Identification of Core Components and Transient Interactors of the Peroxisomal Importomer by Dual-Track Stable Isotope Labeling with Amino Acids in Cell Culture Analysis
Identification of Core Components and Transient Interactors of the Peroxisomal Importomer by Dual-Track Stable Isotope Labeling with Amino Acids in Cell Culture Analysis
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DOI：
10.1021/pr3000333
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发表时间：
2012-04-01
影响因子：
4.4
通讯作者：
Warscheid, Bettina
中科院分区：
文献类型：
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作者：
Oeljeklaus, Silke;Reinartz, Benedikt S.;Warscheid, Bettina
输入体复合物通过介导基质蛋白跨细胞器膜的易位，在过氧化物酶体的生物发生中发挥重要作用。这种高度动态导入机制的核心部分是由 Pex14p、Pex13p 和 Pex17p 组成的对接复合体，通过 Pex8p 连接到环指复合体（Pex2p、Pex10p、Pex12p）。为了详细了解控制过氧化物酶体基质蛋白输入的分子参与者，从而了解过氧化物酶体的完整性和功能性，我们旨在对输入体的核心成分 Pex14p 的稳定和瞬时相互作用伙伴进行最全面的研究。为此，我们基于 Pex14p 表位标记结合细胞培养质谱 (SILAC-MS) 中氨基酸双轨稳定同位素标记对亲和纯化的 Pex14p 复合物进行分析和统计，进行了彻底的定量蛋白质组学研究。结果导致建立了迄今为止最广泛的 Pex14p 相互作用组，包括 9 个核心和另外 12 个瞬态组件。我们确认了几乎所有已知的 Pex14p 相互作用伙伴，包括导入体的核心成分以及 Pex5p、Pex11p、Pex15p 和 Dyn2p。更重要的是，我们确定了新的瞬时相互作用伙伴（Pex25p、Hrr25p、Esl2p、抑制素），为未来研究过氧化物酶体输入体的功能、动力学和调控提供了宝贵的资源。
The importomer complex plays an essential role in the biogenesis of peroxisomes by mediating the translocation of matrix proteins across the organellar membrane. A central part of this highly dynamic import machinery is the docking complex consisting of Pex14p, Pex13p, and Pex17p that is linked to the RING finger complex (Pex2p, Pex10p, Pex12p) via Pex8p. To gain detailed knowledge on the molecular players governing peroxisomal matrix protein import and, thus, the integrity and functionality of peroxisomes, we aimed at a most comprehensive investigation of stable and transient interaction partners of Pex14p, the central component of the importomer. To this end, we performed a thorough quantitative proteomics study based on epitope tagging of Pex14p combined with dual-track stable isotope labeling with amino acids in cell culture-mass spectrometry (SILAC-MS) analysis of affinity-purified Pex14p complexes and statistics. The results led to the establishment of the so far most extensive Pex14p interactome, comprising 9 core and further 12 transient components. We confirmed virtually all known Pex14p interaction partners including the core constituents of the importomer as well as Pex5p, Pex11p, Pex15p, and Dyn2p. More importantly, we identified new transient interaction partners (Pex25p, Hrr25p, Esl2p, prohibitin) that provide a valuable resource for future investigations on the functionality, dynamics, and regulation of the peroxisomal importomer.