CORDIS - EU research results



The objective of this project was to contribute some knowledge about the factors and mechanisms acting in the mitochondrial respiratory chain (MRC) complex assembly. During the development of this work, the assembly of Complex I (CI) and, mainly, Complex III (CIII) were studied. Additionally, the interaction among the MRC complexes as part of the supercomplexes (SC) or respirasomes, was also analysed.
Mammalian CI is the largest and most complicated of the MRC enzymes, composed of 44 subunits, seven of which (the ND subunits) are encoded in the mitochondrial genome. In the last few years there has been a very active research to discover the assembly factors necessary for the process, as well as the way the subunits come together to give rise to the mature and functional complex. Thanks to the analyses performed in mouse cell lines mutated in different ND genes and using mouse liver isolated mitochondria (in organello analyses) we were able to determine the five distinct entry points of the mitochondrially encoded subunits into the assembly intermediates during the CI assembly pathway [1].
Practically, the model used to explain mammalian CIII assembly is based on the one proposed for the yeast Saccharomyces cerevisiae, due to the high conservation of its components. However, for a much better understanding of the mammalian enzyme and its interaction with the rest of the MRC, it is necessary to study it specifically in mammalian mitochondria, as there are significant differences with respect to the yeast system. Two aspects of the CIII assembly process were studied: 1) the importance and order of incorporation of the structural subunits and 2) the proteins (assembly factors) required for the proper maturation of the enzyme. The incorporation of Mt-Cytb, the only CIII subunit encoded in the mtDNA was studied by in organello protein synthesis. The rest of the subunits and some of the CIII assembly factors, all nuclearly encoded, were radioactively labelled and imported into isolated mitochondria. After the in organello synthesis or import, the samples were analysed by Blue-Native Gel Electrophoresis. These experiments showed that CIII was assembled in isolated mitochondria and which of the subunits are incorporated in the complex in these conditions. Also, the incorporation of the assembly factors into high molecular weight complexes was demonstrated. On the other hand, cell lines carrying mutations in MT-CYB that impair CIII assembly were studied to determine which subunits are present when the mitochondrially encoded one is absent. These analyses showed that some of the CIII subunits are present in assembly intermediates when MT-CYB is missing, and suggested that the assembly model currently proposed might not be correct. In the search for new mammalian CIII assembly factors by homology with yeast proteins, we found LYRM7 as an ortholog of Mzm1. Our study of the protein confirmed that in human cells it functioned as a UQCRFS1 (Rieske Iron-Sulphur protein) chaperone and was renamed as MZM1L [2]. The identification of this protein allowed the finding of pathological mutations in LYRM7/MZM1L, associated to CIII deficiency [3].
The MRC complexes I, III and IV interact forming the so-called SC or respirasomes. Studying the SC composition in different mouse strains we were able to discover that a microdeletion in Cox7a2L is responsible for the lack of interaction between CIII and CIV into the SC. Thus, we characterised the function of Cox7a2L as necessary for the correct formation of the whole respirasome, i. e., the incorporation of CIV into the SC and was therefore renamed as Scafi (Supercomplex assembly factor 1). This constituted the genetic proof for the formation of the SC [4].


1. Perales-Clemente, E., et al., Five entry points of the mitochondrially encoded subunits in Mammalian complex I assembly. Mol Cell Biol, 2010. 30(12): p. 3038-47.
2. Sanchez, E., et al., LYRM7/MZM1L is a UQCRFS1 chaperone involved in the last steps of mitochondrial Complex III assembly in human cells. Biochim Biophys Acta, 2013. 1827(3): p. 285-93.
3. Invernizzi, F., et al., A homozygous mutation in LYRM7/MZM1L associated with early onset encephalopathy, lactic acidosis, and severe reduction of mitochondrial complex III activity. Hum Mutat, 2013. 34(12): p. 1619-22.
4. Lapuente-Brun, E., et al., Supercomplex assembly determines electron flux in the mitochondrial electron transport chain. Science, 2013. 340(6140): p. 1567-70.