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首页> 外文期刊>Biochemistry >Membrane insertion of the Bacillus thuringiensis Cry1Ab toxin: Single mutation in domain II block partitioning of the toxin into the brush border membrane
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Membrane insertion of the Bacillus thuringiensis Cry1Ab toxin: Single mutation in domain II block partitioning of the toxin into the brush border membrane

机译：苏云金芽孢杆菌Cry1Ab毒素的膜插入：域II中的单个突变阻止毒素进入刷状缘膜的分区

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摘要

The umbrella and penknife models hypothesize that insecticidal Bacillus thuringiensis Cry toxins partition into the apical membrane of the insect midgut by insertion of only two alpha-helices from domain I of the protein, alpha-helices 4 and 5 in the case of the umbrella model and a.-helices 5 and 6 in the case of the penknife model. Neither model envisages membrane partitioning by domains II and III. In this study, we present data suggesting that mutations in the domain II residue, F371, affect insertion of the whole toxin into Manduca sexta brush border membrane vesicles (BBMVs). Using steady state fluorescence measurements combined with a proteinase K protection assay, we show that mutants of F371 have lost their ability to insert into the BBMV, even though binding to cadherin is almost unaffected. The study also identifies a difference in partitioning of toxins into artificial lipid vesicles (SUVs) as opposed to native BBMVs. While the F371 mutations block insertion of domains I and II into BBMVs, they only block domain II insertion into SUVs. Bioassay and voltage clamping of midguts also confirm the fluorescence data that the noninserting mutants are nontoxic. Our study leads us to propose that, in contrast to previous models of individual free helices inserting into the membrane, the toxin enters into the membrane as a whole molecule or oligomers of the molecule, wherein the domain II residue F371 has a vital role to play in membrane insertion.
机译：伞形和铅笔状模型假设，通过仅从蛋白质结构域I插入两个α-螺旋，将杀虫苏云金芽孢杆菌Cry毒素分配到昆虫中肠的顶膜中，在伞形模型中，α-螺旋为4和5。对于铅笔刀模型，a。螺旋5和6。两种模型均未考虑通过域II和III进行膜分区。在这项研究中，我们提出的数据表明，域II残基F371中的突变会影响整个毒素插入到Manduca sexta刷状缘膜囊泡（BBMVs）中。使用稳态荧光测量结合蛋白酶K保护测定，我们显示F371突变体已经失去了插入BBMV的能力，即使与钙粘蛋白的结合几乎不受影响。这项研究还确定了与天然BBMVs相反，将毒素分配到人造脂质囊泡（SUVs）中的差异。尽管F371突变阻止结构域I和II插入BBMV，但它们仅阻止结构域II插入SUV。中肠的生物测定和电压钳制也证实了荧光数据，即非插入突变体是无毒的。我们的研究使我们提出，与以前的单个游离螺旋插入膜中的模型相反，毒素以整个分子或分子的低聚物形式进入膜，其中结构域II残基F371具有重要作用在膜插入。

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