Summary
A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA+ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA+ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Akiyama Y, Ito K (1987) Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli. EMBO J 6:3465–3470
Ausubel FM, Brentt R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1987) Current protocols in molecular biology, vol 4. John Wiley & Sons, New York
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
Brosius J, Cate RL, Perlmutter AP (1982) Precise location of two promoters for the β-lactamase gene of pBR322. J Biol Chem 257:9205–9216
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the p15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Craig A, Lo RYC (1989) Cloning, nucleotide sequence, and characterization of genes encoding the secretion function of the Pasteurella haemolytica leucotoxin determinant. J Bacteriol 171:916–928
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
Gentschev I, Goebel W (1992) Topological and functional studies on HlyB of Escherichia coli. Mol Gen Genet 232:40–48
Gentschev I, Hess J, Goebel W (1990) Change in the cellular localization of alkaline phosphatase by alteration of its carboxyterminal sequence. Mol Gen Genet 222:211–216
Glaser P, Ladant D, Sezer O, Pichot F, Ulmann A, Danchin A (1988) The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli. Mol Microbiol 2:19–30
Glisin V, Crkvenjakov R, Byus C (1974) Ribonucleic acid isolation by CsCl centrifugation. Biochemistry 13:2633–2637
Gray L, Mackman L, Nicaud JM, Holland IB (1986) The carboxyterminal region of haemolysin 2001 is required for secretion of the toxin from Escherichia coli. Mol Gen Genet 205:127–133
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, Gentschev I, Goebel W, Jarchau T (1990) Analysis of the hemolysin secretion system by PhoA-H1yA fusion proteins. Mol Gen Genet 224:201–208
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
Jarchau T, Chakraborty T, Garcia F, Goebel W Selection for transport competence among C-terminal polypeptides of Escherichia coli hemolysin: 62 amino acids is the smallest size for HlyA peptides capable of hlyB- and hlyD-dependent transport, in preparation
Juarez A, Goebel W (1984) Chromosomal mutation that affects excretion of hemolysin in Escherichia coli. J Bacteriol 159:1083–1085
Kenny B, Haigh R, Holland IB (1991) Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or β-galactosidase fused to the Hly C-terminal signal domain. Mol Microbiol 5:2557–2568
Klein P, Kanehisha M, DeLisi C (1985) The detection and classification of membrane spanning proteins. Biochim Biophys Acta 815:468–476
Koshland D, Botstein D (1980) Secretion of β-lactamase requires the carboxy end of the protein. Cell 20:749–760
Kramer W, Drutsa V, Jansen HW, Kramer B, Pflugfelder M, Fritz HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res 12:9441–9456
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
Ludwig A, Goebel W (1991) Genetic determinants of cytolytic toxins from Gram-negative bacteria. In: Alouf JE and Freer JH (eds) Sourcebook of bacterial protein toxins. Academic Press, London, pp 117–146
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
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 Harbor Laboratory Press, Cold Spring Harbor, New York
Nicaud JM, Mackman N, Gray L, Holland IB (1986) The C-terminal 23 kD peptide of E. coli hemolysin 2001 contains all information necessary for its secretion by the hemolysin (hly) export machinery. FEBS Lett 204:331–335
Osborn MJ, Gander JE, Parisi E, Carson J (1972) Mechanism of assembly of the outer membrane of Salmonella typhimurium: Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem 247:3962–3972
Rao MJK, Argos P (1986) A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta 869:197–214
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulsen AR (1977) DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Stanley P, Koronakis V, Hughes C (1991) Mutational analysis supports a role for multiple structural features in the C-terminal secretion signal of Escherichia coli. Mol Microbiol 5:2931–2403
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 (hlyR) which enhances synthesis and secretion of hemolysin in Escherichia coli. Mol Gen Genet 212:76–84
Wagner W, Vogel M, Goebel W (1983) Transport of hemolysin across the outer membrane of E. coli requires two functions. J Bacteriol 154:200–210
Wandersman C, Delepelaire P (1990) ToIC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci USA 87:4776–4780
Wandersman C, Delepelaire P (1991) Characterization, localization and transmembrane organization of the three proteins PrtD, PrtE and PrtF necessary for protease secretion by the Gramnegative bacterium Erwinia chrysanthemi. Mol Microbiol 5:2427–2434
Wang R, Seror JS, Blight M, Pratt JM, Broome-Smith JK, Holland IB (1991) Analysis of the membrane organization of an Eseherichia coli protein translocator, HlyB, a member of a large family of prokaryote and eukaryote surface transport proteins. J Mol Biol 217:441–454
Welch RA, Pellett S (1988) Transcriptional organization of the Escherichia coli hemolysin genes. J Bacteriol 170:1622–1630
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
Author information
Authors and Affiliations
Additional information
Communicated by E. Bautz
Rights and permissions
About this article
Cite this article
Schülein, R., Gentschev, I., Mollenkopf, HJ. et al. A topological model for the haemolysin translocator protein HlyD. Molec. Gen. Genet. 234, 155–163 (1992). https://doi.org/10.1007/BF00272357
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00272357