These results demonstrate that the gene secY (priA) is essential for protein .... 5. Structure of the promoter-distal part of the spc ribosomal protein operon and the ...
The EMBO Journal vol.3 no.3 pp.631-635, 1984
A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia col: isolation and characterization of a new temperature-sensitive secY mutant
Kiyotaka Shiba, Koreaki Ito*, Takashi Yura and Douglas Pat Cermtti1 Institute for Virus Research, Kyoto University, Kyoto 606, Japan, and lInstitute for Enzyme Research, University of Wisconsin, Madison, WI 53706, USA *To whom reprint requests should be sent Communicated by G.Schatz
We describe the properties of a temperature-sensitive mutant, ts24, of Escherichia coli. The mutant has a conditional defect in export of periplasmic and outer membrane proteins. At 42°C, precursor forms of these proteins accumulate within the cell where they are protected from digestion by extemally added trypsin. The accumulated precursors are secreted and processed very slowly at 42°C. The mutation is complemented by expression of the wild-type secY(orprlA) gene, which has been cloned into a plasmid vector from the promoter-distal part of the spc ribosomal protein operon. The mutant has a single base change in the middle of the secY gene, which would result in the replacement of a glycine residue by aspartic acid in the protein product. These results demonstrate that the gene secY (priA) is essential for protein translocation across the E. coli cytoplasmic membrane. Key words: Escherichia coli secY gene/precursor processing/ protein secretion/ribosomal protein operon/signal peptide Introduction Protein export in bacterial cells is a complex process which may require the functions of a number of different gene products (Michaelis and Beckwith, 1982). In Escherichia coli, several genes have been implicated in the secretion of envelope proteins across the cytoplasmic membrane. The prl series of genes, priA, priB and priC, have been defined by mutations, which extragenically suppress the mutations in the signal sequence part of the structural genes for the envelope proteins (Emr et al., 1981; Emr and Bassford, 1982). The secA and secB genes have been identified as genes whose mutations can make the cell pleiotropically defective in secretion of several envelope proteins (Oliver and Beckwith, 1981; Kumamoto and Beckwith, 1983). In addition, genes for the enzymes that catalyze the signal (leader) peptide cleavage have been located on the chromosome (Silver and Wickner, 1983; Yamagata et al., 1983). We have previously reported a temperature-sensitive mutant, ts215, which exhibits a slow processing of envelope proteins, accumulating precursor proteins at 42°C. The genetic analysis of this mutant led to the conclusion that a gene, secY (or the Y reading frame according to Cerretti et al., 1983), located at the promoter-distal part of the spc ribosomal protein operon, is essential for normal secretion of periplasmic and outer membrane proteins (Ito et al., 1983). Subsequent studies have revealed that ts215 is a nonsense mutant having an amber mutation in the gene rplO (the structural gene for ribosomal protein L15, located just upstream of secY) in IRL Press Limited, Oxford, England.
combination with a temperature-sensitive amber suppressor. The secretion defect is, nevertheless, complemented by a recombinant plasmid carrying only the Y reading frame from the chromosome. These results indicate that the amber mutation in rplO exerts a polar effect on the expression of secY (K.Ito, D.Cerretti, M.Wittekind and M.Nomura, in preparation). Although the above new findings do not affect our previous conclusion on the role of secY in protein secretion (Ito et al., 1983), it is highly desirable to isolate a mutant of the secY gene itself, which exhibits defects in protein secretion. We now show that a new mutant, ts24, is defective in protein translocation across the cytoplasmic membrane, and that the mutant has a single base change in the Y reading frame. Shultz et al. (1982) have shown that priA is located within the spc operon, in a DNA segment downstream of rplO. Our nomenclature of secY (Ito et al., 1983) came from 'secretion' and 'Y reading frame' (Cerretti et al., 1983). It is almost certain that priA and secY actually refer to a single gene. Although the former nomenclature has been well established in the literature, we prefer to continue to use secY in this paper, because sec has been used for genes defined by mutations of general secretion defects (Oliver and Beckwith, 1981; Kumamoto and Beckwith, 1983) and might be more appropriate to describe our mutants. We believe that the genes for the protein export machinery will have a unified system of nomenclature in the future. Results The mutant is temperature-sensitive in growth An E. coli mutant ts24 was isolated after localized mutagenesis of the chromosomal segment around the spc operon (see Materials and methods). The mutant shows the following growth properties. On minimal agar plates, it can grow up to 40°C but not at 42°C. On broth plates, it can form colonies even at 42°C, however, the colonies are very abnormal and become 'ghost-like' after prolonged incubations. Survivors picked up by re-streaking the ghost colonies and incubating at 30°C were all shown to be temperature-resistant revertants. The results indicate that the failure of the mutant cells to grow on minimal agar at 42°C is not due to a specific nutritional requirement. The mutant cell does not show any immediate cessation of growth in either minimal or broth liquid media; growth continues exponentially for at least several hours at 42°C. These observations suggest that the mutation probably does not affect directly general macromolecular synthesis or energy metabolism. The mutant cells eventually die at the high temperature. The loss of viability seems to occur earlier in the minimal media than in the broth media. Temperature-resistant revertants can be found at frequencies of 0.3-7 x 10- 6. Processing of envelope proteins is slowed down at 42 °C in the mutant Synthesis of periplasmic and outer membrane proteins was 631
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Fig. 3. Fractionation of labeled cells by osmotic shock. Cells at 120 min after temperature shift (30 to 42°C) were labeled with [3H]leucine for I min and chased with unlabeled leucine for 30 mlin. Labeled cultures were rapidly chilled with crushed ice, and subjected to fractionation by osmotic shock. Proteins in each fraction were concentrated by trichloroacetic acid precipitation, solubilized in SDS and treated with anti-maltose-binding protein serum. Lanes 1 and 2, shocked cells and shock fluid, respectively, from pulse-labeled wild-type (IQ86) cells; lanes 3 and 4, shocked cells and shock fluid, respectively, from pulse-labeled mutant (IQ85) cells; lanes 5 and 6, shocked cells and shock fluid, respectively, from chased mutant cells.
