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.

Mitochondrialbc1complex from yeast has 10 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. Because 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 because of the 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.

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 mosquito Anopheles stephensi shedding light on the evolution of metazoan protein complexes. CompaCt is available from https://github.com/cmbi/compact-bio.

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