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• W2134637395 abstract "Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher. Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher. An in-depth understanding of cellular proteomes requires knowledge of protein subcellular topology, assembly in macromolecular complexes, and modification and degradation of poplypeptides. Escherichia coli, a model organism for many such studies, is by far the best studied. The genomes of strain K-12 derivatives MG1655 and W3110 have been sequenced (1Blattner F. Plunkett G. Bloch C. Perna N. Burland V. Riley M. Collado-Vides J. Glasner J. Rode C. Mayhew G. Gregor J. Davis N. Kirkpatrick H. Goeden M. Rose D. Mau B. Shao Y. The complete genome sequence of Escherichia coli K-12.Science. 1997; 277: 1453-1474Crossref PubMed Scopus (6008) Google Scholar, 2Hayashi K. Morooka N. Yamamoto Y. Fujita K. Isono K. Choi S. Ohtsubo E. Baba T. Wanner B.L. Mori H. Horiuchi T. Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110.Mol. Syst. Biol. 2006; 22006.0007Crossref PubMed Scopus (362) Google Scholar), and >75% of their genes have been functionally assigned (3Keseler I.M. Collado-Vides J. Santos-Zavaleta A. Peralta-Gil M. Gama-Castro S. Muniz-Rascado L. Bonavides-Martinez C. Paley S. Krummenacker M. Altman T. Kaipa P. Spaulding A. Pacheco J. Latendresse M. Fulcher C. Sarker M. Shearer A.G. Mackie A. Paulsen I. Gunsalus R.P. Karp P.D. EcoCyc: a comprehensive database of Escherichia coli biology.Nucleic Acids Res. 2011; 39: D583-D590Crossref PubMed Scopus (341) Google Scholar). Almost 90% of the K-12 proteome has been identified experimentally, and >73% of its proteins have known structures (4Sayers E.W. Barrett T. Benson D.A. Bolton E. Bryant S.H. Canese K. Chetvernin V. Church D.M. Dicuccio M. Federhen S. Feolo M. Fingerman I.M. Geer L.Y. Helmberg W. Kapustin Y. Krasnov S. Landsman D. Lipman D.J. Lu Z. Madden T.L. Madej T. Maglott D.R. Marchler-Bauer A. Miller V. Karsch-Mizrachi I. Ostell J. Panchenko A. Phan L. Pruitt K.D. Schuler G.D. Sequeira E. Sherry S.T. Shumway M. Sirotkin K. Slotta D. Souvorov A. Starchenko G. Tatusova T.A. Wagner L. Wang Y. Wilbur W.J. Yaschenko E. Ye J. Database resources of the National Center for Biotechnology Information.Nucleic Acids Res. 2012; 40: D13-D25Crossref PubMed Scopus (438) Google Scholar, 5Consortium T.U. Reorganizing the protein space at the Universal Protein Resource (UniProt).Nucleic Acids Res. 2012; 40: D71-D75Crossref PubMed Scopus (1099) Google Scholar). Moreover, the genomes of another 38 E. coli strains have been determined (see EcoliWiki for details). In E. coli, like in all Gram-negative bacteria, the bacterial cell envelope comprises the plasma or inner membrane and the outer membrane, which are separated by the periplasmic space. The inner membrane encloses the cytoplasm and is a dynamic substructure. It harbors a wide variety of proteins that function in vital cell processes such as the trafficking of ions, molecules, and macromolecules; cell division; environmental sensing; lipid, polysaccharide, and peptidoglycan biosynthesis; and metabolism. Inner membrane proteins either fully span the lipid bilayer using one or more hydrophobic transmembrane helices (integral) or are bound either directly to phospholipid components or via protein–protein interactions to the surface of the membrane (peripheral) (6Singer S.J. Nicolson G.L. The fluid mosaic model of the structure of cell membranes.Science. 1972; 175: 720-731Crossref PubMed Scopus (6074) Google Scholar) (Fig. 1A). Peripheral inner membrane proteins exist on either side of the membrane and may be recruited in membrane-associated complexes on demand (7Dowhan W. Bogdanov M. Mileykovskaya E. Chapter 1: functional roles of lipids in membranes.in: Dennis E.V. Jean E.V. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, San Diego2008: 1-ICrossref Scopus (87) Google Scholar). Peripheral inner membrane proteins on the cytoplasmic side constitute a sub-proteome of central importance because of their interaction with the cytoplasmic proteome, the nucleoid, and most of the cell's metabolism. Thanks to their soluble character and the nature of their interactions with the membrane (mostly electrostatic and moderate hydrophobic interactions (7Dowhan W. Bogdanov M. Mileykovskaya E. Chapter 1: functional roles of lipids in membranes.in: Dennis E.V. Jean E.V. