Summary
The topology of HlyB, a protein located in the inner membrane of Escherichia coli and involved in the secretion of α-haemolysin (HlyA), was determined by the generation of HlyB-PhoA and HlyB-LacZ fusion proteins. The data obtained by this biochemical method together with computer predictions suggest that HlyB is inserted in the cytoplasmic membrane by six stable hydrophobic, α-helical transmembrane segments. These segments extend from amino acid positions 158 to 432 of HlyB. The cytoplasmic loops between these transmembrane segments are relatively large and carry an excess of positively charged amino acids, while the periplasmic loops are rather small. In addition to these six transmembrane segments, two additional regions in the 78 N-terminal amino acids of HlyB appear to be also inserted in the cytoplasmic membrane. However, the association of these two segments with the cytoplasmic membrane seems to be less tight, since active PhoA and LacZ fusions were obtained by insertion into the same positions of these segments. A LacZ-HlyAs fusion protein carrying, at the C-terminus of LacZ, the 60-amino acid signal sequence of HlyA was not secreted in the presence of HlyB/HlyD. However, transport of this fusion protein into the cytoplasmic membrane appeared to be initiated, as suggested by the tight association of this protein with the inner membrane. A similar close association of LacZ-HlyAs with the inner membrane was also observed in the presence of HlyB alone but not in its absence. These data suggest that HlyB recognizes the HlyA signal sequence and initiates the transport of HlyA into the membrane.
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Adler HI, Fischer WD, Cohen A, Hardigree AA (1967) Miniature E. coli cells deficient in DNA. Proc Natl Acad Sci USA 57:321–326
Blight MA, Holland IB (1990) Structure and function of haemolysin B, P-glycoprotein and other members of a novel family of membrane translocators. Mol Microbiol 4:873–880
Brickman E, Beckwith J (1975) Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and φ80 transducing phages. J Mol Biol 96:307–316
Broome-Smith J, Spratt B (1986) A vector for the construction of translational fusions to TEM β-lactamase and the analysis of protein export signals and membrane protein topology. Gene 49:341–349
Broome-Smith JK, Tadayyon M, Zhang Y (1990) β-Lactamase as a probe of membrane protein assembly and protein export. Mol Microbiol 4:1637–1644
Eisenberg D, Schwarz E, Komaromy M, Wall R (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol 179:125–142
Felmlee T, Pellett S, Welch RA (1985 a) E. coli hemolysin is released extracellularly without cleavage of signal peptide. J Bacteriol 163:88–93
Felmlee T, Pellett S, Welch RA (1985 b) Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol 163:94–105
Gentschev I, Hess J, Goebel W (1990) Change in the cellular localization of alkaline phosphatase by alteration of its carboxy-terminal sequence. Mol Gen Genet 222:211–216
Goebel W, Hedgpeth J (1982) Cloning and functional characterization of the plasmid-encoded hemolysin determinant of E. coli. J Bacteriol 151:1290–1298
Gray L, Baker K, Kenny B, Mackman N, Haigh R, Holland IB (1989) A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium. J Cell Sci Suppl 11:45–57
Härtlein M, Schiessl S, Wagner W, Rdest U, Kreft J, Goebel W (1983) Transport of haemolysin by E. coli. J Cell Biochem 22:87–97
Hess J, Wels W, Vogel M, Goebel W (1986) Nucleotide sequence of a plasmid-encoded hemolysin determinant and its comparison with a corresponding chromosomal hemolysin sequence. FEMS Microbiol Lett 34:1–11
Hess J, Gentschev I, Goebel W, Jarchau T (1990) Analysis of the hemolysin secretion system by PhoA-HlyA fusion proteins. Mol Gen Genet 224:201–208
Hess (1990) Transport von Hämolysin in Escherichia coli. Ph. D. Thesis, University of Würzburg, FRG
