Identification and functional characterisation of different inner mitochondrial membrane supercomplexes

The inner mitochondria membrane (IMM) is densely packed with proteins necessary for mitochondrial activity, including oxidative phosphorylation (OXPHOS) complexes and transporters. Mitochondrial respiratory chain (MRC) complexes, which generate ATP by OXPHOS, associate in higher-order assemblies, known as supercomplexes (SC), that are structurally interdependent. Numerous patients carrying mutations in a single complex, indeed, present with combined enzyme deficiencies and, in particular, the destabilisation of complex I (CI) has been often described in the absence of complex III (CIII). To clarify the structural and functional relationships between complexes, we have analysed a MTCYB-deficient human cell line, unable to assemble CIII. Our results showed that in this line, CI biogenesis was blocked by preventing the incorporation of the NADH module, the last step of CI assembly, rather than decreasing its stability. Moreover, complex IV (CIV) biogenesis was impaired as well, and CIV subunits appeared sequestered within CIII subassemblies. Therefore, we propose that CIII is central not only for the formation of SC but also for the maturation of the other electron transport chain complexes. These results challenge the previous SC model that described the formation of fully assembled individual complexes before the association in SC. In contrast, they support a cooperative-assembly model in which the main role of CIII in SC is to provide a structural and functional platform for the completion of overall MRC biogenesis. Next, we identified and characterised the interaction between CIV and the mitochondrial calcium uniporter complex (MCUC), responsible for mitochondrial calcium uptake and homeostasis. Our data, indeed, showed a specific physical interaction between this ETC enzyme and various subunits of MCUC in first and second dimension blue native PAGE, SILAC labelling/pulldown, and BioID proteomic-based methods. We then investigated the effects of this association with CIV, measuring enzyme activity and mitochondrial respiration, but also on MCUC and CIV distribution in the IMM, by N-structured illuminated super-resolution microscopy (N-SIM). Our results showed a specific reduction in CIV activity in the absence of MICU1, the main regulator of the uniporter, but surprisingly no effects were observed after MCU downregulation or pharmacological inhibition of mitochondrial Ca2+ entry. Instead, the lack of CIV seems to have an impact on MCU-containing complexes formation and induces a re-localisation of MCU from the cristae membrane to the inner boundary membrane. Further experiments will be performed to shed light on the physiological relevance of this interaction.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Protasoni, Margherita

Advisor dc:contributor.advisor

• Prudent, Julien

• mitochondria
• OXPHOS
• complex assembly
• supercomplexes
• MCU
• mitochondrial medicine
• mitochondrial physiology

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/4434ce04-b672-4413-b5c7-df0cbfa08e3e/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0001-6557-461X

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/316344