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

Result code in 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>

Result on the web

<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

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Result language

angličtina

Original language name

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

Original language description

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.

Czech name

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Czech description

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Type

O - Miscellaneous

CEP classification

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OECD FORD branch

10608 - Biochemistry and molecular biology

Project

<a href="/en/project/LL1205" target="_blank" >LL1205: Exploration of the unique charakters od the Trypanosoma brucei FoF1 ATP synthase complex for future drug development against african sleeping sickness.</a><br>

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2015

Confidentiality

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