Abstract
A number of secY mutants of Escherichia coli showing protein export defects were isolated by a combination of localized mutagenesis and secA-lacZ screening. Most of them were cold sensitive and contained single base substitutions in secY leading to amino acid replacements in various parts of the SecY protein, mainly in the cytoplasmic and the transmembrane domains. A temperature-sensitive mutant with an export defect had the same base substitution as secY24, which was characterized previously. Many cold-sensitive secY mutants exhibited rapid responses to temperature lowering but their apparent defects varied at the permissive temperature. Others exhibited delayed responses to the temperature shift. Some secY mutations, including secY39, interfered with protein export when expressed from a multicopy plasmid, even in the presence of wild-type secY on the chromosome. Such “dominant negative” mutations, including secY −d l, which was studied previously, were all located in either cytoplasmic domain 5 or 6, which is consistent with our previous proposal that the C-terminal region of SecY is important for its function as a protein translocator. We also studied the phenotypes of strains in which one of the secY mutations was combined with the components of the SecD operon. Overexpression of SecD partially suppressed the secY39 mutation, while overexpression of secF exacerbated the export defects of secY122 and secY125 mutations. Overexpression of “yajC”, located within the SecD operon, suppressed sec Y −d1. Although yajC itself proved to be dispensable, its disruption impaired the growth of the secY39 mutant at 42°C. These observations suggest that SecY interacts with SecD, SecF, and the product of yajC.
Similar content being viewed by others
References
Akimaru J, Matsuyama S, Tokuda H, Mizushima S (1991) Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Escherichia coli. Proc Natl Acad Sci USA 88:6545–6549
Akiyama Y, Ito K (1985) The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins. EMBO J 4:3351–3356
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
Akiyama Y, Ito K (1990) SecY protein, a membrane embedded secretion factor of E. coli, is cleaved by the OmpT protease in vitro. Biochem Biophys Res Commun 167:711–715
Baba T, Jacq A, Brickman E, Beckwith J, Taura T, Ueguchi C, Akiyama Y, Ito K (1990) Characterization of cold-sensitive SecY mutants of Escherichia coli. J Bacteriol 172:7005–7010
Bieker KL, Silhavy TJ (1990) PrlA (SecY) and PrlG (SecE) interact directly and function sequentially during protein translocation in E. coli. Cell 61:833–842
Bieker-Brady K, Silhavy TJ (1992) Suppressor analysis suggests a multiple, cyclic mechanism for protein secretion in Escherichia coli. EMBO J 11:3165–3174
Brandl CJ, Deber CM (1986) Hypothesis about the function of membrane-buried proline residues in transport proteins. Proc Natl Acad Sci USA 83:917–921
Brundage L, Hendrick JP, Schiebel E, Driessen AJM, Wickner W (1990) The purified E. coli integral membrane protein SecY/SecE is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell 62:649–657
Derman AI, Puziss JW, Bassford Jr PJ, Beckwith J (1993) A signal sequence is not required for protein export in prlA mutants of Escherichia coli. EMBO J 12:879–888
Douglas SE (1992) A secY homologue is found in the plastid genome of Cryptomonas Φ. FEBS Lett 298:93–96
Francetic O, Hanson MP, Kumamoto CA (1993) prlA suppression of defective export of maltose-binding protein in secB mutants of Escherichia coli. J Bacteriol 175:4036–4044
Gardel C, Benson S, Hunt J, Michaelis S, Beckwith J (1987) secD, a new gene involved in protein export in Escherichia coli. J Bacteriol 169:1286–1290
Gardel C, Johnson K, Jacq A, Beckwith J (1990) The secD locus of E. coli codes for two membrane proteins required for protein export. EMBO J 19:3209–3216
Gorlich D, Prehn S, Hartmann E, Kalies K-U, Rapoport TA (1992) A mammalian homolog of SEC61p and SecYp is associated with ribosomes and nascent polypeptides during translocation. Cell 71:489–503
Ito K (1992) SecY and integral membrane components of the Escherichia coli protein translocation system. Mol Microbiol 6:2423–2428
Ito K, Wittekind M, Nomura M, Shiba K, Yura T, Miura A, Nashimoto H (1983) A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins. Cell 32:789–797
Ito K, Hirota Y, Akiyama Y (1989) Temperature-sensitive mutants of Escherichia coli: inhibition of protein export at the permissive temperature. J Bacteriol 171:1742–1743
Kumamoto CA (1991) Molecular chaperones and protein translocation across the Escherichia coli inner membrane. Mol Microbiol 5:19–22
Matsuyama S, Fujita Y, Sagara K, Mizushima S (1992) Overproduction, purification and characterization of SecD and SecF integral membrane components of the protein translocation machinery of Escherichia coli. Biochim Biophys Acta 1122:77–84
