Endocytotic uptake and retrograde transport of a virally encoded killer toxin in yeast
Endocytotic uptake and retrograde transport of a virally encoded killer toxin in yeast
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DOI：
10.1046/j.1365-2958.2000.02063.x
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发表时间：
2000-08-01
影响因子：
3.6
通讯作者：
Schmitt, MJ
中科院分区：
文献类型：
--
作者：
Eisfeld, K;Riffer, F;Schmitt, MJ
我们证明，病毒编码的酵母“杀手”毒素进入其真核靶细胞的内吞作用，随后旅行酵母分泌途径反向展示其致死作用。K28杀伤毒素是一种分泌的α/β异源二聚体，通过阻断细胞核中的DNA合成以受体介导的方式杀死敏感酵母。毒素前体的体内加工产生一种蛋白质，其β-C-末端携带内质网（ER）滞留信号HDEL，正如我们在这里所展示的，这是逆行毒素转运所必需的。酵母end 3/4突变体以及缺乏HDEL受体（Deltaerd 2）的细胞或高尔基体至ER蛋白再循环缺陷的突变体（erd 1）是毒素抗性的，因为毒素不再能够进入和/或逆行通过细胞。定点诱变进一步表明，毒素的β-HDEL基序确保逆行转运，尽管在毒素分泌酵母中，β-C-末端最初被R残基（β-HDELR）掩蔽，直到Kex 1 p裂解揭示了毒素在晚期高尔基体隔室中的靶向信号。阻止Kex 1 p加工导致高水平分泌一种生物学上无活性的蛋白质，这种蛋白质不能重新进入分泌途径。最后，我们提出的证据表明，ER到胞质溶胶毒素出口介导的Sec 61 p易位，并需要功能性拷贝的内腔ER伴侣Kar 2 p和Cne 1 p。
We demonstrate that a virally encoded yeast 'killer' toxin is entering its eukaryotic target cell by endocytosis, subsequently travelling the yeast secretory pathway in reverse to exhibit its lethal effect. The K28 killer toxin is a secreted alpha/beta heterodimer that kills sensitive yeasts in a receptor-mediated fashion by blocking DNA synthesis in the nucleus. In vivo processing of the toxin precursor results in a protein whose beta-C-terminus carries the endoplasmic reticulum (ER) retention signal HDEL, which, as we show here, is essential for retrograde toxin transport. Yeast end3/4 mutants as well as cells lacking the HDEL receptor (Delta erd2) or mutants defective in Golgi-to-ER protein recycling (erd1) are toxin resistant because the toxin can no longer enter and/or retrograde pass the cell. Site-directed mutagenesis further indicated that the toxin's beta-HDEL motif ensures retrograde transport, although in a toxin-secreting yeast the beta-C-terminus is initially masked by an R residue (beta-HDELR) until Kex1p cleavage uncovers the toxin's targeting signal in a late Golgi compartment. Prevention of Kex1p processing results in high-level secretion of a biologically inactive protein incapable of re-entering the secretory pathway. Finally, we present evidence that ER-to-cytosol toxin export is mediated by the Sec61p translocon and requires functional copies of the lumenal ER chaperones Kar2p and Cne1p.