Both transmembrane domains of SecG contribute to signal sequence recognition by the Escherichia coli protein export machinery.
A chimeric protein containing the uncleaved signal sequence of plasminogen activators inhibitor-2 (PAI2) fused to alkaline phosphatase (AP) interferes with Escherichia coli protein export and arrests growth. Suppressors of this toxicity include secG mutations that define the Thr-41-Leu-42-Phe-43 (TLF) domain of SecG. These mutations slow down the export of PAI2-AP. Another construct encoding a truncated PAI2 signal sequence (hB-AP) is also toxic. Most suppressors exert their effect on both chimeric proteins. We describe here five secG suppressors that only suppress the toxicity of hB-AP and selectively slow down its export. These mutations do not alter the TLF domain: three encode truncated SecG, whereas two introduce Arg residues in the transmembrane domains of SecG. The shortest truncated protein only contains 13 residues of SecG, suggesting that the mutation is equivalent to a null allele. Indeed, a secG disruption selectively suppresses the toxicity of hB-AP. However, the missense mutations are not null alleles. They allow SecG binding to SecYE, although with reduced affinity. Furthermore, these mutated SecG are functional, as they facilitate the export of endogenous proteins. Thus, SecG participates in signal sequence recognition, and both transmembrane domains of SecG contribute to ensure normal signal sequence recognition by the translocase.
Bost
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Department of Pathology, CMU, 1 rue Michel Servet, CH-1211 Geneva, Switzerland.
Silva
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F
Rudaz
C
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Belin
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D
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017396:Plasminogen Activator Inhibitor 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D000069816:SEC Translocation Channels; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D017396:Plasminogen Activator Inhibitor 2; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; C088616:SecG protein, E coli; D000469:Alkaline Phosphatase