Higgins CF, Hiles ID, Salmon GPC, Gill DR, Downie JA, Evans IJ, Holland IB, Gray L, Buckel SD, Bell AW, Hermodson MA (1986) A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature 323:448–450
Holland IB, Wang R, Seror SJ, Light M (1989) Haemolysin secretion and other protein translocation mechanisms in gram-negative bacteria. In: Baumber S, Hunter I, Rhodes M (eds) Microbial products: New approaches. Cambridge University Press, pp 219–254
Jarchau T, Chakraborty T, Garcia F, Goebel W (1991) Selection for transport competence among C-terminal polypeptides of Escherichia coli hemolysin: 62 amino acids is the smallest size for H1yA-peptides capable of hlyB and hlyD dependent transport. Submitted for publication
Kern P, Kanehisa M, DeLisi C (1985) The detection and classification of membrane spanning proteins. Biochim Biophys Acta 815:468–476
Koronakis V, Koronakis E, Hughes C (1989) Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes. EMBO J 8:595–605
Koshland D, Botstein D (1980) Secretion of beta lactamase requires the carboxy end of the protein. Cell 20:749–760
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685
Mackman N, Nicaud J-M, Gray L, Holland IB (1985) Identification of polypeptides required for the export of haemolysin 2001 from E. coli. Mol Gen Genet 201:529–536
Mackman N, Baker K, Gray L, Haigh R, Nicaud J-M, Holland IB (1987) Release of a chimeric protein into the medium from Escherichia coli using the C-terminal signal of hemolysin. EMBO J 6:2835–2841
Manoil C (1990) Analysis of protein localization by use of gene fusions with complementary properties. J Bacteriol 172:1035–1042
Manoil C, Beckwith J (1985) TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci USA 82:8129–8133
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbour Laboratory, Cold Spring Harbour, NY
Noegel A, Rdest U, Springer W, Goebel W (1979) Plasmid cistrons controlling synthesis and excretion of the exotoxin α-haemolysin of Escherichia coli. Mol Gen Genet 175:343–350
Oropeza-Wekerle RL, Speth W, Imhof B, Gentschev I, Goebel W (1990) Translocation and compartmentalization of Escherichia coli hemolysin (HlyA). J Bacteriol 172:3711–3717
Rao MJK, Argos P (1986) A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta 869:197–214
Rüther U, Müller-Hill B (1983) Easy identification of cDNA clones. EMBO J 2:1791–1794
Sanger F, Nicklen S, Coulsen AR (1977) DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Stanssens P, Opsomer C, McKeown YM, Kramer W, Zabeau M, Fritz H-J (1989) Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped-duplex DNA method using alternating selectable markers. Nucleic Acids Res 17:4441–4454
Towbin H, Staehelin H, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Vogel M, Hess J, Then I, Juarez A, Goebel W (1988) Characterization of a sequence (hylR) which enhances synthesis and secretion of hemolysin in Escherichia coli. Mol Gen Genet 212:76–84
von Heijne G (1986) The distribution of positively charged residues in bacterial inner membrane proteins correlates with the transmembrane topology. EMBO J 5:3021–3027
Wagner W, Vogel M, Goebel M (1983) Transport of hemolysin across the outer membrane of E. coli requires two functions. J Bacteriol 154:200–210
Wandersman C, Delepelaire P (1990) TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci USA 87:4776–4780
Wang R, Seror SJ, Blight M, Pratt JM, Broome-Smith JK, Holland IB (1991) Analysis of the membrane organization of an Escherichia coli protein translocator, HlyB, a member of a large family of prokaryote and eukaryote surface transport proteins. J Mol Biol 217:441–454
Yanisch-Perron C, Vieira I, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 1167:103–119
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Gentschev, I., Goebel, W. Topological and functional studies on HlyB of Escherichia coli . Molec. Gen. Genet. 232, 40–48 (1992). https://doi.org/10.1007/BF00299135
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DOI: https://doi.org/10.1007/BF00299135