Protein complexes are an intrinsic aspect of life in the membrane. Knowing which proteins are assembled in these complexes is therefore essential to understanding protein function(s). Unfortunately, recent high throughput protein interaction studies have failed to deliver any significant information on proteins embedded in the membrane, and many membrane protein complexes remain ill defined. In this study, we have optimized the blue native-PAGE technique for the study of membrane protein complexes in the inner and outer membranes of Escherichia coli. In combination with second dimension SDS-PAGE and mass spectrometry, we have been able to identify 43 distinct protein complexes. In addition to a number of well characterized complexes, we have identified known and orphan proteins in novel oligomeric states. For two orphan proteins, YhcB and YjdB, our findings enable a tentative functional assignment. We propose that YhcB is a hitherto unidentified additional subunit of the cytochrome bd oxidase and that YjdB, which co-localizes with the ZipA protein, is involved in cell division. Our reference two-dimensional blue native-SDS-polyacrylamide gels will facilitate future studies of the assembly and composition of E. coli membrane protein complexes during different growth conditions and in different mutant backgrounds.It has been suggested that nearly all biochemical processes are performed by protein complexes (1). This is particularly true in cellular membranes, where many well characterized proteins assemble into complexes that carry out important tasks in energy generation, protein trafficking, and small molecule transport. Many uncharacterized proteins ("orphans") are also predicted to be localized in cell membranes (2, 3), and it is probable that they also often assemble into complexes. Identifying the interacting partners of these proteins is critical to understanding their function.Unfortunately, our knowledge of protein complexes in cellular membranes is poor, because membrane proteins are incompatible with commonly used protein interaction assays. High throughput studies on model systems (4 -11) have therefore consistently disregarded membrane proteins (12). Although genetic tools specific for membrane protein interactions have been developed (13-15), they have not yet been pursued past proof of principle.A related and elusive aspect of membrane biology pertains to how proteins are assembled into complexes following their insertion into the membrane. Although some folding chaperones have been identified for model substrates, the ubiquity of their roles is not known, and little is known about the assembly process. Robust and effective experimental assays are required to tackle the question of membrane protein assembly.Blue native (BN) 3 -PAGE (16, 17) offers an attractive proteomic solution for the analysis of membrane protein complexes. It has been successfully applied to respiratory complexes in mitochondria and Paracoccus denitrificans (18 -24) and photosynthetic complexes of chloroplasts and Synechocystis ...

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.

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Enterococcus faecalis is a gram-positive bacterium that is part of the indigenous microbiotica of humans and animals as well as an opportunistic pathogen. In this study we have fractionated the membrane proteome of E. faecalis and identified many of its constituents by mass spectrometry. We present BN-/SDS-PAGE reference maps that contain 102 proteins. These proteins are important for cellular homeostasis, virulence, and antibiotic intervention. Intriguingly, many proteins with no known function were also identified, indicating that there are substantial gaps in knowledge of this organism’s biology. On a more limited scale we also provide insight into the composition of membrane protein complexes. This study is a first step toward elucidating the membrane proteome of E. faecaliswhich is critical for a better understanding of how this bacterium interacts with a host and with the extracellular milieu.

The growth and conidiation of the aged Trichoderma viride culture grown in the dark, and after an induction by a light pulse, was examined in the presence of selected mono-, di(tri)saccharides, amino acids and alcohols as sole carbon sources. Hexoses and disaccharides, but not pentoses and amino acids, promoted proportionally both growth and conidiation induced by aging or light. All compounds but pentoses promoted the conidiation in aged cultures and photoconidiation in a close correlation. Ethanol, glycerol and ethylene glycol supported both growth and conidiation but these processes were not supported equally. Conidia formation with hexoses and amino acids as sole carbon sources seems to be a function of growth promotion, rather than of growth restriction (starvation, stress, aging). With glucose as sole carbon source the conidiation was not triggered by nutrient limitation, nor by the accumulation of waste metabolites. The aging-induced conidiation can be considered to be triggered by the genetic program of the microorganism rather than by its nutrient status.