LaPointe Paul, Wei Xin, Gariépy Jean
Department of Medical Biophysics, University of Toronto and the Ontario Cancer Institute, University Health Network, Canada.
J Biol Chem. 2005 Jun 17;280(24):23310-8. doi: 10.1074/jbc.M414193200. Epub 2005 Apr 7.
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.
志贺样毒素I（Slt-I）是一种核糖体失活蛋白，它通过逆行转运至内质网，从而对真核细胞发挥细胞毒性作用。其具有催化活性的A(1)结构域随后从内质网（ER）腔迁移至细胞质。为了研究这一最终的逆行转运事件，基于细胞毒性Slt-I A(1)片段在酿酒酵母中的细胞质表达和内质网靶向性，开发了一种自杀测定法。带有和不带有内质网靶向序列的Slt-I A(1)结构域（第1至251位氨基酸残基）的表达对宿主具有致死性，这表明该结构域能够有效地从内质网区室迁移至细胞质溶胶。缺失分析表明，第1至239位氨基酸残基代表了显示出完整酶活性的最小A(1)片段。然而，当该片段被导向内质网区室时，它会在内质网腔中积累。添加第240至251位氨基酸残基可恢复A(1)链在酵母中的转运特性。然而，在251个氨基酸残基长的A(1)结构域的背景下，该区域内的单个突变并不会显著改变此功能，或者在全毒素的背景下，不会影响所得Slt-I变体对Vero细胞的毒性。由于这种逆行转运机制对于其他缺乏与第240至251位氨基酸残基序列相似的肽基序的蛋白质毒素来说是常见的，因此目前的结果表明，内质网输出机制可能涉及对一种更普遍的结构元件的识别，例如位于A(1)链C末端（第240至251位氨基酸残基）的错误折叠或改变的肽结构域，而不是独特的内质网输出信号序列。
