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.

• Klíčová slova
• CRISPR/Cas9, Giardia, gene knockout, multiploid,
• MeSH
• CRISPR-Cas systémy * MeSH
• editace genu metody   MeSH
• Giardia lamblia * genetika   MeSH
• lidé MeSH
• tetraploidie MeSH
• vodící RNA, systémy CRISPR-Cas MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• vodící RNA, systémy CRISPR-Cas MeSH

Formation of mitochondria by the conversion of a bacterial endosymbiont was a key moment in the evolution of eukaryotes. It was made possible by outsourcing the endosymbiont's genetic control to the host nucleus, while developing the import machinery for proteins synthesized on cytosolic ribosomes. The original protein export machines of the nascent organelle remained to be repurposed or were completely abandoned. This review follows the evolutionary fates of three prokaryotic inner membrane translocases Sec, Tat, and YidC. Homologs of all three translocases can still be found in current mitochondria, but with different importance for mitochondrial function. Although the mitochondrial YidC homolog, Oxa1, became an omnipresent independent insertase, the other two remained only sporadically present in mitochondria. Only a single substrate is known for the mitochondrial Tat and no function has yet been assigned for the mitochondrial Sec. Finally, this review compares these ancestral mitochondrial proteins with their paralogs operating in the plastids and the endomembrane system.

• Klíčová slova
• eukaryogenesis, membrane trafficking, neofunctionalization, protein targeting,
• MeSH
• Eukaryota * genetika   metabolismus   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• molekulární evoluce MeSH
• proteiny z Escherichia coli * genetika   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• membránové transportní proteiny MeSH
• mitochondriální proteiny MeSH
• proteiny z Escherichia coli * MeSH

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.

• MeSH
• biologické modely MeSH
• Eukaryota klasifikace   genetika   metabolismus   MeSH
• fylogeneze MeSH
• gramnegativní bakterie klasifikace   genetika   metabolismus   MeSH
• konzervovaná sekvence MeSH
• mitochondriální proteiny klasifikace   genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• molekulární evoluce * MeSH
• molekulární modely MeSH
• Naegleria klasifikace   genetika   metabolismus   MeSH
• peroxizomy metabolismus   MeSH
• protozoální proteiny klasifikace   genetika   metabolismus   MeSH
• sekreční systém typu II klasifikace   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• protozoální proteiny MeSH
• sekreční systém typu II MeSH

BACKGROUND: Bacteria and mitochondria contain translocases that function to transport proteins across or insert proteins into their inner and outer membranes. Extant mitochondria retain some bacterial-derived translocases but have lost others. While BamA and YidC were integrated into general mitochondrial protein transport pathways (as Sam50 and Oxa1), the inner membrane TAT translocase, which uniquely transports folded proteins across the membrane, was retained sporadically across the eukaryote tree. RESULTS: We have identified mitochondrial TAT machinery in diverse eukaryotic lineages and define three different types of eukaryote-encoded TatABC-derived machineries (TatAC, TatBC and TatC-only). Here, we investigate TatAC and TatC-only machineries, which have not been studied previously. We show that mitochondria-encoded TatAC of the jakobid Andalucia godoyi represent the minimal functional pathway capable of substituting for the Escherichia coli TatABC complex and can transport at least one substrate. However, selected TatC-only machineries, from multiple eukaryotic lineages, were not capable of supporting the translocation of this substrate across the bacterial membrane. Despite the multiple losses of the TatC gene from the mitochondrial genome, the gene was never transferred to the cell nucleus. Although the major constraint preventing nuclear transfer of mitochondrial TatC is likely its high hydrophobicity, we show that in chloroplasts, such transfer of TatC was made possible due to modifications of the first transmembrane domain. CONCLUSIONS: At its origin, mitochondria inherited three inner membrane translocases Sec, TAT and Oxa1 (YidC) from its bacterial ancestor. Our work shows for the first time that mitochondrial TAT has likely retained its unique function of transporting folded proteins at least in those few eukaryotes with TatA and TatC subunits encoded in the mitochondrial genome. However, mitochondria, in contrast to chloroplasts, abandoned the machinery multiple times in evolution. The overall lower hydrophobicity of the Oxa1 protein was likely the main reason why this translocase was nearly universally retained in mitochondrial biogenesis pathways.

