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.

• MeSH
• biotinylace MeSH
• delece genu * MeSH
• down regulace MeSH
• Eukaryota genetika   MeSH
• fenotyp MeSH
• fylogeneze MeSH
• genom mitochondriální MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• respirační komplex I metabolismus   MeSH
• respirační komplex IV metabolismus   MeSH
• Trypanosoma brucei brucei metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• protozoální proteiny MeSH
• respirační komplex I MeSH
• respirační komplex IV 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
• buněčný cyklus fyziologie   MeSH
• kinetoplastová DNA genetika   MeSH
• leucylaminopeptidasa genetika   metabolismus   MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie metabolismus   ultrastruktura   MeSH
• protozoální DNA genetika   MeSH
• protozoální proteiny metabolismus   MeSH
• replikace DNA fyziologie   MeSH
• Trypanosoma brucei brucei genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kinetoplastová DNA MeSH
• leucylaminopeptidasa MeSH
• mitochondriální DNA MeSH
• protozoální DNA MeSH
• protozoální proteiny MeSH

UNLABELLED: Mitochondrial chaperones have multiple functions that are essential for proper functioning of mitochondria. In the human-pathogenic protist Trypanosoma brucei, we demonstrate a novel function of the highly conserved machinery composed of mitochondrial heat shock proteins 70 and 40 (mtHsp70/mtHsp40) and the ATP exchange factor Mge1. The mitochondrial DNA of T. brucei, also known as kinetoplast DNA (kDNA), is represented by a single catenated network composed of thousands of minicircles and dozens of maxicircles packed into an electron-dense kDNA disk. The chaperones mtHsp70 and mtHsp40 and their cofactor Mge1 are uniformly distributed throughout the single mitochondrial network and are all essential for the parasite. Following RNA interference (RNAi)-mediated depletion of each of these proteins, the kDNA network shrinks and eventually disappears. Ultrastructural analysis of cells depleted for mtHsp70 or mtHsp40 revealed that the otherwise compact kDNA network becomes severely compromised, a consequence of decreased maxicircle and minicircle copy numbers. Moreover, we show that the replication of minicircles is impaired, although the lack of these proteins has a bigger impact on the less abundant maxicircles. We provide additional evidence that these chaperones are indispensable for the maintenance and replication of kDNA, in addition to their already known functions in Fe-S cluster synthesis and protein import. IMPORTANCE: Impairment or loss of mitochondrial DNA is associated with mitochondrial dysfunction and a wide range of neural, muscular, and other diseases. We present the first evidence showing that the entire mtHsp70/mtHsp40 machinery plays an important role in mitochondrial DNA replication and maintenance, a function likely retained from prokaryotes. These abundant, ubiquitous, and multifunctional chaperones share phenotypes with enzymes engaged in the initial stages of replication of the mitochondrial DNA in T. brucei.

• MeSH
• kinetoplastová DNA genetika   metabolismus   MeSH
• lidé MeSH
• mitochondriální DNA genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP40 genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP70 genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• replikace DNA * MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• trypanozomóza africká parazitologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• kinetoplastová DNA MeSH
• mitochondriální DNA MeSH
• proteiny tepelného šoku HSP40 MeSH
• proteiny tepelného šoku HSP70 MeSH
• protozoální proteiny MeSH

Kinetoplastids are flagellated protozoans, whose members include the pathogens Trypanosoma brucei, T. cruzi and Leishmania species, that are considered among the earliest diverging eukaryotes with a mitochondrion. This organelle has become famous because of its many unusual properties, which are unique to the order Kinetoplastida, including an extensive kinetoplast DNA network and U-insertion/deletion type RNA editing of its mitochondrial transcripts. In the last decade, considerable progress has been made in elucidating the complex machinery of RNA editing. Moreover, our understanding of the structure and replication of kinetoplast DNA has also dramatically improved. Much less however, is known, about the developmental regulation of RNA editing, its integration with other RNA maturation processes, stability of mitochondrial mRNAs, or evolution of the editing process itself. Yet the profusion of genomic data recently made available by sequencing consortia, in combination with methods of reverse genetics, hold promise in understanding the complexity of this exciting organelle, knowledge of which may enable us to fight these often medically important protozoans.

• MeSH
• editace RNA MeSH
• exprese genu MeSH
• genetická transkripce MeSH
• genom protozoální * MeSH
• Kinetoplastida genetika   MeSH
• kinetoplastová DNA chemie   MeSH
• messenger RNA metabolismus   MeSH
• mitochondriální geny * MeSH
• mitochondrie genetika   MeSH
• RNA protozoální metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• kinetoplastová DNA MeSH
• messenger RNA MeSH
• RNA protozoální MeSH