Autocatalytic Processing and Substrate Specificity of Arabidopsis Chloroplast Glutamyl Peptidase
Autocatalytic Processing and Substrate Specificity of Arabidopsis Chloroplast Glutamyl Peptidase
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DOI：
10.1104/pp.20.00752
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发表时间：
2020-09-01
期刊：
影响因子：
7.4
通讯作者：
van Wijk, Klaas J.
中科院分区：
文献类型：
--
作者：
Bhuiyan, Nazmul H.;Rowland, Elden;van Wijk, Klaas J.
叶绿体蛋白质稳态是由一个肽酶网络控制的。作为这个网络的一部分，我们表明，拟南芥（拟南芥）叶绿体谷氨酰肽酶（CGEP）是一个同源寡聚基质丝氨酸型（S9 D）肽酶与外切和内切肽酶活性。拟南芥CGEP无效突变等位基因（cgep）没有可见的表型，但表现出强烈的遗传相互作用与基质CLP蛋白酶系统突变体，导致生长减少。CGEP的损失上调叶绿体蛋白伴侣机制和70 S核糖体蛋白，但蛋白质稳态网络的其他部分不受影响。比较蛋白质组学和基于mRNA的共表达分析都强烈表明CGEP的功能至少部分参与淀粉代谢调节。重组CGEP降解小于25 kD的肽和蛋白质。CGEP特异性地切割Glu的C-末端侧上的底物，而不考虑相邻残基，如使用与重组CGEP一起孵育的肽文库和质谱所示。CGEP显示在体外和体内在E946处经历自催化C-末端切割，去除15个残基。紧挨E946上游的保守基序（A[S/T]GGG[N/G] PE 946）在双子叶植物中被鉴定，但在单子叶植物中没有。结构建模表明，C-末端加工通过改善催化腔的进入而增加了基板尺寸的上限。使用在cgep clpr 2 -1背景中与催化失活的CGEP-S781 R或具有未加工的C-末端的CGEP变体的体内互补来证明CGEP肽酶活性及其自催化加工的生理重要性。光合细菌和非光合细菌的CGEP同源物缺乏C-末端序列，表明它是植物中最近的功能适应。
Chloroplast proteostasis is governed by a network of peptidases. As a part of this network, we show that Arabidopsis (Arabidopsis thaliana) chloroplast glutamyl peptidase (CGEP) is a homo-oligomeric stromal Ser-type (S9D) peptidase with both exo- and endo-peptidase activity. Arabidopsis CGEP null mutant alleles (cgep) had no visible phenotype but showed strong genetic interactions with stromal CLP protease system mutants, resulting in reduced growth. Loss of CGEP upregulated the chloroplast protein chaperone machinery and 70S ribosomal proteins, but other parts of the proteostasis network were unaffected. Both comparative proteomics and mRNA-based coexpression analyses strongly suggested that the function of CGEP is at least partly involved in starch metabolism regulation. Recombinant CGEP degraded peptides and proteins smaller than;25 kD. CGEP specifically cleaved substrates on the C-terminal side of Glu irrespective of neighboring residues, as shown using peptide libraries incubated with recombinant CGEP and mass spectrometry. CGEP was shown to undergo autocatalytic C-terminal cleavage at E946, removing 15 residues, both in vitro and in vivo. A conserved motif (A[S/T]GGG[N/G]PE946) immediately upstream of E946 was identified in dicotyledons, but not monocotyledons. Structural modeling suggested that C-terminal processing increases the upper substrate size limit by improving catalytic cavity access. In vivo complementation with catalytically inactive CGEP-S781R or a CGEP variant with an unprocessed C-terminus in a cgep clpr2-1 background was used to demonstrate the physiological importance of both CGEP peptidase activity and its autocatalytic processing. CGEP homologs of photosynthetic and nonphotosynthetic bacteria lack the C-terminal prosequence, suggesting it is a recent functional adaptation in plants.