Domain II of Pseudomonas Exotoxin A Arrests the Transfer of Translocating Nascent Chains into Mammalian Microsomes

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Abstract
The translocation of PE from the extracytosolic compartment to the cytosol during the intoxication of mammalian cells is mediated by domain II of the toxin. We have shown previously that within domain II amino acids 280-313 of PE promote their own export from mammalian microsomes following signal sequence-directed membrane insertion. In this study, we attempted to target full-length PE into mammalian microsomes using the preprocecropin signal sequence, but found that translocation was arrested to generate a transmembrane protein. "Stop transfer" required the presence of amino acids 280-313 of PE, and the first 313 amino acids of PE were sufficient to generate a transmembrane protein (N-terminus-in/C-terminus-out). The mechanism of stop transfer appears to be different from that described previously because amino acids 280-313 of PE are not highly hydrophobic and do contain many charged residues. In addition, the transmembrane segment appeared to be influenced by the cytoplasmic domain of the transmembrane proteins.

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