Interactions Between Late-Acting Proteins Required for Peptidoglycan Synthesis during Sporulation
Interactions Between Late-Acting Proteins Required for Peptidoglycan Synthesis during Sporulation
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DOI：
10.1016/j.jmb.2010.04.036
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发表时间：
2010-06-18
影响因子：
5.6
通讯作者：
Dworkin, Jonathan
中科院分区：
文献类型：
--
作者：
Fay, Allison;Meyer, Pablo;Dworkin, Jonathan
肽聚糖合成对生长的要求使分析参与该途径的蛋白质之间的相互作用变得复杂。特别是，涉及膜连接底物的后一步骤已被证明在很大程度上难以进行体内分析。在这里，我们利用枯草芽孢杆菌产孢过程中发生的肽聚糖合成来研究SpoVE和SpoVD之间的相互作用。SpoVE是一种非必需的、产孢特异性的同源蛋白，是高度保守的、必需的SEDS（形状延伸、分裂和产孢）蛋白，SpoVD是一种非必需的B类青霉素结合蛋白。我们发现SpoVD的定位依赖于SpoVE，并且SpoVD保护SpoVE免受体内蛋白水解。共免疫沉淀和荧光共振能量转移实验表明，SpoVE和SpoVD相互作用，在大肠杆菌中共亲和纯化表明，这种相互作用是直接的。最后，我们生成了一个由SpoVE-SpoVD融合组成的功能性蛋白，并发现融合的任何一部分的功能缺失点突变都会导致整个融合的功能丧失，而野生型蛋白无法补充。因此，SpoVE与SpoVD具有直接的功能相互作用，这一结论将有助于理解SpoVE及其相关SEDS蛋白（如FtsW和RodA）在细菌生长和分裂中的重要功能。(C) 2010 Elsevier Ltd.版权所有。
The requirement of peptidoglycan synthesis for growth complicates the analysis of interactions between proteins involved in this pathway. In particular, the latter steps that involve membrane-linked substrates have proven largely recalcitrant to in vivo analysis. Here, we have taken advantage of the peptidoglycan synthesis that occurs during sporulation in Bacillus subtilis to examine the interactions between SpoVE, a nonessential, sporulation-specific homolog of the well-conserved and essential SEDS (shape elongation, division, and sporulation) proteins, and SpoVD, a nonessential class B penicillin binding protein. We found that localization of SpoVD is dependent on SpoVE and that SpoVD protects SpoVE from in vivo proteolysis. Co-immunoprecipitations and fluorescence resonance energy transfer experiments indicated that SpoVE and SpoVD interact, and co-affinity purification in Escherichia coli demonstrated that this interaction is direct. Finally, we generated a functional protein consisting of an SpoVE-SpoVD fusion and found that a loss-of-function point mutation in either part of the fusion resulted in loss of function of the entire fusion that was not complemented by a wild-type protein. Thus, SpoVE has a direct and functional interaction with SpoVD, and this conclusion will facilitate understanding the essential function that SpoVE and related SEDS proteins, such as FtsW and RodA, play in bacterial growth and division. (C) 2010 Elsevier Ltd. All rights reserved.