Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors

Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.

Mitochondrial Disease

Primary mitochondrial diseases are a heterogeneous group of disorders that result from defects of the oxidative phosphorylation system of the mitochondria. Often underrecognized, mitochondrial diseases are uncommon (estimated incidence, 1 in 10,000 live births). Mitochondria are double-membrane–bound cytoplasmic organelles whose primary function is to provide energy (ie, adenosine triphosphate [ATP]) from the breakdown of carbohydrates, protein, and lipids by means of the electron transport chain and the oxidative phosphorylation system. The respiratory chain of mitochondria, located in the inner mitochondrial membrane, consists of 5 multimeric protein complexes (complexes I-IV and ATP synthase [complex V]). The structural proteins of these complexes are encoded by both mitochondrial and nuclear genes. Therefore, primary mitochondrial disorders can follow a maternal or mendelian inheritance pattern.

Abstract P-15: Cryo-Electron Microscopy Study of Dehydrogenase Complexes Interaction with Oxidative Phosphorylation System Supercomplex

Background: Electron transport chain (ETC) complexes, pyruvate dehydrogenase complex (PDC), and α-ketoglutarate dehydrogenase complex (KGDC) are important elements in mitochondrial metabolism. The localization of the aforementioned protein complexes differs since oxidative phosphorylation complexes are membrane proteins, while dehydrogenase complexes (DCs) are contained in the mitochondrial matrix. Our previous cryo-electron tomography (cryo-ET) studies showed the existence of a full oxidative phosphorylation system supercomplex consisting of ETC complexes and ATP synthases (Nesterov et al., 2021). Literature data also shows the binding of fatty acid oxidation enzymes to ETC complex I (Wang et al., 2010). Although it has long been shown that PDCs can bind to complex I (Sumegi et al., 1984) in vitro, this has not been visualized directly in mitochondria and the binding mechanisms are still unknown. Methods: The mitochondria were isolated from Wistar rat heart ventricles according to a standard procedure (Nesterov et al., 2021). The dense mitochondrial suspension was diluted to ~0.3mg/ml in a respiration medium. Phosphorylation was started 10 minutes prior to vitrification. Experimental data was obtained by cryo-ET using Titan Krios and processed with IMOD and RELION. Results: The tomograms show that the significant part of DCs is localized near the inner membrane of partially destroyed mitochondria in an array-like fashion. Sole PDCs and KGDCs can be identified on the images and their position appears to be close to ETC complex I. Subtomogram averaging of close to the membrane DCs showed that there is no specific density between them, suggesting that they are not linked with identical proteins or that this link may be soft. Significant damage to the mitochondrial membrane leads to the formation of membrane-unbound DCs fraction. It suggests that coupling of DCs with ETC complexes can be controlled in vivo by the topology of the inner mitochondrial membrane and the volume of the mitochondrial matrix. Conclusion: The obtained results show a possibility of unprecedentedly large multienzyme complex formation, including almost all main mitochondrial metabolic systems. Although cryo-ET of partially destroyed mitochondria showed close localization of PDC and KGDC to complex I, further studies are required in intact mitochondria. The mechanism of their binding also remains an open question.

Ordered Clusters of the Complete Oxidative Phosphorylation System in Cardiac Mitochondria

The existence of a complete oxidative phosphorylation system (OXPHOS) supercomplex including both electron transport system and ATP synthases has long been assumed based on functional evidence. However, no structural confirmation of the docking between ATP synthase and proton pumps has been obtained. In this study, cryo-electron tomography was used to reveal the supramolecular architecture of the rat heart mitochondria cristae during ATP synthesis. Respirasome and ATP synthase structure in situ were determined using subtomogram averaging. The obtained reconstructions of the inner mitochondrial membrane demonstrated that rows of respiratory chain supercomplexes can dock with rows of ATP synthases forming oligomeric ordered clusters. These ordered clusters indicate a new type of OXPHOS structural organization. It should ensure the quickness, efficiency, and damage resistance of OXPHOS, providing a direct proton transfer from pumps to ATP synthase along the lateral pH gradient without energy dissipation.

Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly

Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.

Oligomeric hypercomplex of the complete oxidative phosphorylation system in heart mitochondria

The existence of a complete oxidative phosphorylation system supercomplex including both electron transport system and ATP synthases has long been assumed based on functional evidence. However, no conclusive structural confirmation has been obtained. In this study cryo-electron tomography was used to reveal the supramolecular architecture of the rat heart mitochondria cristae. We show that rows of respiratory chain supercomplexes are connected with rows of ATP synthases forming the oligomeric hypercomplex. The discovered hypercomplexes may increase the effectiveness of oxidative phosphorylation ensuring a direct proton transfer from pumps to ATP synthase along the membrane without energy dissipation.