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, San Diego2008: 1-ICrossref Scopus (87) Google Scholar)), peripheral inner membrane proteins can be extracted using high salt concentrations, extreme pH levels, or chaotropes without disrupting the lipid bilayer (8Adelman M.R. Sabatini D.D. Blobel G. Ribosome-membrane interaction.J. Cell Biol. 1973; 56: 206-229Crossref PubMed Scopus (213) Google Scholar, 9Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1382) Google Scholar, 10Ohlendieck K. Extraction of membrane proteins.in: Cutler P. Protein Purification Protocols. Humana Press, Totowa, NJ2003: 283-290Crossref Google Scholar, 11Kreibich G. Sabatini D.D. Selective release of content from microsomal vesicles without membrane disassembly.J. Cell Biol. 1974; 61: 789-807Crossref PubMed Scopus (42) Google Scholar). In contrast, the solubilization of integral proteins requires amphiphilic detergents in order to displace the membrane phospholipids and maintain them as soluble in aqueous solutions (12Speers A.E. Wu C.C. Proteomics of integral membrane proteins: theory and application.Chem. Rev. 2007; 107: 3687-3714Crossref PubMed Scopus (256) Google Scholar). Unlike the cytoplasmic proteome of E. coli, which has been extensively characterized (13Han M.J. Lee S.Y. The Escherichia coli proteome: past, present, and future prospects.Microbiol. Mol. Biol. R. 2006; 70: 362-439Crossref PubMed Scopus (148) Google Scholar), its membrane sub-proteome is still poorly defined. Of 1133 predicted integral inner membrane proteins, only half were experimentally identified through proteomics approaches (14Bernsel A. Daley D. Exploring the inner membrane proteome of Escherichia coli: which proteins are eluding detection and why?.Trends Microbiol. 2009; 17: 444-449Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). These figures are constantly being re-evaluated, 2Papanastasiou, M., Orfanoudaki, G., Koukaki, M., Kountourakis, N., Tsolis, K., Sardis, M. F., Aivaliotis, M., Karamanou, S., Economou, A., manuscript in preparation. but most protein identifications appear robust. In contrast to integral inner membrane proteins, bioinformatics prediction of peripheral inner membrane proteins is currently not possible because they are not known to possess any specific features. Despite the occasional designation of partner proteins identified as peripheral in studies that target inner membrane complexes (15Spelbrink R.E.J. Kolkman A. Slijper M. Killian J.A. de Kruijff B. Detection and identification of stable oligomeric protein complexes in Escherichia coli inner membranes.J. Biol. Chem. 2005; 280: 28742-28748Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 16Stenberg F. Chovanec P. Maslen S.L. Robinson C.V. Ilag L.L. von Heijne G. Daley D.O. Protein complexes of the Escherichia coli cell envelope.J. Biol. Chem. 2005; 280: 34409-34419Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 17Huang C.Z. Lin X.M. Wu L.N. Zhang D.F. Liu D. Wang S.Y. Peng X.X. Systematic identification of the subproteome of Escherichia coli cell envelope reveals the interaction network of membrane proteins and membrane-associated peripheral proteins.J. Proteome Res. 2006; 5: 3268-3276Crossref PubMed Scopus (39) Google Scholar, 18Lasserre J.P. Beyne E. Pyndiah S. Lapaillerie D. Claverol S. Bonneu M. A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis.Electrophoresis. 2006; 27: 3306-3321Crossref PubMed Scopus (92) Google Scholar, 19Pan J.Y. Li H. Ma Y. Chen P. Zhao P. Wang S.Y. Peng X.X. Complexome of Escherichia coli envelope proteins under normal physiological conditions.J. Proteome Res. 2010; 9: 3730-3740Crossref PubMed Scopus (21) Google Scholar, 20Li H. Pan J.Y. Liu X.J. Gao J.X. Wu H.K. Wang C. Peng X.X. Alterations of protein complexes and pathways in genetic information flow and response to stimulus contribute to Escherichia coli resistance to balofloxacin.Mol. Biosyst. 