SecA-independent translocation of the periplasmic N-terminal tail of an Escherichia coli inner membrane protein. Position-specific effects on translocation of positively charged residues and construction of a protein with a C-terminal translocation signal.
We have shown previously that the 100-residue-long periplasmic N-terminal tail of the Escherichia coli inner membrane protein ProW can be translocated across the inner membrane in a sec-independent manner and that its translocation is blocked by the introduction of three positively charged residues near its C-terminal end (Whitley, P., Zander, T., Ehrmann, M., Haardt, M., Bremer, E., and von Heijne, G. (1994) EMBO J. 13, 4653-4661). We have now further analyzed the requirements for translocation of the N-terminal tail and found that the introduction of even a single arginine can block translocation. Position-specific differences in the effects on translocation of arginine insertions suggest that the C-terminal end of the N-terminal tail is more critical for translocation than the central and N-terminal regions. We also show that the N-terminal tail is translocated in a truncation mutant where a stop codon is placed immediately after the first transmembrane segment, provided that the transmembrane segment is flanked on its C-terminal end by positively charged residues. Thus, sec-independent translocation of a relatively large domain can be induced by a translocation signal located at the extreme C terminus of a protein.
Department of Biochemistry, Arrhenius Laboratory, Stockholm University, Sweden.
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Whitley
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Gafvelin
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von Heijne
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D018528:ATP-Binding Cassette Transporters; D001386:Azides; D001426:Bacterial Proteins; D003062:Codon; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C490210:ProW protein, E coli; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins