Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry.
The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1 with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild-type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.
Trujillo
Carolina
C
Department of Microbiology, Boston University School of Medicine, Boston, MA 02118, USA.
Taylor-Parker
Julian
J
Harrison
Robert
R
Murphy
John R
JR
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D002460:Cell Line; D020755:Coat Protein Complex I / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D021381:Protein Transport
D020755:Coat Protein Complex I; D004167:Diphtheria Toxin; D008239:Lysine
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