Biochemical Studies of Bacterial Protein Export

  • P. C. Tai
Part of the Current Topics in Microbiology and Immunology book series (CT MICROBIOLOGY, volume 125)


Studies on the mechanisms of protein secretion and transport have been, until recently, confined to animal cells. In the past few years genetic and biochemical studies with bacteria have contributed greatly to our knowledge of mechanisms of protein secretion and localization (reviews by Davis and Tai 1980; Wickner 1980; Osborn and Wu 1980; Michaelis and Beckwith 1982; Randall and Hardy 1984; Benson et al. 1985). In particular, selection of mutants has permitted the definition of the function of the cleavable signal sequence and the identification of many gene products that may be involved in protein export (see Beckwith and Ferro-Novick, this volume; Lunn et al., this volume). In addition, several signal peptidases have been purified and characterized and their genes identified (Date and Wickner 1981; Innis et al. 1984; Yu et al. 1984; Zwizinski and Wickner 1980; Ray et al., this volume). Moreoever, since the outer surface of the bacterial membrane is readily accessible to manipulation, we have been able to use extracellular radioactive labeling or protease digestions of protruding secreted nascent chains to demonstrate directly the contranslational secretion (Smith et al. 1977, 1978) that had been inferred from the earlier in vitro work. The study of accessibility of nascent chains accumulates on the inner surface of the cytoplasmic membrane before the chains reach the outer surface (Randall 1983).


Membrane Vesicle Protein Translocation Signal Peptidase Protein Export Nascent Chain 
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  1. Adler LA, Arvidson S (1984) Immunological crossreaction between proteins supposed to be involved in protein secretion in Staphylococcus aureus.FEMS Microbiol Lett 23: 17–20CrossRefGoogle Scholar
  2. Bakker EP, Randall LL (1984) The requirement for energy during export of ß-lactamase in Escherichia coli is fulfilled by the total protonmotive force. EMBO J 3: 895–900PubMedGoogle Scholar
  3. Benson S, Hall MN, Silhavy TJ (1985) Genetic analysis of protein export in Escherichia coli K12.Annu Rev Biochem 54: 101 - 134PubMedCrossRefGoogle Scholar
  4. Caulfield MP, Tai PC, Davis BD (1983) Association of penicillin binding proteins and other enzymes with the ribosome-free membrane fraction of Bacillus subtilis. J Bacteriol 156: 1–5PubMedGoogle Scholar
  5. Caulfield MP, Tai PC, Davis BD (1984) The 64-kilodalton membrane protein of Bacillus subtilis is also present as a multiprotein complex on membrane-free ribosomes. Proc Natl Acad Sci USA 81: 7772–7776PubMedCrossRefGoogle Scholar
  6. Caulfleld MP, Furlong D, Tai PC, Davis BD (1985) The secretory S complex of Bacillus subtilis forms a large, organized structure when released from ribosomes. Proc Natl Acad Sci USA 82: 4031–4035CrossRefGoogle Scholar
  7. Chang CN, Model P, Blobel G (1979) Membrane biogenesis: cotranslational integration of the bacteriophage f1 coat protein into an Escherichia coli membrane fraction. Proc Natl Acad Sci USA 76: 1251–1255PubMedCrossRefGoogle Scholar
  8. Chen LL, Tai PC (1985) ATP is essential for protein translocation into Escherichia coli membrane vesicles. Proc Natl Acad Sci USA 82: 4384–4388PubMedCrossRefGoogle Scholar
  9. Chen LL, Rhoads D, Tai PC (1985) Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli. J Bacteriol 161: 973–980PubMedGoogle Scholar
  10. Daniels CJ, Anderson JJ, Landick R, Oxender DL (1980) The in vitro synthesis and processing of the branched-chain amino acid binding proteins. J Supramol Struct 14: 305–311PubMedCrossRefGoogle Scholar
  11. Daniels CJ, Bole DG, Quay SC, Oxender DL (1981) A role for membrane potential in the secretion of protein into the periplasm of Escherichia coli. Proc Natl Acad Sci USA 77: 4669–4673Google Scholar
  12. Date T, Wickner W (1981) Isolation of the Escherichia coli leader peptidase gene and effects of leader peptidase overproduction in vivo. Proc Natl Acad Sci USA 78: 6106–6110PubMedCrossRefGoogle Scholar
