Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

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Wnt/␤-catenin signaling is fundamental in embryogenesis and tissue homeostasis in metazoans. Upon Wnt stimulation, cognate coreceptors LRP5 and LRP6 ([LRP5/6] low-density lipoprotein receptor-related proteins 5 and 6) are activated via phosphorylation at key residues. Although several kinases have been implicated, the LRP5/6 activation mechanism remains unclear. Here, we report that transmembrane protein 198 (TMEM198), a previously uncharacterized seven-transmembrane protein, is able to specifically activate LRP6 in transducing Wnt signaling. TMEM198 associates with LRP6 and recruits casein kinase family proteins, via the cytoplasmic domain, to phosphorylate key residues important for LRP6 activation. In mammalian cells, TMEM198 is required for Wnt signaling and casein kinase 1-induced LRP6 phosphorylation. During Xenopus embryogenesis, maternal and zygotic tmem198 mRNAs are widely distributed in the ectoderm and mesoderm. TMEM198 is required for Wnt-mediated neural crest formation, antero-posterior patterning, and particularly engrailed-2 expression in Xenopus embryos. Thus, our results identified TMEM198 as a membrane scaffold protein that promotes LRP6 phosphorylation and Wnt signaling activation.

Here we identified a hydrophobic 6.4kDa protein, Cox26, as a novel component of yeast mitochondrial supercomplex comprising respiratory complexes III and IV. Multi-dimensional native and denaturing electrophoretic techniques were used to identify proteins interacting with Cox26. The majority of the Cox26 protein was found non-covalently bound to the complex IV moiety of the III-IV supercomplexes. A population of Cox26 was observed to exist in a disulfide bond partnership with the Cox2 subunit of complex IV. No pronounced growth phenotype for Cox26 deficiency was observed, indicating that Cox26 may not play a critical role in the COX enzymology, and we speculate that Cox26 may serve to regulate or support the Cox2 protein. Respiratory supercomplexes are assembled in the absence of the Cox26 protein, however their pattern slightly differs to the wild type III-IV supercomplex appearance. The catalytic activities of complexes III and IV were observed to be normal and respiration was comparable to wild type as long as cells were cultivated under normal growth conditions. Stress conditions, such as elevated temperatures resulted in mild decrease of respiration in non-fermentative media when the Cox26 protein was absent.

DDX RNA helicases promote RNA processing but DDX3X is also known to activate casein kinase 1 ϵ (CK1ϵ). Here we show that not only is protein kinase stimulation a latent property of other DDX proteins towards CK1ϵ, but that this extends to casein kinase 2 (CK2α2) as well. CK2α2 enzymatic activity is stimulated by a variety of DDX proteins and we identify DDX1/24/41/54 as physiological activators required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X reveals that CK1 and CK2 kinase stimulation engages its RNA binding- but not catalytic motifs. Mathematical modelling of enzyme kinetics and stopped-flow spectroscopy converge that DDX proteins function as nucleotide exchange factor towards CK2α2 that reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as a new principle in kinase regulation and an evolved function of DDX proteins.

DDX RNA helicases promote RNA processing, but DDX3X also activates casein kinase 1 (CK1ε). We show that other DDX proteins also stimulate the protein kinase activity of CK1ε and that this extends to casein kinase 2 (CK2). CK2 enzymatic activity was stimulated by various DDX proteins at high substrate concentrations. DDX1, DDX24, DDX41, and DDX54 were required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X indicated that CK1 and CK2 kinase stimulation engages its RNA binding but not catalytic motifs. Mathematical modeling of enzyme kinetics and stopped-flow spectroscopy showed that DDX proteins function as nucleotide exchange factors toward CK2 and reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as important for kinase regulation and as a generic function of DDX proteins.