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Complexome profiling allows large-scale, untargeted, and comprehensive characterization of protein complexes in a biological sample using a combined approach of separating intact protein complexes e.g., by native gel electrophoresis, followed by mass spectrometric analysis of the proteins in the resulting fractions. Over the last decade, its application has resulted in a large collection of complexome profiling datasets. While computational methods have been developed for the analysis of individual datasets, methods for large-scale comparative analysis of complexomes from multiple species are lacking. Here, we present Comparative Clustering (CompaCt), that performs fully automated integrative analysis of complexome profiling data from multiple species, enabling systematic characterization and comparison of complexomes. CompaCt implements a novel method for leveraging orthology in comparative analysis to allow systematic identification of conserved as well as taxon-specific elements of the analyzed complexomes. We applied this method to a collection of 53 complexome profiles spanning the major branches of the eukaryotes. We demonstrate the ability of CompaCt to robustly identify the composition of protein complexes, and show that integrated analysis of multiple datasets improves characterization of complexes from specific complexome profiles when compared to separate analysis. We identified novel candidate interactors and complexes in a number of species from previously analyzed datasets, like the emp24, the V-ATPase and mitochondrial ATP synthase complexes. Lastly, we demonstrate the utility of CompaCt for the automated large-scale characterization of the complexome of the mosquitoAnopheles stephensishedding light on the evolution of metazoan protein complexes. CompaCt is available fromhttps://github.com/cmbi/compact-bio.Author summaryProteins carry out essential functions in the majority of processes in life, often by binding with other proteins to form multiprotein complexes. State of the art experimental techniques such as complexome profiling enable large-scale identification of protein complexes in a biological sample. With the increase in use of this method in recent years these experiments have been performed on a variety of species, of which the results are publicly available. Combining the results from these experiments presents a computational challenge, but could identify novel protein complexes and provide insights into their evolution. Here, we introduce CompaCt as a method to integrate complexome profiles from multiple species enabling automatic large-scale characterization of protein complexes. It identifies commonalities as well as the differences between species. By applying CompaCt to a collection of complexome profiles, we identified candidate complexes and interacting proteins in a number of species that were not detected in previous separate analyses of these datasets. In doing so we shed light on the evolutionary origin of several protein complex members, pinpointed the function of biomedically relevant proteins, whose role was previously unknown, and performed the first investigation of theAnopheles stephensicomplexome, a mosquito that transmits the malaria parasite.

Mitochondrialbc1complex from yeast has ten subunits, but only Cytochromeb(Cytb) subunit is encoded in the mitochondrial genome. Cytbhas eight transmembrane helices containing two hemesbfor electron transfer. Cbp3 and Cbp6 assist Cytbsynthesis, and together with Cbp4 induce Cytbhemylation. Subunits Qcr7/Qcr8 participate in the first steps of assembly, and lack of Qcr7 reduces Cytbsynthesis through an assembly-feedback mechanism involving Cbp3/Cbp6. Since Qcr7 resides near the Cytbcarboxyl-region, we wondered whether this region is important for Cytbsynthesis/assembly. Although deletion of the CytbC-region did not abrogate Cytbsynthesis, the assembly-feedback regulation was lost, so Cytbsynthesis was normal even if Qcr7 was missing. Mutants lacking the CytbC-terminus were non-respiratory due to absence of fully assembledbc1complex. By performing complexome profiling, we showed the existence of aberrant early-stage subassemblies in the mutant. In this work we demonstrate that the C-terminal region of Cytbis critical for regulation of Cytbsynthesis andbc1complex assembly.

Pentameric ligand-gated ion channels represent a large family of receptors comprising an extracellular domain, four transmembrane helices and a cytosolic intracellular domain (ICD). ICDs play important roles in receptor localization and trafficking, thus regulating synaptic activity and plasticity. Glycine and GABA type A receptor ICDs bind to the scaffolding protein gephyrin, a master regulator of inhibitory synapses. Here we report the use of yeast lumazine synthase as soluble pentameric protein scaffold for the study of receptor ICDs derived from GlyR α1− and β-subunits. We were able to create ICDs assemblies in a homo- (LS-βICD) and hetero-pentameric state (LS-αβICD) and provide first-in-class structural insights on their high structural flexibility using small angle X-ray scattering. We report a high-affinity interaction between the LS-αβICD and gephyrin leading to thein vitroformation of high-molecular mega-Dalton complexes composed of three gephyrin trimers and three pentamers as basic building block. Depending on the stoichiometric ratios between gephyrin and LS-ICDs the formed complexes grow or shrink in size. In cells, LS-ICDs efficiently recruited gephyrin and were able to accumulate gephyrin at GABAergic synapses in neurons. Our findings collectively propose a new, potentially general, mechanistic concept for a gephyrin-dependent bridging of GlyRs at the inhibitory synapse.

The tricarboxylic acid (TCA) cycle, or Krebs cycle, is the central pathway of energy production in eukaryotic cells and plays a key part in aerobic respiration throughout all kingdoms of life. The enzymes involved in this cycle generate the reducing equivalents NADH and FADH2 by a series of enzymatic reactions, which are utilized by the electron transport chain to produce ATP. One of the pivotal enzymes in this cycle is 2-oxoglutarate dehydrogenase complex (OGDHC), which generates NADH by oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. OGDHC is a megadalton protein complex originally thought to be assembled just from three catalytically active subunits (E1o, E2o, E3). In fungi and animals, however, the protein MRPS36 has more recently been proposed as a putative additional component. Based on extensive XL-MS data obtained from measurements in mice and bovine heart mitochondria, supported by phylogenetic analyses, we provide evidence that MRPS36 is an essential member of OGDHC, albeit only in eukaryotes. Comparative sequence analysis and computational structure predictions reveal that in eukaryotic OGDHC, E2o does not contain the peripheral subunit-binding domain (PSBD), present in bacterial and archaeal E2o’s. We propose that in eukaryotes MRPS36 evolved as an E3 adaptor protein, functionally replacing the PSBD. We further provide a refined structural model of the complete eukaryotic OGDHC containing 16 E1o, 12 E3, and 6 subunits of MRPS36 accommodated around the OGDHC core composed of 24 E2o subunits (3.45 MDa). The model provides new insights into the OGDH complex topology and stipulates putative mechanistic implications.

SummaryMitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multi-proteomic approach to demonstrate regulation of the m-AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca2+/H+ exchanger in the mitochondrial inner membrane, which binds to and inhibits the m-AAA protease. TMBIM5 ensures cell survival and respiration, allowing Ca2+ efflux from mitochondria and limiting mitochondrial hyperpolarization. Persistent hyperpolarization, however, triggers degradation of TMBIM5 and activation of the m-AAA protease. The m-AAA protease broadly remodels the mitochondrial proteome and mediates the proteolytic breakdown of respiratory complex I to confine ROS production and oxidative damage in hyperpolarized mitochondria. TMBIM5 thus integrates mitochondrial Ca2+ signaling and the energetic status of mitochondria with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism.

Combining mass spectrometry based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis inducing factor 1 (AIFM1) forms a defined complex with ~10% of monomeric cytochromecoxidase (COX), but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 inter-crosslinks engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM12complex. Application of two complementary proteomic approaches thus provided unexpected insight into the macromolecular organization of the mitochondrial complexome. Our structural model excludes direct electron transfer between AIFM1 and COX. Notably however, the binding site of cytochromecremains accessible allowing formation of a ternary complex. The discovery of the previously overlooked COX-AIFM12complex and clues provided by the structural model hint at a role of AIFM1 in OXPHOS biogenesis and in programmed cell death.