  13. Davis BD, Tai PC (1980) The mechanism of protein secretion across membranes. Nature 283: 433–437PubMedCrossRefGoogle Scholar
  14. DiRienzo JM, Inouye M (1979) Lipid fluidity-dependent biosynthesis and assembly of the outer membrane proteins of E. coli. Cell 17: 155–161PubMedCrossRefGoogle Scholar
  15. Enequist HG, Hirst TR, Hardy SJS, Harayama S, Randall LL (1981) Energy is required for maturation of exported proteins in Escherichia coli. Eur J Biochem 116: 227–233PubMedCrossRefGoogle Scholar
  16. Engelmen DM, Steitz TA (1981) The spontaneous insertion of proteins into and across membranes:the helical hairpin hypothesis. Cell 23: 411–422CrossRefGoogle Scholar
  17. Fecycz IT, Campbell N (1985) Mechanisms of activation and secretion of a cell-associated precursor of an exocellular proteases of Pseudomonas aeruginosa 34362A. Eur J Biochem 146: 35–42PubMedCrossRefGoogle Scholar
  18. Fishman Y, Rottem S, Citri N (1978) Evidence linking penicillinase formation and secretion to lipid metabolism in Bacillus licheniformis. J Bacteriol 134: 434–439PubMedGoogle Scholar
  19. Fishman Y, Rottem S, Citri N (1980) Preferential suppression of normal exoenzyme formation by membrane-modifying agents. J Bacteriol 141: 1435–1438PubMedGoogle Scholar
  20. Garwin JL, Beckwith J (1982) Secretion and processing of ribose-binding protein in Escherichia coli. J Bacteriol 149: 789–792PubMedGoogle Scholar
  21. Gasser SM, Daum G, Schatz G (1982) Import of proteins into mitochondria. J Biol Chem 257: 13034–13041PubMedGoogle Scholar
  22. Gilmore R, Walter P, Blobel G (1982) Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor. J Cell Biol 95: 470–477PubMedCrossRefGoogle Scholar
  23. Goodman JM, Watts C, Wickner W (1981) Membrane assembly: posttranslational insertion of Ml3 procoat protein into E. coli membranes and its proteolytic conversion to coat protein in vitro. Cell 24: 437–444PubMedCrossRefGoogle Scholar
  24. Grossman A, Bartlett S, Chua NH (1980) Energy-dependent uptake of cytoplasmically synthesized polypeptides by chloroplasts. Nature 285: 625–628CrossRefGoogle Scholar
  25. Hirst TR, Sanchez J, Kaper JB, Hardy SJ, Holmgren J (1984) Mechanism of toxin secretion by Vibrio cholerae investigated in strains harboring plasmids that encode heat-labile enterotoxins of Escherichia coli. Proc Natl Acad Sci USA 81: 7752–7756PubMedCrossRefGoogle Scholar
  26. Horiuchi S, Marty-Mazars D, Tai PC, Davis BD (1983 a) Localization and quantitation of proteins characteristic of the complexed membrane of Bacillus subtilis. J Bacteriol 154: 1215–1221PubMedGoogle Scholar
  27. Horiuchi S, Tai PC, Davis BD (1983 b) A 64-kilodalton membrane protein of Bacillus subtilis covered by secreting ribosomes. Proc Natl Acad Sci USA 80: 3287–3291PubMedCrossRefGoogle Scholar
  28. Howard SP, Buckley JT (1985a) Protein export by a gram-negative bacterium: production of aeroly-sin by Aeromonas hydrophila. J Bacteriol 161: 1118–1124PubMedGoogle Scholar
  29. Howard SP, Buckley JT (1985 b) Activation of the hole-forming toxin aerolysin by extracellular processing. J Bacteriol 163: 336–340PubMedGoogle Scholar
  30. Innis MA, Tokunaga M, Williams ME, Loranger JM, Chang SY, Chang S, Wu HC (1984) Nucleotide sequence of Escherichia coli prolipoprotein signal peptidase ( Isp) gene. Proc Natl Acad Sci USA 81: 3708–3712PubMedCrossRefGoogle Scholar
  31. Josefsson L, Randall LL (1981) Different exported proteins in Escherichia coli show differences in the temporal mode of processing in vivo. Cell 25: 151–157PubMedCrossRefGoogle Scholar
  32. Kimura K, Izui K (1976) Importance of membrane fluidity in the induction of alkaline phosphatase, a periplasmic enzyme in Escherichia coli. Biochem Biophys Res Commun 70: 900–906PubMedCrossRefGoogle Scholar
  33. Koshland D, Botstein D (1982) Evidence for post-translational translocation of beta-lactamase across the bacterial inner membrane. Cell 30: 893–902PubMedCrossRefGoogle Scholar