Matsuyama S, Fujita Y, Mizushima S (1993) SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli. EMBO J 12:265–270
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Oliver DB (1993) SecA protein: autoregulated ATPase catalyzing preprotein insertion and translocation across the Escherichia coli inner membrane. Mol Microbiol 7:159–165
Osborne RS, Silhavy TJ (1993) PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains. EMBO J 12:3391–3398
Pogliano KJ, Beckwith J (1993) The Cs sec mutants of Escherichia coli reflect the cold sensitivity of protein export itself. Genetics 133:763–773
Puziss JW, Strobel SM, Bassford Jr PJ (1992) Export of maltosebinding protein species with altered change distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations. J Bacteriol 174:92–101
Reuter K, Slany R, Ullrich F, Kersten H (1991) Structure and organization of Escherichia coli genes involved in biosynthesis of the deazaguanine derivative queuine, a nutrient factor for eukaryotes. J Bacteriol 173:2256–2264
Riggs PD, Derman AI, Beckwith J (1988) A mutation affecting the regulation of a secA-lacZ fusion defines a new sec gene. Genetics 118:571–579
Sako T, Iino T (1988) Distinct mutation sites in prlA suppressor mutant strains of Escherichia coli respond either to suppression of signal peptide mutations or to blockage of staphylokinase processing. J Bacteriol 170: 5389–5391
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Scaramuzzi CD, Stokes HW, Hiller RG (1992) Characterization of a chloroplast-encoded secY homologue and atpH from a chromophytic alga. Evidence for a novel chloroplast genome organization. FEBS Lett 304:119–123
Schatz PJ, Beckwith J (1990) Genetic analysis of protein export in Escherichia coli. Annu Rev Genet 24:215–248
Schatz PJ, Riggs PD, Jacq A, Fath MJ, Beckwith J (1989) The secE gene encodes an integral membrane protein required for protein export in Escherichia coli. Genes Dev 3:1035–1044
Schatz P, Bieker KL, Ottemann KM, Silhavy TJ, Beckwith J (1991) One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coli secretion machinery. EMBO J 10:1749–1757
Shiba K, Ito K, Yura T, Cerretti DP (1984) A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant. EMBO J 3: 631–635
Shimoike T, Akiyama Y, Baba T, Taura T, Ito K (1992) secY variants that interfere with E. coli protein export in the presence of normal secY Mol Microbiol 6:1205–1210
Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Stahl FW, Kobayashi I, Thaler D, Stahl MM (1986) Direction of travel of RecBC recombinase through bacteriophage lambda DNA. Genetics 113:215–227
Stirling CJ, Rothblatt J, Hosobuchi M, Deshaies R, Schekman R (1992) Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum. Mol Biol Cell 3:129–142
Takeshita S, Sato M, Toba M, Masahashi W, Hashimoto-Gotoh T (1987) High-copy-number and low-copy-number plasmid vectors for lacZ α-complementation and chloramphenicol- or kanamycin-resistance selection. Gene 61:63–74
Taura T, Ueguchi C, Shiba K, Ito K (1992) Insertional disruption of the nusB (ssyB) gene leads to cold-sensitive growth of Escherichia coli and suppression of the secY24 mutation. Mol Gen Genet 234:429–432
Taura T, Baba T, Akiyama Y, Ito K (1993) Determinants of the quantity of the stable SecY complex in the Escherichia coli cell. J Bacteriol 175:7771–7775
Tschauder S, Driessen AJM, Freudl R (1992) Cloning and molecular characterization of the secY genes from Bacillus licheniformis and Staphylococcus carnosus: comparative analysis of nine members of the SecY family. Mol Gen Genet 235:147–152
Ueguchi C, Ito K (1990) Escherichia coli sec mutants accumulate a processed immature form of maltose-binding protein (MBP), a late-phase intermediate in MBP export. J Bacteriol 172:5643–5649
Ueguchi C, Ito K (1992) Multicopy suppression: an approach to the intracellular functioning of the protein export system. J Bacteriol 174:1454–1461
Ueguchi C, Wittekind M, Nomura M, Akiyama Y, Ito K (1989) The secY-rpmJ region of the spc ribosomal protein operon in Escherichia coli: Structural alterations affecting secY expression. Mol Gen Genet 217:1–5
Vieira J, Messing J (1982) The pUC plasmids, an M131mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Wicker W, Driessen AJM, Hartl F-U (1991) The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochim 60:101–124
Williams KA, Deber CM (1991) Proline residues in transmembrane helices: structural or dynamic role? Biochemistry 30:8919–8923
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Taura, T., Akiyama, Y. & Ito, K. Genetic analysis of SecY: additional export-defective mutations and factors affecting their phenotypes. Molec. Gen. Genet. 243, 261–269 (1994). https://doi.org/10.1007/BF00301061
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00301061