Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel
Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel
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DOI：
10.1073/pnas.0405754101
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发表时间：
2004-11-30
影响因子：
11.1
通讯作者：
Collier, RJ
中科院分区：
文献类型：
--
作者：
Zhang, S;Finkelstein, A;Collier, RJ
炭疽毒素的酶组分[致死因子（LF）和水肿因子]进入哺乳动物细胞的胞质溶胶取决于活化的保护性抗原（PA（63））组分在酸性细胞内区室的膜中形成通道（孔）的能力。为了研究易位的机制，我们表征了LF（LFN）的PA（63）结合结构域的N-末端截短形式。删除27或36个残基强烈抑制了酸触发的LFN穿过CHO-K1细胞质膜的易位，并在小的正电压（+20 mV）下消除了蛋白质阻断平面脂质双层中PA 63通道的能力。融合的H-6-标签的IN末端的截短的蛋白质恢复易位和通道阻断活动。在+20 mV下，N-末端H-6和生物素标签分别可接近添加到平面双层的反式隔室中的Ni 2+和链霉亲和素。基于这些发现，我们提出PA（63）结合LF或水肿因子的N末端在酸性pH和正跨膜电位的影响下进入PA（63）通道，并启动N-至C-末端方向的易位。
Entry of the enzymatic components of anthrax toxin [lethal factor (LF) and edema factor] into the cytosol of mammalian cells depends on the ability of the activated protective antigen (PA(63)) component to form a channel (pore) in the membrane of an acidic intracellular compartment. To investigate the mechanism of translocation, we characterized N-terminally truncated forms of the PA(63)-binding domain of LF (LFN). Deleting 27 or 36 residues strongly inhibited acid-triggered translocation of LFN across the plasma membrane of CHO-K1 cells and ablated the protein's ability to block PA63 channels in planar lipid bilayers at a small positive voltage (+20 mV). Fusing a H-6-tag to the IN terminus of the truncated proteins restored both translocation and channel-blocking activities. At +20 mV, N-terminal H-6 and biotin tags were accessible to Ni2+ and streptavidin, respectively, added to the trans compartment of a planar bilayer. On the basis of these findings, we propose that the N terminus of PA(63)-bound LF or edema factor enters the PA(63)-channel under the influence of acidic pH and a positive transmembrane potential and initiates translocation in an N- to C-terminal direction.