2012; 8: 2303-2311Crossref PubMed Scopus (7) Google Scholar, 21Maddalo G. Stenberg-Bruzell F. Götzke H. Toddo S. Björkholm P. Eriksson H. Chovanec P. Genevaux P. Lehtiö J. Ilag L.L. Daley D.O. Systematic analysis of native membrane protein complexes in Escherichia coli.J. Proteome Res. 2011; 10: 1848-1859Crossref PubMed Scopus (48) Google Scholar), no systematic effort has been undertaken to analyze the peripheral inner membrane proteome. Here we have used a multi-pronged strategy employing bioinformatics, biochemistry, proteomics, and complexomics to systematically determine the peripheral inner membrane proteome of E. coli. We focus exclusively on the peripheral inner membrane proteome that faces the cytoplasm, referred to hereinafter as PIM, 1The abbreviations used are:CMCcritical micellar concentrationDDMn-dodecyl β-D-maltosideEDTAethylenediaminetetraacetic acidIMVinverted inner membrane vesicleN-PAGEnative poly-acrylamide gel electrophoresisPIMperipheral inner membrane. 1The abbreviations used are:CMCcritical micellar concentrationDDMn-dodecyl β-D-maltosideEDTAethylenediaminetetraacetic acidIMVinverted inner membrane vesicleN-PAGEnative poly-acrylamide gel electrophoresisPIMperipheral inner membrane. and do not analyze peripheral inner membrane proteins residing on the periplasm. Manually curated and re-evaluated topology of the E. coli K-12 proteome was extrapolated to the non-K-12 strain BL21(DE3) (95% proteome homology to K-12) (22Jeong H. Barbe V. Lee C.H. Vallenet D. Yu D.S. Choi S.H. Couloux A. Lee S.W. Yoon S.H. Cattolico L. Hur C.G. Park H.S. Segurens B. Kim S.C. Oh T.K. Lenski R.E. Studier F.W. Daegelen P. Kim J.F. Genome sequences of Escherichia coli B strains REL606 and BL21(DE3).J. Mol. Biol. 2009; 394: 644-652Crossref PubMed Scopus (252) Google Scholar). By combining various biochemical treatments, we determined experimentally that several cytoplasmic proteins are also novel PIM proteins, and many of them participate in protein complexes associated with the membrane. Collectively, we demonstrate that a significant, previously unsuspected percentage of the expressed polypeptides constitute the PIM proteome. critical micellar concentration n-dodecyl β-D-maltoside ethylenediaminetetraacetic acid inverted inner membrane vesicle native poly-acrylamide gel electrophoresis peripheral inner membrane. critical micellar concentration n-dodecyl β-D-maltoside ethylenediaminetetraacetic acid inverted inner membrane vesicle native poly-acrylamide gel electrophoresis peripheral inner membrane. The PIM sub-proteome is largely elusive. The complete absence of bioinformatic predictor tools for PIM proteins renders experimental approaches, such as proteomics, essential. To curate the currently available PIM proteome, we performed an exhaustive in-depth analysis combining data from the literature, a variety of bioinformatics tools (Fig. 1B), and topological information for the E. coli K-12 proteins available in public databases such as EchoLOCATION (23Horler R.S.P. Butcher A. Papangelopoulos N. Ashton P.D. Thomas G.H. EchoLOCATION: an in silico analysis of the subcellular locations of Escherichia coli proteins and comparison with experimentally derived locations.Bioinformatics. 2009; 25: 163-166Crossref PubMed Scopus (24) Google Scholar), Uniprot (5Consortium T.U. Reorganizing the protein space at the Universal Protein Resource (UniProt).Nucleic Acids Res. 2012; 40: D71-D75Crossref PubMed Scopus (1099) Google Scholar), and EcoCyc (3Keseler I.M. Collado-Vides J. Santos-Zavaleta A. Peralta-Gil M. Gama-Castro S. Muniz-Rascado L. Bonavides-Martinez C. Paley S. Krummenacker M. Altman T. Kaipa P. Spaulding A. Pacheco J. Latendresse M. Fulcher C. Sarker M. Shearer A.G. Mackie A. Paulsen I. Gunsalus R.P. Karp P.D. EcoCyc: a comprehensive database of Escherichia coli biology.Nucleic Acids Res. 2011; 39: D583-D590Crossref PubMed Scopus (341) Google Scholar). We then extrapolated the results to the E. coli BL21(DE3) strain (supplemental Table S1A). The nomenclature of protein classes used here was based on that of EchoLOCATION (Fig. 1A). A total of 138 K-12 proteins were annotated as PIM (indicated with “F1” in supplemental Table S1A) in EchoLOCATION and Uniprot, but this number was subsequently reduced to 123 after manual validation (supplemental Table S1B). These corresponded to 133 homologues in BL21(DE3). PIM proteins of strain BL21(DE3) that were curated for the first time in the present study (138 proteins, indicated with “F1*” in supplemental Table S1A) were derived mainly from a thorough search of the E. coli literature, comparison to homologues from other organisms, and a multi-pronged iterative curation approach (supplemental Table S1C). We reexamined any proposed topology classification for cytoplasmic or integral inner membrane proteins using several bioinformatic predictors (SignalP, TatP, LipoP, TMHMM, Phobius, Protscale, SOSUI, and AmphipaSeek) and homology (BLAST) tools. The results were subsequently refined using the available databases (EcoGene, EcoCyc) and the literature (supplemental Table S1A). The collective use of these approaches increased the total number of curated PIM proteins to 278. To experimentally identify PIM proteins, we used inverted inner membrane vesicles (IMVs), in which the cytoplasmic face of the membrane is exposed and accessible and the periplasmic side faces the sealed lumen (24Futai M. Orientation of membrane vesicles from Escherichia coli prepared by different procedures.J. Membrane Biol. 1974; 15: 15-28Crossref PubMed Scopus (124) Google Scholar). The surfaces of intact IMVs were treated with trypsin, and soluble tryptic peptides were analyzed via nanoLC-MS/MS. Multiple technical and biological repeats were performed to ensure statistical robustness (supplemental Table S3A). This method is highly specific for surface-attached proteins, because trypsin (27 kDa) cannot penetrate IMVs, and lumenally trapped cytoplasmic proteins are therefore neither trypsined nor analyzed here (supplemental Fig. S1A). Two major challenges were the discrimination of PIM from non-specifically bound cytoplasmic proteins and overcoming “peptide noise” derived from abundant membrane-bound ribosomes. In the untreated IMVs, ∼70% of identified proteins corresponded to cytoplasmic and ribosomal polypeptides (Fig. 1D), indicating that these are present on the IMV surface. We systematically probed the degree of their extraction from the membrane through the consecutive use of chemical agents that are traditionally used to characterize peripherally attached proteins (Fig. 1C) and to remove unspecifically bound cytoplasmic proteins (25Steck T.L. The organization of proteins in the human red blood cell membrane.J. Cell Biol. 1974; 62: 1-19Crossref PubMed Scopus (1052) Google Scholar, 26Pieper R. Huang S.T. Clark D.J. Robinson J.M. Alami H. Parmar P.P. Suh M.J. Kuntumalla S. Bunai C.L. Perry R.D. Fleischmann R.D. Peterson S.N. Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes.Proteome Sci. 2009; 7: 16Crossref PubMed Scopus (25) Google Scholar). Such treatments included KCl, EDTA, Na2CO3 at pH 11, and urea combined with mild sonication and sucrose density centrifugations (see supplemental Materials and Methods) (8Adelman M.R. Sabatini D.D. Blobel G. Ribosome-membrane interaction.J. Cell Biol. 1973; 56: 206-229Crossref PubMed Scopus (213) Google Scholar, 9Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1382) Google Scholar, 10Ohlendieck K. Extraction of membrane proteins.in: Cutler P. Protein Purification Protocols. Humana Press, Totowa, NJ2003: 283-290Crossref Google Scholar, 11Kreibich G. Sabatini D.D. Selective release of content from microsomal vesicles without membrane disassembly.J. Cell Biol. 1974; 61: 789-807Crossref PubMed Scopus (42) Google Scholar). These treatments did not functionally compromise IMVs, which remained fully competent for protein translocation (supplemental Fig. S1B). Finally, the IMVs were treated with low levels of the non-ionic detergent n-dodecyl β-D-maltoside (DDM) (0.1–2.0 critical micellar concentration (CMC)) (supplemental Fig. S1C) to disrupt hydrophobic protein–protein interactions. The chemical agents used gradually removed cytoplasmic, ribosomal, and known PIM proteins from the membrane periphery, as indicated by the significant decrease of the total number of soluble proteins identified (Fig. 1D) and by the decrease of their relative amounts across consecutive treatments (supplemental Table S3). Compared with the untreated IMVs, new proteins were also identified across treatments, indicating that their identification was obstructed by contaminating polypeptides residing on the membrane surface. The relative protein amounts during the multiple consecutive treatments described above (Fig. 1D) were determined using label-free quantitation (27Silva J.C. Gorenstein M.V. Li G.Z. Vissers J.P.C. Geromanos S.J. Absolute quantification of proteins by LCMSE: a virtue of parallel MS Acquisition.Mol. Cell. Proteomics. 2006; 5: 144-156Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar). Some known peripheral proteins such as DhsA and DhsB (of the succinate dehydrogenase heterotetramer), SecA (the motor of the Sec translocase), and the FtsE component of the cell division ring remained membrane associated even after extensive treatments. All of these proteins were highly abundant and were detected in almost all technical repeats of a given sample preparation condition (columns F–H and I–M in supplemental Table S3B). In contrast, the intensity of some other subunits of these complexes decreased upon IMV treatment. For example, SecB was detected at low abundance only in the untreated IMV samples and was removed completely upon treatment with KCl and EDTA. Another example was FtsZ, which was gradually removed during the various treatments as indicated by its abundance values (∼90-fold decrease) and detection rate (found in all repeats in the untreated IMVs and in only 1 out of the 13 repeats in the DDM-treated IMVs). Remarkably, several proteins classified previously as cytoplasmic, suchas HemG/HemH/YbbO (involved in heme biosynthesis), DeoB/DeoC/DeoD (involved in the synthesis of nucleoside catabolic enzymes), and AccA/AccD (of the acetyl coenzyme A carboxylase), survived several IMV treatments (supplemental Table S3B), indicating that they are avid membrane binders and could therefore be considered legitimate PIM proteins. In order to assign a cytoplasmic protein as PIM in a systematic fashion, a number of criteria were applied (supplemental Fig. S2). For each sample preparation condition (not treated, salt and then DDM, etc.), a threshold was set based on the confidence with which a protein was detected (how many times a protein was detected in technical repeats). The threshold was higher for the untreated IMVs (which were anticipated to contain nonspecific bound proteins, meaning their detection would be random) and lower for the treated and DDM-treated IMVs (nonspecific bound proteins would have been removed by the various treating agents). Proteins that fulfilled this criterion were directly annotated as peripheral (supplemental Fig. S2). For example, HemG and YbbO were detected in all sample preparation conditions, but they fulfilled the criteria only for the washed IMVs (and DDM-washed IMVs) (supplemental Table S3B). If these thresholds were not met, then the abundance of a protein in the cell was taken into account. Low-abundance proteins (<500 copies per cell) were assigned as peripheral even if they were detected in fewer repeats (in more than three) in the IMV fraction (i.e. YggL, which has ∼400 molecules per cell (supplemental Table S1) and was detected in six repeats in the untreated IMVs only), whereas high abundance proteins were considered as lower confidence PIM proteins if they had the same detection score, because it could not be excluded that their detection was due to nonspecific, low-affinity associations (i.e. AmpM with ∼2000 molecules per cell that was detected in four repeats in the untreated IMVs only). A protein that did not meet any of the above criteria but which was part of a known membrane-associated protein complex (3Keseler I.M. Collado-Vides J. Santos-Zavaleta A. Peralta-Gil M. Gama-Castro S. Muniz-Rascado L. Bonavides-Martinez C. Paley S. Krummenacker M. Altman T. Kaipa P. Spaulding A. Pacheco J. Latendresse M. Fulcher C. Sarker M. Shearer A.G. Mackie A. Paulsen I. Gunsalus R.P. Karp P.D. EcoCyc: a comprehensive database of Escherichia coli biology.Nucleic Acids Res. 2011; 39: D583-D590Crossref PubMed Scopus (341) Google Scholar), for which a minimum of one peripheral protein subunit was detected in this study, was also considered peripheral. In this way we reduced the possibility of false positive identifications. Therefore, proteins such as CspC (∼379,000 molecules per cell), which was identified in two repeats, were not assigned as peripheral proteins. In conclusion, this analysis proposes that a total of 169 of the cytoplasmic proteins identified on IMVs are novel PIM proteins. Our analysis suggested that several of the PIM proteins might associate with the membrane as part of complexes rather than directly. Given the interfacial location of PIM proteins and the dynamic equilibria with the cytoplasmic proteome pool, it was of interest to determine interacting partners. Based on interactomics (28Butland G. Peregrin-Alvarez J.M. Li J. Yang W. Yang X. Canadien V. Starostine A. Richards D. Beattie B. Krogan N. Davey M. Parkinson J. Greenblatt J. Emili A. Interaction network containing conserved and essential protein complexes in Escherichia coli.Nature. 2005; 433: 531-537Crossref PubMed Scopus (937) Google Scholar, 29Arifuzzaman M. Maeda M. Itoh A. Nishikata K. Takita C. Saito R. Ara T. Nakahigashi K. Huang H.C. Hirai A. Tsuzuki K. Nakamura S. Altaf-Ul-Amin M. Oshima T. Baba T. Yamamoto N. Kawamura T. Ioka-Nakamichi T. Kitagawa M. Tomita M. Kanaya S. Wada C. Mori H. Large-scale identification of protein-protein interaction of Escherichia coli K-12.Genome Res. 2006; 16: 686-691Crossref PubMed Scopus (335) Google Scholar) and gel-based complexome (15Spelbrink R.E.J. Kolkman A. Slijper M. Killian J.A. de Kruijff B. Detection and identification of stable oligomeric protein complexes in Escherichia coli inner membranes.J. Biol. Chem. 2005; 280: 28742-28748Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 16Stenberg F. Chovanec P. Maslen S.L. Robinson C.V. Ilag L.L. von Heijne G. Daley D.O. Protein complexes of the Escherichia coli cell envelope.J. Biol. Chem. 2005; 280: 34409-34419Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 17Huang C.Z. Lin X.M. Wu L.N. Zhang D.F. Liu D. Wang S.Y. Peng X.X. Systematic identification of the subproteome of Escherichia coli cell envelope reveals the interaction network of membrane proteins and membrane-associated peripheral proteins.J. Proteome Res. 2006; 5: 3268-3276Crossref PubMed Scopus (39) Google Scholar, 18Lasserre J.P. Beyne E. Pyndiah S. Lapaillerie D. Claverol S. Bonneu M. A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis.Electrophoresis. 2006; 27: 3306-3321Crossref PubMed Scopus (92) Google Scholar, 19Pan J.Y. Li H. Ma Y. Chen P. Zhao P. Wang S.Y. Peng X.X. Complexome of Escherichia coli envelope proteins under normal physiological conditions.J. Proteome Res. 2010; 9: 3730-3740Crossref PubMed Scopus (21) Google Scholar, 21Maddalo G. Stenberg-Bruzell F. Götzke H. Toddo S. Björkholm P. Eriksson H. Chovanec P. Genevaux P. Lehtiö J. Ilag L.L. Daley D.O. Systematic analysis of native membrane protein complexes in Escherichia coli.J. Proteome Res. 2011; 10: 1848-1859Crossref PubMed Scopus (48) Google Scholar, 30Regonesi M.E. Del Favero M. Basilico F. Briani F. Benazzi L. Tortora P. Mauri P. Deho G. Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach.Biochimie (Paris). 2006; 88: 151-161Crossref PubMed Scopus (62) Google Scholar, 31Wu T. McCandlish A.C. Gronenberg L.S. Chng S.S. Silhavy T.J. Kahne D. Identification of a protein complex that assembles lipopolysaccharide in the outer membrane of Escherichia coli.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11754-11759Crossref PubMed Scopus (267) Google Scholar) data, >90% of the known and PIM proteins curated in this study have known interactors (supplemental Table S6) (32Kerrien S. Aranda B. Breuza L. Bridge A. Broackes-Carter F. Chen C. Duesbury M. Dumousseau M. Feuermann M. Hinz U. Jandrasits C. Jimenez R.C. Khadake J. Mahadevan U. Masson P. Pedruzzi I. Pfeiffenberger E. Porras P. Raghunath A. Roechert B. Orchard S. Hermjakob H. The IntAct molecular interaction database in 2012.Nucleic Acids Res. 2012; 40: D841-D846Crossref PubMed Scopus (806) Google Scholar), but these are not necessarily membrane-associated proteins. In order to experimentally extract stable PIM complexes that employ either electrostatic or hydrophobic interactions, the IMVs were treated with mild methods: either KCl (1 m) or DDM (1–2 times CMC), or a sequential combination of both (supplemental Fig." @default.
• W2134637395 created "2016-06-24" @default.
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• W2134637395 date "2013-03-01" @default.
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• W2134637395 title "The Escherichia coli Peripheral Inner Membrane Proteome" @default.
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