Protein Complexes of the Escherichia coli Cell Envelope

Abstract

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 ...

Involvement of Neisseria meningitidis Lipoprotein GNA2091 in the Assembly of a Subset of Outer Membrane Proteins

“…These findings are consistent with several studies that addressed the composition of outer and inner membrane complexes in E. coli. Using various approaches, these studies identified all the known Bam complex components consistently together in one complex, but none of them reported on the presence of GNA2091 homolog YraP in any complex (31)(32)(33). In this respect, it is noteworthy that also a stable interaction between BamA and BamD was recently reported to be unnecessary for proper functioning of the Bam complex in E. coli (34).…”

Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS

“…In E. coli there was only a limited overlap of 9 integral membrane proteins between the datasets obtained after SDS-TFE-SDS-PAGE and BN-SDS-PAGE separation [5,12,13], indicating that in this organism the approaches are complementary. In yeast mitochondria, only one TFEsensitive protein not integral to the membrane could be identified using our approach.…”

“…When applied to E. coli inner membrane preparations, 23 integral membrane proteins and 16 peripheral membrane proteins were identified in off-diagonal spots, while only 14 soluble proteins were found. Comparison to the results of two BN-SDS-PAGE studies on E. coli [12,13], revealed an overlap of only 9 integral membrane proteins and 11 peripheral proteins, indicating that, particularly for integral membrane proteins, the two techniques are complementary. For two of the proteins identified in the SDS-TFE-SDS-PAGE study, the preprotein translocase SecA and the mechanosensitive channel MscS, the oligomeric state and, in the latter case, its dissociation by TFE was confirmed in one dimensional SDS-PAGE gels using immunodetection [8,14].…”

Preparation and Characterization of Bacterial Protein Complexes for Structural Analysis

“…An alternative approach, the use of blue native/SDS-PAGE, followed by the identification of the protein complex by LC-MS/MS, has been used to identify hetero-oligomeric complexes from E. coli (Lasserre et al, 2006). Other studies have focused on characterizing another, although more experimentally challenging set of protein complexes, those found in the membrane (Stenberg et al, 2005).…”