The atypical subunit composition of respiratory complexes I and IV is associated with original extra structural domains in Euglena gracilis

H V Miranda-Astudillo,F Bouillenne,K N S Yadav,H Degand,P Cardol,L Colina-Tenorio,P Morsomme,E J Boekema

doi:10.1038/s41598-018-28039-z

Abstract

In mitochondrial oxidative phosphorylation, electron transfer from NADH or succinate to oxygen by a series of large protein complexes in the inner mitochondrial membrane (complexes I–IV) is coupled to the generation of an electrochemical proton gradient, the energy of which is utilized by complex V to generate ATP. In Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes, these respiratory complexes totalize more than 40 Euglenozoa-specific subunits along with about 50 classical subunits described in other eukaryotes. In the present study the Euglena proton-pumping complexes I, III, and IV were purified from isolated mitochondria by a two-steps liquid chromatography approach. Their atypical subunit composition was further resolved and confirmed using a three-steps PAGE analysis coupled to mass spectrometry identification of peptides. The purified complexes were also observed by electron microscopy followed by single-particle analysis. Even if the overall structures of the three oxidases are similar to the structure of canonical enzymes (e.g. from mammals), additional atypical domains were observed in complexes I and IV: an extra domain located at the tip of the peripheral arm of complex I and a “helmet-like” domain on the top of the cytochrome c binding region in complex IV.

Highlights

Mitochondria generate most of the energy in eukaryotic cells via oxidative phosphorylation (OXPHOS)
In a previous study we reported that the respiratory complexes of E. gracilis comprise many subunits which were described in trypanosomatid respiratory complexes and, many subunits were lacking which are conserved among mammals and fungi[27]
The fraction containing complex I was almost pure, as judged by the main 1.4 MDa complex observed after BN-PAGE analysis, while the fractions enriched in complex III (500 kDa) or complex IV (460 kDa) were slightly contaminated

Summary

Introduction

Mitochondria generate most of the energy in eukaryotic cells via oxidative phosphorylation (OXPHOS). The core subunits (Cox[1], Cox[2], and Cox3) are highly hydrophobic integral membrane proteins without substantial extramembrane domains Their structure is highly-conserved in α-proteobacteria and eukaryotes. The remaining 11 mammalian accessory subunits are located surrounding the catalytic core[10,11] This complex exists as a 420 kDa dimer in its crystalline state, the fully active monomeric form can be isolated[12,13,14]. A model organism among Euglenids, is a secondary photosynthetic unicellular eukaryote that arises from an endosymbiosis between a green alga and an ancient phagotroph euglenozoan species[17,18] This flagellate has a remarkable adaptability to various environmental conditions.

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Conclusion