Critical regions of secM that control its translation and secretion and promote secretion-specific secA regulation

Abstract

SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins. secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM. The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation. In addition, mutational analysis of the 3' end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system.

FIG. 1.

secM signal sequence alleles used in this study. The N- and H-regions of the secM signal peptide are shown. The codon and amino acid substitutions for each allele are also shown. Empty brackets indicate deletions. An arrow indicates the presumed signal peptide processing site.

FIG. 2.

Effects of secM signal sequence mutations on processing. (A) MC4100.2 containing pSS1 (WT) or an allelic derivative was grown in M63 minimal medium containing 0.4% glucose, 2 μg of thiamine per ml, 20 μg of each of 18 amino acids (not including methionine and cysteine) per ml, and 20 μg of ampicillin per ml at 37°C until the mid-logarithmic phase. Sodium azide (NaN3) was not added (−) or was added to a final concentration of 2 mM (+), and labeling was initiated after 5 min. A 0.5-ml aliquot of each culture was pulse-labeled with 10 μCi of Tran ³⁵S label (>1,000 Ci mmol⁻¹) for 1 min, and then an equal volume of ice-cold 10% trichloroacetic acid was added to terminate labeling. Samples were processed for immunoprecipitation with antisera to alkaline phosphatase and OmpA and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography as described previously (36). The positions of the precursor and mature forms of the SecM-PhoA fusion proteins (pSecM-PhoA and SecM-PhoA, respectively) and OmpA (pOmpA and OmpA, respectively) are indicated on the left. Both the precursor and mature forms of OmpA migrated as two bands, which were the heat-modifiable and non-heat-modifiable forms (22). (B) Similar to panel A, except that a mixture of methionine and cysteine (final concentration of each, 200 μg/ml) was added after 1 min of labeling to initiate the chase (0 min) and aliquots were removed at different times and mixed with an equal volume of ice-cold 10% trichloroacetic acid to terminate the chase.

FIG. 3.

Effects of secM signal sequence mutations on translational pausing. GN40(pSTD343) containing pNH22 (WT) or an allelic derivative was grown in M63 minimal medium supplemented with 0.4% glucose, 2 μg of thiamine per ml, 20 μg of each of 18 amino acids (not including methionine and cysteine) per ml, 20 μg of ampicillin per ml, and 10 μg of chloramphenicol per ml at 37°C until the mid-logarithmic phase, when IPTG and cyclic AMP were added at concentrations of 1 and 5 mM, respectively. Thirty minutes later an aliquot of each culture was pulse-labeled with 100 μCi of Tran ³⁵S label (>1,000 Ci mmol⁻¹) per ml for 1 min. Then a mixture of methionine and cysteine (final concentration of each, 200 μg/ml) was added to initiate the chase (0 min), and aliquots were removed at different times and mixed with an equal volume of ice-cold 10% trichloroacetic acid to terminate the chase. Samples were immunoprecipitated with a mixture of antisera against N- and C-terminal synthetic peptides of SecM (33) and analyzed on sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis gels by fluorography as described previously (35). p, preSecM-Met₆; A, translationally paused SecM; m, mature SecM-Met₆.

FIG. 4.

Effects of prl suppressors on the phenotypes of secM signal sequence mutations. A strain containing pPhIF (wild type) or its allelic derivative was grown in LB broth containing 100 μg of ampicillin per ml and 5 μg of chloramphenicol per ml, when necessary, at 37°C to the mid-logarithmic phase. β-Galactosidase assays were performed as described in Table 2, footnote a. (A) prlA suppressor; (B) prlD suppressor.

FIG. 5.

Proposed RNA secondary structure for helix I. secM mutants are indicated by codons and single-letter codes for amino acids. The structure was taken from reference .

FIG. 6.

Genetic interaction of secM signal sequence and TPE alleles. CG155 or CG29 carrying pPhIF (wild type) or an allelic derivative was grown and assayed for β-galactosidase activity as described in Table 2, footnote a.

FIG. 7.

Effects of secM TPE mutations on translational pausing. GN40(pSTD343) containing pNH22 (WT) or an allelic derivative was grown and pulse-labeled with 100 μCi of Tran ³⁵S label per ml for 1 min, and samples were processed and visualized as described in the legend to Fig. 3. Where indicated (+), sodium azide was added 5 min prior to labeling at a final concentration of 2 mM. p, preSecM-Met₆; A, translationally paused SecM; m, mature SecM-Met₆. The data are representative of the data obtained in three separate experiments.

FIG. 8.

Alignment of SecM proteins. SecM proteins from Escherichia coli (secMec) (43), Salmonella enterica serovar Typhi (secMst), Klebsiella pneumoniae (secMkp), and Yersinia pestis (secMyp) were multiply aligned by the Clustal X method (49). The S. enterica and Y. pestis sequences are available at ftp://ftp.sanger.ac.uk/pub/pathogens, and the K. pneumoniae sequence was produced by the Genome Sequencing Center at Washington University, St. Louis, Mo. (personal communication). Asterisks indicate completely conserved amino acid residues, while the arrow indicates the predicted signal peptide processing site.

FIG. 9.

Model for secA regulation. The proposed structure of the secA repressor helix (helix II) and its disruption by the secM translational pause are shown. The positions of the TPE mutations in codons 163 and 164 are indicated, along with the positions of 3′ nucleotides that would be sequestered by the translating ribosome (boldface type and box). The termination codon of secM (UAA) and the secA Shine-Dalgarno (SD) and the initiation codon (AUG) are enclosed in boxes. 30S, 30S ribosomal subunit. The structure of the secA repressor helix was taken from reference .