  34. Kreibich G, Czako-Graham M, Grebenau R, Mok W, Rodriguez-Boulan E, Sabatini D (1978 a) Characterization of the ribosomal binding site in rat liver rough microsomes: ribophorins I and II, two integral membrane proteins related to ribosome binding. J Supramol Struct 8: 279–302PubMedCrossRefGoogle Scholar
  35. Kreibich G, Ulrich BL, Sabatini D ( 1978 b) Proteins of rough microsomal membranes related to ribosome binding I. Identification of ribophorins I and II, membrane proteins characteristic of rough microsomes. J Cell Biol 77: 464–487PubMedCrossRefGoogle Scholar
  36. Kumamoto C, Beckwith J (1985) Evidence for specificity at an early step in protein export in Escherichia coli. J Bacteriol 163: 267–274PubMedGoogle Scholar
  37. Lazdunski C, Baty D, Pages JM (1979) Procaine, a local anesthetic interacting with the cell membrane, inhibits the processing of precursor forms of periplasmic proteins in Escherichia coli. Eur J Biochem 96: 49–57PubMedCrossRefGoogle Scholar
  38. Lin JJ-C, Kanazawa H, Ozols J, Wu HC (1978) Escherichia coli mutant with an amino-acid alteration with signal sequence of outer membrane prolipoprotein. Proc Natl Acad Sci USA 75: 4891–4895PubMedCrossRefGoogle Scholar
  39. Lory S, Tai PC, Davis BD (1983) Mechanisms of protein excretion by gram-negative bacteria:Pseudomonas aeruginosa exotoxin A. J Bacteriol 156: 695–702PubMedGoogle Scholar
  40. Marty-Mazars D, Horiuchi S, Tai PC, Davis BD (1983) Proteins of ribosome-bearing and-free membrane domains in Bacillus subtilis. J Bacteriol 154: 1381–1388PubMedGoogle Scholar
  41. Meyer DI (1985) Signal recognition particle (SRP) does not mediate a translational arrest of nascent secretory proteins in mammalian cell-free systems. EMBO J 4: 2031–2033PubMedGoogle Scholar
  42. Meyer DI, Krause E, Dobberstein B (1982) Secretory protein translocating across membranes-the role of the “docking protein.” Nature 297: 647–650PubMedCrossRefGoogle Scholar
  43. Michaelis S, Beckwith J (1982) Mechanism of incorporation of cell envelope proteins in Escherichia coli. Annu Rev Microbiol 36: 435 - 465PubMedCrossRefGoogle Scholar
  44. Müller M, Blobel G (1984 a) In vitro translocation of bacterial proteins across the plasma membrane of Escherichia coli. Proc Natl Acad Sei USA 81:7421–7425CrossRefGoogle Scholar
  45. Müller M, Blobel G (1984b) Protein export in Escherichia coli requires a soluble activity. Proc Natl Acad SCi USA 81: 7737–7741PubMedCrossRefGoogle Scholar
  46. Nakamura S, Racker E (1984) Inhibitory effect of duramycin on partial reactions catalyzed by(Na+, K+)-adenosine triphosphatase from dog kidney. Biochemistry 23: 385–389PubMedCrossRefGoogle Scholar
  47. Nesmeyanova MA (1982) On the possible participation of acid phospholipids in the translocation of secreted proteins through the bacterial cytoplasmic membrane. FEBS Lett 142: 189–193PubMedCrossRefGoogle Scholar
  48. Oliver D, Beckwith J (1981) E. coli mutant pleiotropically defective in the export of secreted proteins. Cell 25: 765–772PubMedCrossRefGoogle Scholar
  49. Osborn MJ, Wu H (1980) Proteins of the outer membrane of Gram-negative bacteria. Annu Rev Microbiol 34: 369–422PubMedCrossRefGoogle Scholar
  50. Pages JM, Lazdunski C (1982) Maturation of exported proteins in Escherichia coli. Requirement of energy, site and kinetics of processing. Eur J Biochem 124: 561–566PubMedCrossRefGoogle Scholar
  51. Pages JM, Piovant M, Varronne S, Lazdunski C (1978) Mechanistic aspects of the transfer of nascent periplasmic proteins across the cytoplasmic membranes in Escherichia coli. Eur J Biochem 80: 589–602CrossRefGoogle Scholar
  52. Pages JM, Anba J, Bernadac A, Shinagawa H, Nakata A, Lazdunski C (1984) Normal precursors of periplasmic proteins accumulated in the cytoplasm are not exported posttranslationally in Escherichia coli. J Biochem 143: 499–505Google Scholar
  53. Palva ET, Hirst TR, Hardy SJS, Holmgren J, Randall LL (1981) Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli. J Bacteriol 146: 325–340PubMedGoogle Scholar
  54. Randall LL (1983) Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation. Cell 33: 231–240PubMedCrossRefGoogle Scholar
