Evidence against the double-arginine motif as the only determinant for protein translocation by a novel Sec-independent pathway in Escherichia coli.
Proteins which are synthesized with a signal peptide containing a 'double-arginine' motif may be translocated across the bacterial cytoplasmic membrane by a mechanism that is different from the known Sec and signal recognition particle pathways. The function of the double-arginine motif as a determinant for this novel pathway was studied by expressions of gene constructs coding for the high potential iron-sulfur protein (HiPIP) from Chromatium vinosum D in Escherichia coli. When the protein was produced with its original double-arginine motif-containing signal peptide, it was in part translocated into the periplasm and thereby processed, as shown by immunoblots after cell fractionation and N-terminal sequencing of purified HiPIP. Processing was not inhibited significantly by 3 mM sodium azide, indicating that translocation of HiPIP occurs by a SecA-independent pathway. Translocation of HiPIP could be altered to the SecA-dependent mode when its signal peptide was substituted by that of PelB from Erwinia carotovora. When the HiPIP double-arginine motif (SRRDAVK) was introduced into the corresponding position of the PelB signal peptide, the transport pathway remained SecA-dependent. This indicates that additional determinants are required for translocation by the Sec-independent pathway.
Institut für Mikrobiologie and Biotechnologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Germany. uzsba4@ibm.rhrz.uni-bonn.de
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001120:Arginine; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002844:Chromatium / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015151:Immunoblotting; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016973:Pectobacterium carotovorum; D045322:Photosynthetic Reaction Center Complex Proteins; D011133:Polysaccharide-Lyases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D019810:Sodium Azide / Q000494:pharmacology
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D026901:Membrane Transport Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; C007943:high potential iron-sulfur protein; D001120:Arginine; D019810:Sodium Azide; D000251:Adenosine Triphosphatases; D011133:Polysaccharide-Lyases; C074124:pectin lyase B; D000081416:SecA Proteins