Cholera toxin is exported from microsomes by the Sec61p complex.
After endocytosis cholera toxin is transported to the endoplasmic reticulum (ER), from where its A1 subunit (CTA1) is assumed to be transferred to the cytosol by an as-yet unknown mechanism. Here, export of CTA1 from the ER to the cytosol was investigated in a cell-free assay using either microsomes loaded with CTA1 by in vitro translation or reconstituted microsomes containing CTA1 purified from V. cholerae. Export of CTA1 from the microsomes was time- and adenosine triphosphate-dependent and required lumenal ER proteins. By coimmunoprecipitation CTA1 was shown to be associated during export with the Sec61p complex, which mediates import of proteins into the ER. Export of CTA1 was inhibited when the Sec61p complexes were blocked by nascent polypeptides arrested during import, demonstrating that the export of CTA1 depended on translocation-competent Sec61p complexes. Export of CTA1 from the reconstituted microsomes indicated the de novo insertion of the toxin into the Sec61p complex from the lumenal side. Our results suggest that Sec61p complex-mediated protein export from the ER is not restricted to ER-associated protein degradation but is also used by bacterial toxins, enabling their entry into the cytosol of the target cell.
Schmitz
A
A
Institut für Zellbiologie, Rheinische Friedrich-Wilhelms-Universität Bonn, 53121 Bonn, Germany. anton-schmitz@uni-bonn.de
Herrgen
H
H
Winkeler
A
A
Herzog
V
V
D000818:Animals; D002772:Cholera Toxin / Q000235:genetics / Q000302:isolation & purification / Q000493:pharmacokinetics*; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D005089:Exocytosis; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism* / Q000648:ultrastructure*; D010179:Pancreas / Q000648:ultrastructure; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000493:pharmacokinetics; D012270:Ribosomes / Q000378:metabolism; D000069816:SEC Translocation Channels; D013552:Swine
D008565:Membrane Proteins; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D002772:Cholera Toxin