiosis. Mitosomes are the simplest and the least well-studied type of anaerobic mitochondria. Transport of proteins via TIM complexes, composed of three proteins of the Tim17 protein family (Tim17/22/23), is one of the key unifying aspects of mitochondrial and mitochondria-derived organelles. However, multiple experimental and bioinformatic attempts have so far failed to identify the nature of TIM in mitosomes of the anaerobic metamonad protist, Giardia intestinalis, one of the few experimental models for mitosome biology. Here, we present the identification of a single G. intestinalis Tim17 protein (GiTim17), made possible only by the implementation of a metamonad-specific hidden Markov model. While very divergent in primary sequence and in predicted membrane topology, experimental data suggest that GiTim17 is an inner membrane mitosomal protein, forming a disulphide-linked dimer. We suggest that the peculiar GiTim17 sequence reflects adaptation to the unusual, detergent resistant, inner mitosomal membrane. Specific pull-down experiments indicate interaction of GiTim17 with mitosomal Tim44, the tethering component of the import motor complex. Analysis of TIM complexes across eukaryote diversity indicates that a “single Tim” translocase is a convergent adaptation of mitosomes in anaerobic protists, with Tim22 and Tim17 (but not Tim23), providing the protein backbone

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis