Deep Proteomic Analysis of Trypanosoma brucei FoF1--ATP synthase reveals unique features

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60077344%3A_____%2F15%3A00488330" target="_blank" >RIV/60077344:_____/15:00488330 - isvavai.cz</a>

Výsledek na webu

<a href="http://www.parazitologie.cz/protozoologie/Protodny2015/JPD_sbornik_2015.pdf" target="_blank" >http://www.parazitologie.cz/protozoologie/Protodny2015/JPD_sbornik_2015.pdf</a>

DOI - Digital Object Identifier

—

Jazyk výsledku

angličtina

Název v původním jazyce

Deep Proteomic Analysis of Trypanosoma brucei FoF1--ATP synthase reveals unique features

Popis výsledku v původním jazyce

Membrane-bound FoF1-ATP synthases are central enzymes in the energetic metabolism of bacteria and eukaryotic organelles. We established procedures to isolate either the catalytic F1-ATPase moiety or the entire FoF1-ATP synthase from procyclic stage T. brucei. The first approach involves two purification steps – ion exchange followed by gel filtration chromatography. The second strategy relies on a GST-tagged inhibitory peptide (TbIF1) to tightly bind the FoF1-ATP synthase, which is subsequently purified using a GST-Trap column. Both methods have been optimized to yield very pure complexes that are currently undergoing structural studies using X-ray crystallography and high-resolution cryo-EM. The purification of functional F1-ATPase reveals that it is comprised of all the usual eukaryotic components, plus a multicopy subunit p18 that is essential. The silencing of p18 results in dramatic losses of F1 complexes. Furthermore, we mapped two cleavage sites in the sequence of subunit, which is split into two fragments in vivo. In addition to the F1 components, the isolated FoF1-ATP synthase contains only two homologous subunits of the eukaryotic Fo-moiety: the proton channel subunit c and OSCP. Additional identified subunits have no homology outside the Euglenozoa, suggesting that the peripheral stalk and membrane-bound subunits are either extremely divergent or replaced by other proteins. Quantitative mass spectrometry with isotope-labelled standards is being implemented to assess the stoichiometry of p18 and selected Fo subunits. Our data are in line with accumulating evidence from other non-model eukaryotes that the compositional diversity of functionally conserved F1Fo-ATP synthases is significantly higher than previously thought.

Název v anglickém jazyce

Deep Proteomic Analysis of Trypanosoma brucei FoF1--ATP synthase reveals unique features

Popis výsledku anglicky

Membrane-bound FoF1-ATP synthases are central enzymes in the energetic metabolism of bacteria and eukaryotic organelles. We established procedures to isolate either the catalytic F1-ATPase moiety or the entire FoF1-ATP synthase from procyclic stage T. brucei. The first approach involves two purification steps – ion exchange followed by gel filtration chromatography. The second strategy relies on a GST-tagged inhibitory peptide (TbIF1) to tightly bind the FoF1-ATP synthase, which is subsequently purified using a GST-Trap column. Both methods have been optimized to yield very pure complexes that are currently undergoing structural studies using X-ray crystallography and high-resolution cryo-EM. The purification of functional F1-ATPase reveals that it is comprised of all the usual eukaryotic components, plus a multicopy subunit p18 that is essential. The silencing of p18 results in dramatic losses of F1 complexes. Furthermore, we mapped two cleavage sites in the sequence of subunit, which is split into two fragments in vivo. In addition to the F1 components, the isolated FoF1-ATP synthase contains only two homologous subunits of the eukaryotic Fo-moiety: the proton channel subunit c and OSCP. Additional identified subunits have no homology outside the Euglenozoa, suggesting that the peripheral stalk and membrane-bound subunits are either extremely divergent or replaced by other proteins. Quantitative mass spectrometry with isotope-labelled standards is being implemented to assess the stoichiometry of p18 and selected Fo subunits. Our data are in line with accumulating evidence from other non-model eukaryotes that the compositional diversity of functionally conserved F1Fo-ATP synthases is significantly higher than previously thought.

Druh

O - Ostatní výsledky

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/LL1205" target="_blank" >LL1205: Charakterizace unikátních vlastností esenciální FoF1 ATP syntázy u původce africké spavé nemoci Trypanosoma bucei za účelem vývoje inhibitorů tohoto komplexu.</a><br>

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2015

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů