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The Escherichia coli Peripheral Inner Membrane Proteome*

  • Malvina Papanastasiou
    Affiliations
    Institute of Molecular Biology and Biotechnology-FORTH, Iraklio, Crete 711110, Greece
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  • Georgia Orfanoudaki
    Affiliations
    Institute of Molecular Biology and Biotechnology-FORTH, Iraklio, Crete 711110, Greece

    Department of Biology-University of Crete, P.O. Box 1385, Iraklio, Crete 711110, Greece
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  • Marina Koukaki
    Affiliations
    Institute of Molecular Biology and Biotechnology-FORTH, Iraklio, Crete 711110, Greece
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  • Nikos Kountourakis
    Affiliations
    Institute of Molecular Biology and Biotechnology-FORTH, Iraklio, Crete 711110, Greece
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  • Marios Frantzeskos Sardis
    Affiliations
    Institute of Molecular Biology and Biotechnology-FORTH, Iraklio, Crete 711110, Greece

    Department of Biology-University of Crete, P.O. Box 1385, Iraklio, Crete 711110, Greece
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  • Michalis Aivaliotis
    Affiliations
    Institute of Molecular Biology and Biotechnology-FORTH, Iraklio, Crete 711110, Greece
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  • Spyridoula Karamanou
    Affiliations
    Institute of Molecular Biology and Biotechnology-FORTH, Iraklio, Crete 711110, Greece
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  • Anastassios Economou
    Correspondence
    To whom correspondence should be addressed. Tel.: +30-2810-391166; Fax:+30-2810-391950; E-mail: [email protected]
    Affiliations
    Institute of Molecular Biology and Biotechnology-FORTH, Iraklio, Crete 711110, Greece

    Department of Biology-University of Crete, P.O. Box 1385, Iraklio, Crete 711110, Greece

    Rega Institute, Katholieke Universiteit Leuven, Molecular Bacteriology Laboratory, Minderbroedersstraat 10, B-3000 Leuven, Belgium
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  • Author Footnotes
    * The research leading to these results has received funding from the European Commission (EC) through Seventh Framework Programme Agreement No. 229823; Capacities-FP7-REGPOT-2008-1/project “ProFI”; the project “IRAKLITOS II - University of Crete” of the Operational Programme for Education and Lifelong Learning 2007–2013 (E.P.E.D.V.M.) of the NSRF (2007–2013), which is co-funded by the European Union (European Social Fund); the Onassis foundation pre-doctoral program; the Operational Programme “Competitiveness and Entrepreneuriship” (OPCE II - EPAN II) - National Strategic Reference Framework (NSRF 2007–2013), Grant No. 09SYN-13-705; and an Excellence grant (Grant No. 1473) from the General Secretariat of Research (to A.E.).
    This article contains supplemental material.
    2 Papanastasiou, M., Orfanoudaki, G., Koukaki, M., Kountourakis, N., Tsolis, K., Sardis, M. F., Aivaliotis, M., Karamanou, S., Economou, A., manuscript in preparation.
    3 G. L. Waldrop, personal communication.
Open AccessPublished:December 10, 2012DOI:https://doi.org/10.1074/mcp.M112.024711
      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 (
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      The complete genome sequence of Escherichia coli K-12.
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      • Isono K.
      • Choi S.
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      • Baba T.
      • Wanner B.L.
      • Mori H.
      • Horiuchi T.
      Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110.
      ), and >75% of their genes have been functionally assigned (
      • Keseler I.M.
      • Collado-Vides J.
      • Santos-Zavaleta A.
      • Peralta-Gil M.
      • Gama-Castro S.
      • Muniz-Rascado L.
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      • Altman T.
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      • Mackie A.
      • Paulsen I.
      • Gunsalus R.P.
      • Karp P.D.
      EcoCyc: a comprehensive database of Escherichia coli biology.
      ). Almost 90% of the K-12 proteome has been identified experimentally, and >73% of its proteins have known structures (
      • Sayers E.W.
      • Barrett T.
      • Benson D.A.
      • Bolton E.
      • Bryant S.H.
      • Canese K.
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      • Schuler G.D.
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      • 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.
      ,
      • Consortium T.U.
      Reorganizing the protein space at the Universal Protein Resource (UniProt).
      ). 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) (
      • Singer S.J.
      • Nicolson G.L.
      The fluid mosaic model of the structure of cell membranes.
      ) (Fig. 1A). Peripheral inner membrane proteins exist on either side of the membrane and may be recruited in membrane-associated complexes on demand (
      • Dowhan W.
      • Bogdanov M.
      • Mileykovskaya E.
      Chapter 1: functional roles of lipids in membranes.
      ). 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 (
      • Dowhan W.
      • Bogdanov M.
      • Mileykovskaya E.
      Chapter 1: functional roles of lipids in membranes.
      )), peripheral inner membrane proteins can be extracted using high salt concentrations, extreme pH levels, or chaotropes without disrupting the lipid bilayer (
      • Adelman M.R.
      • Sabatini D.D.
      • Blobel G.
      Ribosome-membrane interaction.
      ,
      • Fujiki Y.
      • Hubbard A.L.
      • Fowler S.
      • Lazarow P.B.
      Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.
      ,
      • Ohlendieck K.
      Extraction of membrane proteins.
      ,
      • Kreibich G.
      • Sabatini D.D.
      Selective release of content from microsomal vesicles without membrane disassembly.
      ). 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 (
      • Speers A.E.
      • Wu C.C.
      Proteomics of integral membrane proteins: theory and application.
      ).
      Figure thumbnail gr1
      Fig. 1Bioinformatics and experimental workflow for characterizing peripheral inner membrane proteins. A, schematic representation of the subcellular localization of the E. coli inner membrane peripherome. Protein topology assignment is based on the cellular compartment: A, cytoplasmic; B, integral inner membrane proteins; F1, peripheral inner membrane proteome; r, ribosome. B, schematic diagram for PIM protein annotation. 130 cytoplasmic and PIM E. coli K-12 proteins were downloaded from Uniprot (November 2010) (
      • UniProt
      The Universal Protein Resource (UniProt) in 2010.
      ) and EchoLOCATION (
      • Horler 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.
      ). A set of bioinformatics tools was used to predict topologies and features of the unassigned and differently assigned proteins and to further validate existing protein annotations (see supplemental text). For the annotation of additional peripheral membrane proteins, the literature was extensively searched. Additional, other E. coli K-12 databases containing gene ontology annotations (
      • Keseler I.M.
      • Bonavides-Martínez C.
      • Collado-Vides J.
      • Gama-Castro S.
      • Gunsalus R.P.
      • Johnson D.A.
      • Krummenacker M.
      • Nolan L.M.
      • Paley S.
      • Paulsen I.T.
      • Peralta-Gil M.
      • Santos-Zavaleta A.
      • Shearer A.G.
      • Karp P.D.
      EcoCyc: a comprehensive view of Escherichia coli biology.
      ,
      • Rudd K.E.
      EcoGene: a genome sequence database for Escherichia coli K-12.
      ) and protein homologies through BLAST (
      • Johnson M.
      • Zaretskaya I.
      • Raytselis Y.
      • Merezhuk Y.
      • McGinnis S.
      • Madden T.L.
      NCBI BLAST: a better web interface.
      ) were employed. Homologues of curated E. coli K-12 proteins were identified in E. coli BL21(DE3) (). C, preparation strategy for detecting the E. coli inner membrane peripherome via nanoLC-MS/MS. Inverted membrane vesicles (IMVs) were isolated and washed extensively with the indicated chemical agents to extract cytoplasmic and PIM proteins (“IMVs washed”), and then their surface was trypsinized (gray arrow). Following digestion, soluble peptides were analyzed via nanoLC-MS/MS. D, protein enrichment at different sample preparation conditions. Top: Relative percentage of proteins detected with the proteolysis approach. Proteins are classified here in three major categories: cytoplasmic (A), ribosomal (r), and peripheral (F1). The bar graphs indicate the percentage of proteins in each category relative to the proteins in other categories at a given sample preparation condition. Bottom: Heat maps showing relative quantities of individual proteins at different sample preparation conditions. Perseus (version 1.2.0.16), a part of the MaxQuant bioinformatics platform, was used for the construction of the heat map (
      • Cox J.
      • Mann M.
      MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
      ). A top-three label-free quantitative method was employed (
      • Silva 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.
      ). Individual protein values across the various treatments are given in .
      Unlike the cytoplasmic proteome of E. coli, which has been extensively characterized (
      • Han M.J.
      • Lee S.Y.
      The Escherichia coli proteome: past, present, and future prospects.
      ), its membrane sub-proteome is still poorly defined. Of 1133 predicted integral inner membrane proteins, only half were experimentally identified through proteomics approaches (
      • Bernsel A.
      • Daley D.
      Exploring the inner membrane proteome of Escherichia coli: which proteins are eluding detection and why?.
      ). These figures are constantly being re-evaluated,
      Papanastasiou, 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 (
      • Spelbrink 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.
      ,
      • Stenberg 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.
      ,
      • Huang 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.
      ,
      • Lasserre 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.
      ,
      • Pan 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.
      ,
      • Li 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.
      ,
      • Maddalo 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.
      ), 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,
      The abbreviations used are:
      CMC
      critical micellar concentration
      DDM
      n-dodecyl β-D-maltoside
      EDTA
      ethylenediaminetetraacetic acid
      IMV
      inverted inner membrane vesicle
      N-PAGE
      native poly-acrylamide gel electrophoresis
      PIM
      peripheral inner membrane.
      1The abbreviations used are:CMC
      critical micellar concentration
      DDM
      n-dodecyl β-D-maltoside
      EDTA
      ethylenediaminetetraacetic acid
      IMV
      inverted inner membrane vesicle
      N-PAGE
      native poly-acrylamide gel electrophoresis
      PIM
      peripheral 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) (
      • Jeong 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).
      ). 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.

      DISCUSSION

      We present a systematic analysis of the poorly characterized bacterial PIM proteome. Our analysis relied on experimental proteomics data, as current in silico approaches cannot predict the cell topology of cytoplasmic proteins that also associate with membranes. Proteomics was coupled to complete re-annotation of E. coli protein subcellular localization. We determined that the E. coli BL21(DE3) proteome comprises a total of at least 503 PIM proteins accounting for ∼17% of the basic proteome and a remarkable ∼27% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. Our analysis doubled the number of PIM proteins available in Uniprot and EchoLOCATION.
      PIM protein interactions with the membrane appear to be multiple and complex. They associate with the lipid bilayer non-covalently and reversibly without penetrating the hydrocarbon hydrophobic core (
      • Dowhan W.
      • Bogdanov M.
      • Mileykovskaya E.
      Chapter 1: functional roles of lipids in membranes.
      ). Moreover, the indirect association of soluble components with the membrane may also occur through interactions with PIM proteins physically attached to the membrane. Though these components are not directly associated with the membrane, we propose that they should also be considered as PIM proteins. For example, FtsE, which is involved in cell division, interacts directly with the integral inner membrane FtsX, but also with FtsZ, which in turn recruits other known division proteins that do not interact directly with the membrane (Fig. 4) (
      • De Leeuw E.
      • Graham B.
      • Phillips G.J.
      • Ten Hagen-Jongman C.M.
      • Oudega B.
      • Luirink J.
      Molecular characterization of Escherichia coli FtsE and FtsX.
      ,
      • Lutkenhaus J.
      • Addinall S.G.
      Bacterial cell division and the Z ring.
      ). Some PIM proteins form associations with the membrane that are easily abrogated. These can be weak electrostatic interactions with lipid head groups (i.e. PspA) or with other proteins (e.g. SecB, HflD, and NuoEFG). Another example is AccA and AccD subunits of acetyl-CoA carboxylase, a metabolic complex that catalyzes the synthesis of fatty acids. In E. coli, the complex is ascribed to the cytoplasm (
      • Consortium T.U.
      Reorganizing the protein space at the Universal Protein Resource (UniProt).
      ), whereas in B. subtilis evidence has been provided regarding its membrane proximity (
      • Meile J.
      • Wu L.
      • Ehrlich S.
      • Errington J.
      • Noirot P.
      Systematic localisation of proteins fused to the green fluorescent protein in Bacillus subtilis: identification of new proteins at the DNA replication factory.
      ), consistent with systematic anecdotal evidence for the co-fractionation of these enzymes with membranes.
      G. L. Waldrop, personal communication.
      Here, we identified AccA, AccC, and AccD as PIM proteins that associate tightly with membranes electrostatically and can be removed by high ionic strength (supplemental Table S3B). These proteins were also identified in the size-exclusion chromatography/N-PAGE approach as subunits of acetyl-CoA carboxylase sub-complexes (supplemental Table S4) (in E. coli, the acetyl-CoA carboxylase complex is known to be rather unstable (
      • Choi-Rhee E.
      • Cronan J.E.
      The biotin carboxylase-biotin carboxyl carrier protein complex of Escherichia coli acetyl-CoA carboxylase.
      ), which explains why some subunits eluded detection before). Most important, the organization of these proteins in membrane-associated complexes suggested a possible function, although unknown at present. One obvious possibility is that such complexes might facilitate metabolic channeling of small molecule substrates (
      • Perez-Bercoff A.
      • McLysaght A.
      • Conant G.C.
      Patterns of indirect protein interactions suggest a spatial organization to metabolism.
      ,
      • Huthmacher C.
      • Gille C.
      • Holzhutter H.-G.
      A computational analysis of protein interactions in metabolic networks reveals novel enzyme pairs potentially involved in metabolic channeling.
      ).
      In other cases, interactions with the membrane surface are extremely tight and resistant to several chemical agents. These are suggestive of a multitude of interactions, almost certainly with proteinaceous receptors (e.g. SecA on SecYEG, SRP on FtsY, SeqA on DnaA). Some of these interactions (e.g. MinD, MlaB) can be disrupted only with the use of non-ionic detergent, indicating the importance of strong hydrophobic interactions. This tight interaction behavior is reminiscent of that of integral membrane proteins. Clearly, the biochemical means traditionally used for assigning PIM protein status (
      • Adelman M.R.
      • Sabatini D.D.
      • Blobel G.
      Ribosome-membrane interaction.
      ,
      • Fujiki Y.
      • Hubbard A.L.
      • Fowler S.
      • Lazarow P.B.
      Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.
      ,
      • Ohlendieck K.
      Extraction of membrane proteins.
      ,
      • Kreibich G.
      • Sabatini D.D.
      Selective release of content from microsomal vesicles without membrane disassembly.
      ,
      • Steck T.L.
      The organization of proteins in the human red blood cell membrane.
      ) are inadequate when annotating these tight interactions and should be revised.
      Membrane proteomics studies tend to consider cytoplasmic proteins that co-purify with membrane as “contaminants” (
      • Pieper 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.
      ,
      • Klein C.
      • Garcia-Rizo C.
      • Bisle B.
      • Scheffer B.
      • Zischka H.
      • Pfeiffer F.
      • Siedler F.
      • Oesterhelt D.
      The membrane proteome of Halobacterium salinarum.
      ,
      • Alexandersson E.
      • Saalbach G.
      • Larsson C.
      • Kjellbom P.
      Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking.
      ,
      • Aivaliotis M.
      • Haase W.
      • Karas M.
      • Tsiotis G.
      Proteomic analysis of chlorosome-depleted membranes of the green sulfur bacterium Chlorobium tepidum.
      ,
      • Aivaliotis M.
      • Karas M.
      • Tsiotis G.
      An alternative strategy for the membrane proteome analysis of the green sulfur bacterium Chlorobium tepidum using blue native PAGE and 2-D PAGE on purified membranes.
      ), as it is difficult to conclude whether these represent real interacting partners or nonspecific associations. Especially for novel proteins of unknown subcellular localization and/or function, more biological, biochemical, and cell biological evidence is required. In this context, the purity of inner membranes is crucial. Many of the PIM proteins we identified participate in multimeric complexes and/or have known interactors. The combination of mild membrane treatment (to leave complexes intact) with an orthogonal complexome purification approach identified several PIM complexes comprising abundant proteins (e.g. ATP synthase, NADH dehydrogenase, and succinate dehydrogenase). A future application of our PIM complexomics pipeline will be to identify unknown complexes and discover how these change in various growth regimes.
      Our approach relied heavily on subcellular fractionation. This is a way of eliminating contaminants and detecting low-copy-number proteins. Its inherent limitation is that topological information for proteins in the real cellular context is lost, as are unstable interactions. Also lost are the dynamics of complex formation and the kinetics of membrane-surface occupancy. In order to overcome these limitations, future approaches should employ tools such as monitoring green-fluorescent-protein-tagged proteins in the cell. Given the dual location of PIM proteins in both the cytoplasm and the membrane periphery, advanced kinetic- and FRET-based studies together with single molecule approaches and Total internal reflection fluorescence-based high resolution microscopy will have to be used to strengthen signals from membrane-associated interactions and distinguish them from an extensive cytoplasmic labeling background. Transient or weak interactions may be stabilized using chemical cross-linking prior to MS-based identification (
      • Sinz A.
      Investigation of protein–protein interactions in living cells by chemical crosslinking and mass spectrometry.
      ,
      • Bruce J.E.
      In vivo protein complex topologies: sights through a cross-linking lens.
      ,
      • Rappsilber J.
      The beginning of a beautiful friendship: cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes.
      ).
      The dynamic aspect of the peripherome is particularly fascinating, as it suggests that the membrane can act as a “temporary storage pool” to remove factors from cytoplasmic circulation and thus reduce their effective concentration or deliver them only upon external stimulation. This is the case with transcription factors NadR (
      • Raffaelli N.
      • Lorenzi T.
      • Mariani P.L.
      • Emanuelli M.
      • Amici A.
      • Ruggieri S.
      • Magni G.
      The Escherichia coli NadR regulator is endowed with nicotinamide mononucleotide adenylyltransferase activity.
      ), RpoE, and PutA (
      • Ostrovsky de Spicer P.
      • Maloy S.
      PutA protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator.
      ) detected here, as well as BglG, which was not identified (
      • Lopian L.
      • Nussbaum-Shochat A.
      • O'Day-Kerstein K.
      • Wright A.
      • Amster-Choder O.
      The BglF sensor recruits the BglG transcription regulator to the membrane and releases it on stimulation.
      ). Similarly, LacI, identified here as associating strongly with the membrane, is normally ascribed to a cytoplasmic location but was consistently found here in slightly overexpressed conditions as a membrane-associated tetramer (supplemental Fig. S3A, band K10). These transcription factors may be stored until needed—for example, when the relevant small molecule ligand enters the cell. Likewise, the RNA degradosome component RNE remains bound to the membrane during cell growth and is released and becomes active only in the late stationary phase (
      • Lopez-Campistrous A.
      • Semchuk P.
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      Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth.
      ,
      • Khemici V.
      • Poljak L.
      • Luisi B.F.
      • Carpousis A.J.
      The RNase E of Escherichia coli is a membrane-binding protein.
      ). Another example of dynamic behavior is that of PspA, which either forms oligomeric ring structures on the membrane surface thought to be ion leakage sites (
      • Kobayashi R.
      • Suzuki T.
      • Yoshida M.
      Escherichia coli phage-shock protein A (PspA) binds to membrane phospholipids and repairs proton leakage of the damaged membranes.
      ) or interacts as a monomer with other Psp proteins in solution and at the membrane (
      • Adams H.
      • Teertstra W.
      • Demmers J.
      • Boesten R.
      • Tommassen J.
      Interactions between phage-shock proteins in Escherichia coli.
      ).
      Here, we focused only on stable complexes, unambiguously annotated. As a result, we estimate that the PIM interactome is more complex. Many cytoplasmic proteins are expected to form fleeting interactions with the membrane or with PIM proteins attached to the membrane, as discussed earlier. Rather than static pair-wise interactions, the physiological interactions in the cell are likely to be multidimensional and might even involve “moonlighting” proteins (
      • Jeffery C.J.
      Moonlighting proteins.
      ) (supplemental Fig. S6). Thus the same PIM protein could be a partner in multiple different complexes, as in the cases of MukB (
      • Li Y.
      • Stewart N.K.
      • Berger A.J.
      • Vos S.
      • Schoeffler A.J.
      • Berger J.M.
      • Chait B.T.
      • Oakley M.G.
      Escherichia coli condensin MukB stimulates topoisomerase IV activity by a direct physical interaction.
      ,
      • Petrushenko Z.M.
      • Lai C.H.
      • Rybenkov V.V.
      Antagonistic interactions of kleisins and DNA with bacterial Condensin MukB.
