Proteomic analysis of the 26S proteasome reveals its direct interaction with transit peptides of plastid protein precursors for their degradation.
Proteomic analysis of the 26S proteasome reveals its direct interaction with transit peptides of plastid protein precursors for their degradation.
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DOI：
10.1021/pr401245g
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发表时间：
2014-06
影响因子：
4.4
通讯作者：
Kaori Sako;Y. Yanagawa;Tomoyuki Kanai;Takeo Sato;M. Seki;Masayuki Fujiwara;Y. Fukao;J. Yamaguchi
中科院分区：
文献类型：
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作者：
Kaori Sako;Y. Yanagawa;Tomoyuki Kanai;Takeo Sato;M. Seki;Masayuki Fujiwara;Y. Fukao;J. Yamaguchi
26S蛋白酶体是一种atp依赖的蛋白酶复合物，在真核细胞中负责调节多泛素化蛋白的蛋白水解。本文报道了通过LC-MS/MS分析在拟南芥中发现的新的26S蛋白酶体相互作用蛋白。我们进行了两步筛选过程，包括使用表达flag标记的RPT2a亚基的拟南芥植物对26S蛋白酶体进行亲和纯化，并通过甘油密度梯度离心（GDG）从培养细胞中部分纯化26S蛋白酶体。通过亲和纯化和GDG鉴定的两种质体蛋白LTA2和PDH E1α在体外和体内均与26S蛋白酶体相互作用，并且LTA2和PDH E1α的传递肽是相互作用所必需的。此外，蛋白酶体抑制剂MG132可抑制LTA2和PDH E1α的降解。与这两种蛋白类似，26S蛋白酶体亚基RPT2a/b和RPT5a与其他三种叶绿体蛋白的转运肽相互作用，这些蛋白已知是泛素-26S蛋白酶体系统的底物。这些结果表明，26S蛋白酶体和转运肽之间的直接相互作用对于降解非输入的质体蛋白前体以维持细胞稳态是重要的。
The 26S proteasome is an ATP-dependent proteinase complex that is responsible for regulated proteolysis of polyubiquitinated proteins in eukaryotic cells. Here, we report novel 26S proteasome interacting proteins in Arabidopsis as revealed by LC-MS/MS analysis. We performed a two-step screening process that involved affinity purification of the 26S proteasome using Arabidopsis plants expressing a FLAG-tagged RPT2a subunit and partial purification of the 26S proteasome from cultured cells by glycerol density gradient centrifugation (GDG). Two plastid proteins, LTA2 and PDH E1α, which were commonly identified by both affinity purification and GDG, interacted with the 26S proteasome both in vitro and in vivo, and the transit peptides of LTA2 and PDH E1α were necessary for the interaction. Furthermore, the degradation of both LTA2 and PDH E1α was inhibited by MG132, a proteasome inhibitor. Similar to those two proteins, 26S proteasome subunits RPT2a/b and RPT5a interacted with the transit peptides of three other chloroplast proteins, which are known to be substrates of the ubiquitin-26S proteasome system. These results suggest that a direct interaction between the 26S proteasome and a transit peptide is important for the degradation of unimported plastid protein precursors to maintain cellular homeostasis.