SUMMARY Cellular processes often depend on stable physical associations between proteins. Despite recent progress, knowledge of the composition of human protein complexes remains limited. To close this gap, we applied an integrative global proteomic profiling approach, based on chromatographic separation of cultured human cell extracts into more than one thousand biochemical fractions which were subsequently analyzed by quantitative tandem mass spectrometry, to systematically identify a network of 13,993 high-confidence physical interactions among 3,006 stably-associated soluble human proteins. Most of the 622 putative protein complexes we report are linked to core biological processes, and encompass both candidate disease genes and unnanotated proteins to inform on mechanism. Strikingly, whereas larger multi-protein assemblies tend to be more extensively annotated and evolutionarily conserved, human protein complexes with 5 or fewer subunits are far more likely to be functionally un-annotated or restricted to vertebrates, suggesting more recent functional innovations.

Vibrio cholerae encodes a small RNA with homology to Escherichia coli RyhB. Like E. coli ryhB, V. cholerae ryhB is negatively regulated by iron and Fur and is required for repression of genes encoding the superoxide dismutase SodB and multiple tricarboxylic acid cycle enzymes. However, V. cholerae RyhB is considerably longer (>200 nucleotides) than the E. coli RNA (90 nucleotides), and it regulates the expression of a variety of genes that are not known to be regulated by RyhB in E. coli, including genes involved in motility, chemotaxis, and biofilm formation. A mutant with a deletion in ryhB had reduced chemotactic motility in low-iron medium and was unable to form wild-type biofilms. The defect in biofilm formation was suppressed by growing the mutant in the presence of excess iron or succinate. The wild-type strain showed reduced biofilm formation in iron-deficient medium, further supporting a role for iron in normal biofilm formation. The ryhB mutant was not defective for colonization in a mouse model and appeared to be at a slight advantage when competing with the wild-type parental strain. Other genes whose expression was influenced by RyhB included those encoding the outer membrane porins OmpT and OmpU, several iron transport systems, and proteins containing heme or iron-sulfur clusters. These data indicate that V. cholerae RyhB has diverse functions, ranging from iron homeostasis to the regulation of biofilm formation.Iron plays a critical role in the cellular metabolism of almost all living organisms. Iron is required for processes as diverse as the tricarboxylic acid (TCA) cycle, electron transport, DNA metabolism, and response to oxidative stress. Because iron has the potential for catalyzing production of reactive oxygen species, excess iron can also pose a significant problem. The influx and intracellular fate of iron must therefore be tightly regulated. This is achieved in part through the action of the irondependent negative regulator Fur, which functions to coordinate the iron status of the cell with the expression of genes involved in iron transport, storage, and metabolism. Under iron-replete conditions, Fur complexes with the ferrous ion and blocks transcription of its regulon by binding to conserved regions termed Fur boxes within the promoter region of these genes. There is another layer of complexity in the scheme of iron-and Fur-dependent regulation. In Escherichia coli, certain genes involved in iron storage, iron metabolism, and antioxidant defense appear to be positively regulated by Fur (6,36,42), and this was recently shown to be mediated through the action of a small RNA (sRNA), RyhB (26). RyhB negatively regulates the expression of sodB (encoding superoxide dismutase), ftn and bfr (encoding ferritin and bacterioferritin), and several iron-sulfur cluster-containing TCA cycle enzyme genes, including the sdh operon (encoding succinate dehydrogenase) and acnA (encoding aconitase). Because RyhB is itself negatively regulated by Fur, the net effect is positive regulation of these genes un...

Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post-transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large-scale protein and mRNA expression datasets from seven different species: two bacteria, yeast, nematode, fly, human, and plant. We find that steady-state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge.

Vibrio cholerae responds to environmental changes by altering the protein composition of its outer membrane. In rich medium, V. cholerae expresses almost exclusively the outer membrane porin OmpU, whereas in minimal medium, OmpT is the dominant porin. The supplementation of a minimal medium with a mixture of asparagine, arginine, glutamic acid, and serine (NRES) promotes OmpU production and OmpT repression at levels similar to those seen with rich media. Here we show that the altered Omp profile is not due to an increase in the growth rate in the presence of supplemental amino acids but requires the addition of specific amino acids. The effects of the NRES mix on Omp production were mediated by ToxR, a known regulator of omp gene expression. No changes in the Omp profile were detected in a toxR mutant. Supplementation with the NRES mix resulted in significantly higher levels of ToxR, and the elevated ToxR levels were sufficient to cause a switch in Omp synthesis. The increase in the level of the ToxR protein correlated with an increase in toxR mRNA levels and was observed only when toxR was expressed from its native promoter. ToxS, which is required for ToxR activity, was necessary for NRES-mediated omp gene regulation but not for the increase in ToxR levels. The growth of V. cholerae in the presence of bile acids also resulted in Omp switching, and this required ToxR. However, unlike the NRES mix, bile acids did not increase either ToxR protein or toxR mRNA levels, suggesting a different mechanism of omp gene regulation by bile than that by amino acids.

The transcription factor Fur regulates the expression of a number of genes in Vibrio cholerae in response to changes in the level of available iron. Fur usually acts as a repressor, but here we show that Fur positively regulates the expression of ompT, which encodes a major outer membrane porin. OmpT levels increased when the bacteria were grown in medium containing relatively high levels of iron, and this effect required Fur. The level of ompT mRNA also is increased in the presence of iron and Fur. The effect of iron on OmpT levels was independent of the known ompT regulators ToxR and Crp, and it did not require RyhB, which has been shown to be responsible for positive regulation by iron of some V. cholerae genes. Electrophoretic mobility shift assays showed that Fur binds upstream of the ompT transcription start site in a region overlapping known binding sites for ToxR and Crp. These data suggest that Fur and iron positively regulate ompT expression through the direct binding of Fur to the ompT promoter.