      ), MurG (
      • de Boer P.A.
      Advances in understanding E. coli cell fission.
      ), RpoE (
      • Hayden J.D.
      • Ades S.E.
      The extracytoplasmic stress factor, sigma(E), is required to maintain cell envelope integrity in Escherichia coli.
      ,
      • Bordes P.
      • Lavatine L.
      • Phok K.
      • Barriot R.
      • Boulanger A.
      • Castanie-Cornet M.P.
      • Dejean G.
      • Lauber E.
      • Becker A.
      • Arlat M.
      • Gutierrez C.
      Insights into the extracytoplasmic stress response of Xanthomonas campestris pv. campestris: role and regulation of sigma(E)-dependent activity.
      ), MreB (
      • van den Ent F.
      • Johnson C.M.
      • Persons L.
      • de Boer P.
      • Lowe J.
      Bacterial actin MreB assembles in complex with cell shape protein RodZ.
      ,
      • Bendezu F.O.
      • Hale C.A.
      • Bernhardt T.G.
      • de Boer P.A.
      RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in.
      ,
      • Salje J.
      • van den Ent F.
      • de Boer P.
      • Lowe J.
      Direct membrane binding by bacterial actin MreB.
      ,
      • Kruse T.
      • Blagoev B.
      • Lobner-Olesen A.
      • Wachi M.
      • Sasaki K.
      • Iwai N.
      • Mann M.
      • Gerdes K.
      Actin homolog MreB and RNA polymerase interact and are both required for chromosome segregation in Escherichia coli.
      ), PyrG (
      • Ingerson-Mahar M.
      • Briegel A.
      • Werner J.N.
      • Jensen G.J.
      • Gitai Z.
      The metabolic enzyme CTP synthase forms cytoskeletal filaments.
      ), and GlpD (
      • Pan 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.
      ,
      • Schryvers A.
      • Lohmeier E.
      • Weiner J.H.
      Chemical and functional properties of the native and reconstituted forms of the membrane-bound, aerobic glycerol-3-phosphate dehydrogenase of Escherichia coli.
      ,
      • Cozzarelli N.R.
      • Koch J.P.
      • Hayashi S.
      • Lin E.C.
      Growth stasis by accumulated L-alpha-glycerophosphate in Escherichia coli.
      ). To make things more complex, the same protein can have strikingly different structural behaviors and functions, such as the actin-like protein MreB (detected in all our experiments), which can determine cell polarity in bacteria (
      • Gitai Z.
      • Dye N.
      • Shapiro L.
      An actin-like gene can determine cell polarity in bacteria.
      ), function as a structural filament, interact with elongation factor EF-Tu to define cell morphology (
      • Soufo H.
      • Reimold C.
      • Linne U.
      • Knust T.
      • Gescher J.
      • Graumann P.L.
      Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein.
      ), and also interact with RNA polymerase to initiate chromosome segregation (
      • Kruse T.
      • Blagoev B.
      • Lobner-Olesen A.
      • Wachi M.
      • Sasaki K.
      • Iwai N.
      • Mann M.
      • Gerdes K.
      Actin homolog MreB and RNA polymerase interact and are both required for chromosome segregation in Escherichia coli.
      ).
      In conclusion, a remarkable number of cytoplasmic proteins interact with the bacterial inner membrane. The PIM proteome acts as a dynamic and extensive liaison that connects the inner membrane with most cell processes. Our pipeline is applicable to investigations of peripheromes in other cells such as pathogenic bacteria. We expect future studies to elaborate on these networks of cytoplasmic-membrane cross talk, identify the role and dynamics of PIM proteins of unknown function, and determine the way in which they exert their central role in the cell's biology.

      Acknowledgments

      We are grateful to P. Mavroudis for preliminary experiments, W. Studier for insight in the gene annotation of E. coli K-12 and B strains, G. Waldrop for discussions on AccA, I. Tsamardinos for advice on bioinformatics approaches, E. Coudert (Uniprot) for E. coli protein annotation, K. Tsarhopoulos and P. Rigas (Rigas Labs, S.A.) for support with the LTQ-Orbitrap mass spectrometer, S. Ludwigsen (Proteome Software) for advice on Scaffold, and I. Kouklinos for computing infrastructure support.

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