A role for the protease-sensitive loop region of Shiga-like toxin 1 in the retrotranslocation of its A1 domain from the endoplasmic reticulum lumen.
Shiga-like toxin I (Slt-I) is a ribosome-inactivating protein that undergoes retrograde transport to the endoplasmic reticulum to exert its cytotoxic effect on eukaryotic cells. Its catalytically active A(1) domain subsequently migrates from the endoplasmic reticulum (ER) lumen to the cytoplasm. To study this final retrotranslocation event, a suicide assay was developed based on the cytoplasmic expression and ER-targeting of the cytotoxic Slt-I A(1) fragment in Saccharomyces cerevisiae. Expression of the Slt-I A(1) domain (residues 1-251) with and without an ER-targeting sequence was lethal to the host and demonstrated that this domain can efficiently migrate from the ER compartment to the cytosol. Deletion analyses revealed that residues 1-239 represent the minimal A(1) segment displaying full enzymatic activity. This fragment, however, accumulates in the ER lumen when directed to this compartment. The addition of residues 240-251 restores the translocation property of the A(1) chain in yeast. However, single mutations within this region do not significantly alter this function in the context of the 251-residue long A(1) domain or affect the toxicity of the resulting Slt-I variants toward Vero cells in the context of the holotoxin. Since this mechanism of retrotranslocation is common to other protein toxins lacking a peptide motif similar in sequence to residues 240-251, the present results suggest that the ER export mechanism may involve the recognition of a more universal structural element, such as a misfolded or altered peptide domain localized at the C terminus of the A(1) chain (residues 240-251) rather than a unique ER export signal sequence.
LaPointe
Paul
P
Department of Medical Biophysics, University of Toronto and the Ontario Cancer Institute, University Health Network, Canada.
Wei
Xin
X
Gariépy
Jean
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D022622:Shiga Toxin 1 / Q000737:chemistry* / Q000378:metabolism; D014709:Vero Cells
D017931:DNA Primers; D010455:Peptides; D012333:RNA, Messenger; D022622:Shiga Toxin 1