Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we then generated a draft conservation map consisting of >1 million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering revealed a spectrum of conservation, ranging from ancient Eukaryal assemblies likely serving cellular housekeeping roles for at least 1 billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, by affinity-purification and by functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic significance and adaptive value to animal cell systems.

SUMMARY Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the Membrane Yeast Two-Hybrid (MYTH) and the Mammalian Membrane Two-Hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.

G‐protein‐coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR‐mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two‐hybrid (MYTH) approach and identified interacting partners for 48 selected full‐length human ligand‐unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5‐HT4d, and adenosine ADORA2A receptors. Our data represent the first large‐scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.

The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmidbased NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.T he repair of DNA double-strand breaks (DSBs) is an essential process required for the preservation of genome integrity and the normal functioning of the cell (1). These cytotoxic lesions are repaired by major DSB repair pathways, including the homologous recombination (HR) (2) and nonhomologous end-joining (NHEJ) systems (1). While the former is the prevalent pathway in the unicellular budding yeast Saccharomyces cerevisiae (3), the latter is more prevalent in mammalian cells, especially those that are quiescent (4), and can repair DNA lesions even if there is no homologous strand (5). Notably, the impairment of NHEJ in mammalian cells is frequently linked to genomic instability, cancer, and lymphoid V(D)J (i.e., variable, diversity, and joining gene segments) recombination defects. Therefore, a detailed molecular understanding of this pathway would provide critical insight into the genetic risk factors related to carcinogenesis or immunological disorders (6).As in mammalian cells, the core components of the classical NHEJ pathway in S. cerevisiae depends on three major complexes, YKu (Ku), MRX, and DNL4, which are rapidly recruited to DSBs (7). Initially, the yeast Ku heterodimer (Ku70/80) binds to each end of a DSB, serving as an anchor for protein complexes involved in securing and annealing the break, also suppressing the competing HR pathway (8). After this, the DSB processing complex MRX (Mre11-Rad50-Xrs2), which acts at an early stage of both the NHEJ and HR repair pathways (9), spans the lesions so that the DNA ligase complex, DNL4 (i.e., Dnl4-Lif1-Nej1), can rejoin the DSB ends (10).While the actions of these core protein complexes in y...

Highlights d BraInMap is a global proteomic survey of over 1,000 multiprotein brain complexes d Near-native complex identification by CF-MS and reconstruction by computer learning d Technique interrogates complexes in normal and pathophysiological context d Allows study of functional modules that are adversely affected in neurological diseases

SUMMARY The prevalence of autism spectrum disorders (ASDs) is rapidly growing, yet its molecular basis is poorly understood. We used a systems approach in which ASD candidate genes were mapped onto the ubiquitous human protein complexes and the resulting complexes were characterized. The studies revealed the role of histone deacetylases (HDAC1/2) in regulating the expression of ASD orthologs in the embryonic mouse brain. Proteome-wide screens for the co-complexed subunits with HDAC1 and six other key ASD proteins in neuronal cells revealed a protein interaction network, which displayed preferential expression in fetal brain development, exhibited increased deleterious mutations in ASD cases, and were strongly regulated by FMRP and MECP2 causal for Fragile X and Rett syndromes, respectively. Overall, our study reveals molecular components in ASD, suggests a shared mechanism between the syndromic and idiopathic forms of ASDs, and provides a systems framework for analyzing complex human diseases.

SummaryMitochondrial protein (MP) assemblies undergo alterations during neurogenesis, a complex process vital in brain homeostasis and disease. Yet which MP assemblies remodel during differentiation remains unclear. Here, using mass spectrometry-based co-fractionation profiles and phosphoproteomics, we generated mitochondrial interaction maps of human pluripotent embryonal carcinoma stem cells and differentiated neuronal-like cells, which presented as two discrete cell populations by single-cell RNA sequencing. The resulting networks, encompassing 6,442 high-quality associations among 600 MPs, revealed widespread changes in mitochondrial interactions and site-specific phosphorylation during neuronal differentiation. By leveraging the networks, we show the orphan C20orf24 as a respirasome assembly factor whose disruption markedly reduces respiratory chain activity in patients deficient in complex IV. We also find that a heme-containing neurotrophic factor, neuron-derived neurotrophic factor [NENF], couples with Parkinson disease-related proteins to promote neurotrophic activity. Our results provide insights into the dynamic reorganization of mitochondrial networks during neuronal differentiation and highlights mechanisms for MPs in respirasome, neuronal function, and mitochondrial diseases.

Mitochondrial protein (MP) dysfunction has been linked to neurodegenerative disorders (NDs); however, the discovery of the molecular mechanisms underlying NDs has been impeded by the limited characterization of interactions governing MP function. Here, using mass spectrometry (MS)-based analysis of 210 affinity-purified mitochondrial (mt) fractions isolated from 27 epitope-tagged human ND-linked MPs in HEK293 cells, we report a high-confidence MP network including 1,964 interactions among 772 proteins (>90% previously unreported). Nearly three-fourths of these interactions were confirmed in mouse brain and multiple human differentiated neuronal cell lines by primary antibody immunoprecipitation and MS, with many linked to NDs and autism. We show that the SOD1-PRDX5 interaction, critical for mt redox homeostasis, can be perturbed by amyotrophic lateral sclerosis-linked SOD1 allelic variants and establish a functional role for ND-linked factors coupled with IκBɛ in NF-κB activation. Our results identify mechanisms for ND-linked MPs and expand the human mt interaction landscape.