  55. Randall LL, Hardy SJS (1984) Export of protein in bacteria. Microbiol Rev 48: 290–298PubMedGoogle Scholar
  56. Rhoads DB, Tai PC, Davis BD (1984) Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into membrane vesicles. J Bacteriol 159: 63–70PubMedGoogle Scholar
  57. Salisbury JL, Condeelis JS, Satir P (1983) Receptor-mediated endocytosis: machinery and regulation of the clathrin-coated vesicle pathway. Int Rev Exp Pathol 24: 2–63Google Scholar
  58. Schleyer M, Schmidt B, Neupert W (1982) Requirement of a membrane potential for the posttransla-tional transfer of proteins into mitochondria. Eur J Biochem 125: 109–116PubMedCrossRefGoogle Scholar
  59. Silhavy TJ, Benson SA, Emr SD (1983) Mechanisms of protein localization. Microbiol Rev 47: 313–344PubMedGoogle Scholar
  60. Smith WP (1980) Cotranslational secretion of diphtheria toxin and alkaline phosphatase in vitro:involvement of membrane protein(s). J Bacteriol 141: 1142–1147PubMedGoogle Scholar
  61. Smith WP, Tai PC, Thompson RC, Davis BD (1977) Extracellular labeling of nascent polypeptides traversing the membrane of Escherichia coli. Proc Natl Acad SCi USA 74: 2830–2834PubMedCrossRefGoogle Scholar
  62. Smith WP, Tai PC, Davis BD (1978) Interaction of secreted chains with surrounding membrane in Bacillus subtilis. Proc Natl Acad Sei USA 75: 814–817CrossRefGoogle Scholar
  63. Smith WP, Tai PC, Davis BD (1979) Extracellular labeling of growing secreted polypeptide chains in Bacillus subtilis with diazoiodosulfanic acid. Biochemistry 18: 198–203PubMedCrossRefGoogle Scholar
  64. Smith WP, Tai PC, Davis BD (1981) Bacillus licheniformis penicillinase: cleavages and attachment of lipid during cotranslational secretion. Proc Natl Acad Sci USA 78: 3501–3505PubMedCrossRefGoogle Scholar
  65. Stone DK, Xie X-S, Racker E (1984) Inhibition of clathrin-coated vesicle acidification by duramycin.J Biol Chem 259: 2701–2703PubMedGoogle Scholar
  66. Tribhuwan RC, Pradhan DS (1977) Induction of alkaline phosphatase in Escherichia coli: effect of procaine-hydrochloride. J Bacteriol 131: 431–437PubMedGoogle Scholar
  67. Tribhuwan RC, Pilgaokar AK, Pradhan DS, Screenivasan DS (1970) Effect of phenethylalcohol on induction of alkaline phosphatase in Escherichia coli. Biochem Biophys Res Commun 41: 244–250CrossRefGoogle Scholar
  68. Varenne S, Piovant M, Pages JM, Lazdunski C (1978) Evidence for synthesis of alkaline phosphatase on membrane-bound polysomes in Escherichia coli. Eur J Biochem 86: 603–606PubMedCrossRefGoogle Scholar
  69. Von Heijne G, Blomberg C (1979) Transmembrane translocation of protein. Eur J Biochem 97: 175–181CrossRefGoogle Scholar
  70. Walter P, Blobel G (1980) Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum. Proc Natl Acad Sci USA 77: 7112–7116PubMedCrossRefGoogle Scholar
  71. Walter P, Blobel G (1981) Translocation of proteins across the endoplasmic reticulum. III. Signal recognition protein ( SRP) causes signal-sequence dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J Cell Biol 91: 557–561PubMedCrossRefGoogle Scholar
  72. Wickner W (1980) Assembly of proteins into membranes. Science 210: 861–868PubMedCrossRefGoogle Scholar
  73. Yu F, Yamada H, Daishima K, Mizushima S (1984) Nucleotide sequence of the IspA gene, the structural gene for lipoprotein peptidase of Escherichia coli. FEBS Lett 173: 264–268PubMedCrossRefGoogle Scholar
  74. Zimmermann R, Wickner W (1983) Energy and intermediates of the assembly of protein OmpA into the outer membrane of Escherichia coli. J Biol Chem 258:3920–3925PubMedGoogle Scholar
  75. Zwizinski C, Wickner W (1980) Purification and characterization of leader (signal) peptidase from Escherichia coli. J Biol Chem 255: 7973–7977PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin · Heidelberg 1986

Authors and Affiliations

  • P. C. Tai
    • 1
    • 2
  1. 1.Department of Metabolic RegulationBoston Biomedical Research InstituteBostonUSA
  2. 2.Department of Microbiology and Molecular GeneticsHarvard Medical SchoolBostonUSA

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