Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

Abstract

Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I-III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly. mitochondria | mass spectrometry | interactome | cross-linking | protein interaction reporter M itochondrial proteins play a diverse role in cellular biology and disease. Mitochondrial dysfunction directly causes multiple inherited diseases (1) and is implicated in common diseases, including neurological developmental disorders (2, 3), neurodegenerative and cardiovascular diseases (4-6), diabetes (7), asthma (8), cancer (9), and age-related disease (10). In mammals, these organelles have evolved to retain more than 1,000 proteins that interact within a complex, i.e., dual membrane architecture (11,12). Within the mitochondrial proteome, the "powerhouse" functions are carried out by the core constituents of the oxidative phosphorylation (OXPHOS) system [complexes I-IV of the electron transport chain (ETC) and ATP synthase (complex V)]. These proteins are necessary for creation of the mitochondrial electrochemical gradient that powers synthesis of ATP. This system includes critical protein-protein interactions within individual OXPHOS complexes as well as "supercomplex" interactions between ETC complexes I, III, and IV in the respirasome (13). Deficient supercomplex formation has been proposed as a critical mitochondrial defect in failing hearts (5,6,14,15), and dynamic rearrangement of supercomplexes has been implicated in noncanonical mitochondrial functions such as antibacterial innate immune responses (16). Assessing these interactions is further complicated by regulatory posttranslational modification and conformational changes of mitochondrial proteins (17)(18)(19)(20). Advances in this area have been impeded, in part, by the lack of large-scale detection of dynamic, sometimes transient, interactions between membrane proteins. Thus, large-scale determination of the protein interactome within mitochondria would provide a valuable tool to adv...

Cross‐linked peptide identification: A computational forest of algorithms

“…The different types of visualization are illustrated on RNA polymerase I of Saccharomyces cerevisiae, a dataset that is available through Xlink Analyzer 16 studies through cross-linking. Examples are the elucidation of the murine mitochondrial protein interactome, 89 mapping of the disulfide proteome, 90 analysis of HeLa cells 47 and Shewanella oneidensis. 91 This low number finds its origin in the challenges met when analysing crosslinked peptides by mass spectrometry.…”

Mitochondrial protein interaction landscape of SS-31

“…Chemical cross-linking mass spectrometry (XL-MS) has evolved to be a valuable and widely used tool for studying protein structures and interactions [20][21][22][23]. Recent development of protein interaction reporter (PIR) based XL-MS technology in our group has further enabled large-scale identification of protein interactions in complex mixtures from living cells [24][25][26] and isolated functional organelles [27,28]. Previous studies have shown that SS-31, when added to permeabilized cells or isolated mitochondria, can decrease generation of H2O2 , increase O2 consumption and ATP production [18,19,[29][30][31].…”

Impact of modular mitochondrial epistatic interactions on the evolution of human subpopulations

“…In particular, a large portion of COX genes co-mutate in AS and AM populations whereas in AF, EU and OC populations, there was greater co-mutation bias in functional regions of HVS. Although mitochondrial genome epistasis is largely described in the context of mitochondrial-nuclear interaction due to the closed assembly of OXPHOS complexes (Fonseca, et al, 2008;Picard, et al, 2018;Connallon, et al, 2018;Schweppe, et al, 2017), there are many reports describing the presence of mitochondrial-mitochondrial epistasis, for example, shared family features in Han Chinese family (Wang, et al, 2015) and homoplasy guided by mt-tRNA genes (Moreno-Loshuertos, et al, 2011). Furthermore, few co-mutations were found to be mapped with previously known potential disease alleles, implicating association of disease phenotypes within mt-genome sequences.…”