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.

SummaryToluene is anoxically degraded to CO 2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99-), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox-active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (␣), 90 (␣Ј), 12 (␤) and 10 kDa (␥) were detected on sodium dodecyl sulphate (SDS) gels. The N-terminal sequences of the ␣-and ␣Ј-subunits were identical, suggesting a C-terminal degradation of half of the ␣-subunits to give the ␣Ј-subunit. The composition of native enzyme therefore appears to be ␣ 2 ␤ 2 ␥ 2 . A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical-containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the ␣Ј-subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7-promotor/polymerase system.

Images of entire cells are preceding atomic structures of the separate molecular machines that they contain. The resulting gap in knowledge can be partly bridged by protein-protein interactions, bioinformatics, and electron microscopy. Here we use interactions of known three-dimensional structure to model a large set of yeast complexes, which we also screen by electron microscopy. For 54 of 102 complexes, we obtain at least partial models of interacting subunits. For 29, including the exosome, the chaperonin containing TCP-1, a 3'-messenger RNA degradation complex, and RNA polymerase II, the process suggests atomic details not easily seen by homology, involving the combination of two or more known structures. We also consider interactions between complexes (cross-talk) and use these to construct a structure-based network of molecular machines in the cell.

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