The protein complement of cellular membranes is notoriously resistant to standard proteomic analysis and structural studies. As a result, membrane proteomes remain ill-defined. Here, we report a global topology analysis of the Escherichia coli inner membrane proteome. Using C-terminal tagging with the alkaline phosphatase and green fluorescent protein, we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins. By constraining a topology prediction algorithm with this data, we derived high-quality topology models for the 601 proteins, providing a firm foundation for future functional studies of this and other membrane proteomes. We also estimated the overexpression potential for 397 green fluorescent protein fusions; the results suggest that a large fraction of all inner membrane proteins can be produced in sufficient quantities for biochemical and structural work.

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 ...

A central component of the endosymbiotic theory for the bacterial origin of the mitochondrion is that many of its genes were transferred to the nucleus. Most of this transfer occurred early in mitochondrial evolution; functional transfer of mitochondrial genes has ceased in animals. Although mitochondrial gene transfer continues to occur in plants, no comprehensive study of the frequency and timing of transfers during plant evolution has been conducted. Here we report frequent loss (26 times) and transfer to the nucleus of the mitochondrial gene rps10 among 277 diverse angiosperms. Characterization of nuclear rps10 genes from 16 out of 26 loss lineages implies that many independent, RNA-mediated rps10 transfers occurred during recent angiosperm evolution; each of the genes may represent a separate functional gene transfer. Thus, rps10 has been transferred to the nucleus at a surprisingly high rate during angiosperm evolution. The structures of several nuclear rps10 genes reveal diverse mechanisms by which transferred genes become activated, including parasitism of pre-existing nuclear genes for mitochondrial or cytoplasmic proteins, and activation without gain of a mitochondrial targeting sequence.

We describe a generic, GFP-based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for high-throughput applications. The GFP-based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important reference for the characterization of other membrane proteomes.Keywords: Escherichia coli; membrane protein overexpression; membrane protein isolation; membrane protein characterization; GFP Membrane proteins (MPs) account for 20%-25% of all open reading frames in sequenced genomes, and fulfill a wide range of central functions in the cell (Wallin and von Heijne 1998). However, our knowledge of this important class of proteins is still poor, mainly because of a lack of generally applicable approaches to the overexpression and purification steps that precede functional and structural analysis. Novel approaches in these areas are required to facilitate and speed up MP research.The bacterium Escherichia coli is still the most widely used vehicle for MP overexpression. Overexpression in the cytoplasmic membrane is preferred to overexpression in inclusion bodies, since the isolation of functional MPs from the membrane is usually more successful than refolding from inclusion bodies (Drew et al. 2003). Green fluorescent protein (GFP) fusions can be used to facilitate the monitoring of MP overexpression in the cytoplasmic membrane (Drew et al. 2001). If the fusion protein ends up in inclusion bodies, GFP does not fold and is therefore not fluorescent; in contrast, if the fusion is expressed in the cytoplasmic membrane, GFP folds properly and is fluorescent. GFP is only fluorescent in the cytoplasm of Escherichia coli (Drew et al. 2002), which means that GFP-based screens work only for MPs that have their C terminus located in the cytoplasm. Recently, nearly all E. coli cytoplasmic MPs were fused to GFP for a membrane proteome topology screen (Daley et al. 2005). Approximately 80% of all E. coli cytoplasmic MPs have a cytoplasmic C terminus, and thus GFP can be used to monitor the overexpression levels of the majority of E. coli MPs (Daley et al. 2005).Here, we present a generic pipeline for rapid overexpression screening, detergent extraction, and purification of MPs based on a simple MP-GFP fusion approach. We show that milligram amounts of pure functional MP can Reprint requests to: Jan-Willem de Gier, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; e-mail: degier@dbb.su.se; fax: +46-8-153679.Article published online ahead of print. Article and publication date are at