• Klíčová slova
• Hydrophobicity, Mitochondrial evolution, Protein transport, TAT translocase,
• MeSH
• Escherichia coli genetika   MeSH
• Eukaryota genetika   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární evoluce * MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové transportní proteiny MeSH
• proteiny z Escherichia coli MeSH

Bacterial division initiates at the site of a contractile Z-ring composed of polymerized FtsZ. The location of the Z-ring in the cell is controlled by a system of three mutually antagonistic proteins, MinC, MinD, and MinE. Plastid division is also known to be dependent on homologs of these proteins, derived from the ancestral cyanobacterial endosymbiont that gave rise to plastids. In contrast, the mitochondria of model systems such as Saccharomyces cerevisiae, mammals, and Arabidopsis thaliana seem to have replaced the ancestral α-proteobacterial Min-based division machinery with host-derived dynamin-related proteins that form outer contractile rings. Here, we show that the mitochondrial division system of these model organisms is the exception, rather than the rule, for eukaryotes. We describe endosymbiont-derived, bacterial-like division systems comprising FtsZ and Min proteins in diverse less-studied eukaryote protistan lineages, including jakobid and heterolobosean excavates, a malawimonad, stramenopiles, amoebozoans, a breviate, and an apusomonad. For two of these taxa, the amoebozoan Dictyostelium purpureum and the jakobid Andalucia incarcerata, we confirm a mitochondrial localization of these proteins by their heterologous expression in Saccharomyces cerevisiae. The discovery of a proteobacterial-like division system in mitochondria of diverse eukaryotic lineages suggests that it was the ancestral feature of all eukaryotic mitochondria and has been supplanted by a host-derived system multiple times in distinct eukaryote lineages.

• Klíčová slova
• Min proteins, MinCDE, mitochondria, mitochondrial division, mitochondrial fission,
• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• Arabidopsis genetika   MeSH
• Bacteria cytologie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčné dělení MeSH
• cytoskeletální proteiny genetika   MeSH
• databáze genetické MeSH
• Dictyostelium metabolismus   MeSH
• DNA bakterií genetika   MeSH
• fylogeneze MeSH
• mitochondriální dynamika * MeSH
• mitochondrie metabolismus   MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• plastidy metabolismus   MeSH
• pravděpodobnostní funkce MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• bakteriální proteiny MeSH
• cytoskeletální proteiny MeSH
• DNA bakterií MeSH
• FtsZ protein, Bacteria MeSH Prohlížeč
• MinC protein, Bacteria MeSH Prohlížeč
• MinD protein, E coli MeSH Prohlížeč
• MinE protein, E coli MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• proteiny z Escherichia coli MeSH

Upon infection, Legionella pneumophila uses the Dot/Icm type IV secretion system to translocate effector proteins from the Legionella-containing vacuole (LCV) into the host cell cytoplasm. The effectors target a wide array of host cellular processes that aid LCV biogenesis, including the manipulation of membrane trafficking. In this study, we used a hidden Markov model screen to identify two novel, non-eukaryotic soluble NSF attachment protein receptor (SNARE) homologs: the bacterial Legionella SNARE effector A (LseA) and viral SNARE homolog A proteins. We characterized LseA as a Dot/Icm effector of L. pneumophila, which has close homology to the Qc-SNARE subfamily. The lseA gene was present in multiple sequenced L. pneumophila strains including Corby and was well distributed among L. pneumophila clinical and environmental isolates. Employing a variety of biochemical, cell biological and microbiological techniques, we found that farnesylated LseA localized to membranes associated with the Golgi complex in mammalian cells and LseA interacted with a subset of Qa-, Qb- and R-SNAREs in host cells. Our results suggested that LseA acts as a SNARE protein and has the potential to regulate or mediate membrane fusion events in Golgi-associated pathways.

• MeSH
• bakteriální proteiny metabolismus   MeSH
• buněčné linie MeSH
• epitelové buňky mikrobiologie   MeSH
• faktory virulence metabolismus   MeSH
• interakce hostitele a patogenu * MeSH
• Legionella pneumophila fyziologie   MeSH
• lidé MeSH
• makrofágy mikrobiologie   MeSH
• molekulární mimikry * MeSH
• myši MeSH
• rozpustné proteiny pro vazbu faktoru citlivého k N-ethylmaleimidu metabolismus   MeSH
• sekvenční homologie aminokyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• faktory virulence MeSH
• rozpustné proteiny pro vazbu faktoru citlivého k N-ethylmaleimidu MeSH