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Fig. 1. Accumulation of precursor proteins in the mutant. Cells were in amino acid-supplemented medium E with maltose at 30°C. Temperature was shifted to 42°C at an early exponential phase. Portions of the cultures were labeled with [3H]leucine for 1 min at various time points and labeling was terminated by trichloroacetic acid. Proteins were solubilized with SDS and treated with antibodies against maltose-binding protein (A), or a combination of antibodies against OmpA and OmpF proteins (B), before electrophoresis. To see the outer membrane lipoprotein (C), a part of the sample was directly electrophoresed on a 19.37o gel containing 6 M urea; only the bands previously shown (Ito et al., 1981) to correspond to precursor and mature forms of lipoprotein are shown. Lanes 1 and 2, wild-type strain (IQ86) at 30°C and at 120 min after temperature shift, respectively; lanes 3-8, ts24 (IQ85) at 0, 15, 30, 60, 90 and 120 min after temperature shift, respectively. p and m indicate precursor and mature forms, respectively. MBP, maltose-binding protein; LP, lipoprotein. grown
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Fig. 2. Processing of precursor proteins during chase. Cells of IQ85 at 120 min after temperature shift, were labeled with [3H]leucine for 1 min, and chased with unlabeled leucine. Samples were withdrawn at various time points and treated with a combination of antisera against maltosebinding protein and OmpA protein. Lane 1, no chase; lanes 2-6, at 1, 5, 10, 20 and 30 min after initiation of chase, respectively.
examined by pulse-labeling the mutant cells with [3H]leucine for 1 min. The labeled proteins were analyzed by electrophoresis on SDS gels directly (for the outer membrane lipoprotein) or after antibody precipitation (for the periplasmic 632
Fig. 4. Trypsin accessibility of precursor and mature forms of OmpA protein. IQ85 cells, at 60 min after temperature shift (30 to 42°C) were pulselabeled for 1 min. The culture was chilled rapidly by crushed ice, and chloramphenicol (100 /Ag/ml) and NaN3 (10 mM) were added. Spheroplasts were prepared and one half of them were lysed. The intact (lanes 1-3) and the lysed (lanes 4-6) spheroplasts were treated with 0 (lanes 1 and 4), 10 (lanes 2 and 5), and 50 (lanes 3 and 6) Ag/ml of trypsin. Samples were immunoprecipitated using anti-OmpA protein serum.
maltose-binding protein and the outer membrane OmpA and OmpF proteins). The fluorogram shown in Figure 1 indicates that the ts24 mutant has a defect in the conversion of precursors to the mature proteins at 42°C, and that this defect can be first noticed 30 min after the temperature shift to 42°C and becomes stronger thereafter (lanes 4-8). At 2 h after the temperature shift, radioactive proteins detected in the mutant cells by the pulse-labeling technique are almost entirely the precursors (lane 8), whereas no such precursors were detected in the wild-type cells (lanes 1 and 2). The radioactive precursor proteins detected in the mutant cells are apparently converted to the mature forms during subsequent chase (at 42°C) with non-radioactive leucine. This is illustrated in Figure 2 for maltose-binding protein and OmpA protein. The conversion of the precursor form of maltose-binding protein to the mature form is slow and a substantial amount of the precursor form remains after a 30 min chase (Figure 2, lane 6). This species of the precursor could be a dead-end product that has failed to be processed by the slow post-translational mechanism operating in the mutant cell. In contrast, the precursor form of OmpA protein was completely converted to the mature form within 5 min. OmpF protein showed a slower conversion than OmpA protein (data not shown). Thus, with respect to the precursor processing, different proteins are apparently affected to different extents by the
E. coli mutant defective in protein export PstI
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Wild type
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rpsM
5'. GTT GAG CGT GAT CAA CGC CGC ATT GTG
ts24
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val glu arg asp gln arg arg ile val
pN01025 pN01576 pN01576-1 I
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