Defective translocation of a signal sequence mutant in a prlA4 suppressor strain of Escherichia coli.
In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem.269, 5564-5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position -10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon.
Adams
Hendrik
H
Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, The Netherlands.
Scotti
Pier A
PA
Luirink
Joen
J
Tommassen
Jan
J
D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019286:Endopeptidase K / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D007425:Intracellular Membranes / Q000378:metabolism; D007930:Leucine / Q000737:chemistry; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D021381:Protein Transport; D000069816:SEC Translocation Channels; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D000069816:SEC Translocation Channels; C057842:SecB protein, Bacteria; C057841:SecY protein, E coli; D019286:Endopeptidase K; D007930:Leucine; D005998:Glycine