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.

叶绿体蛋白质稳态由肽酶网络控制。作为该网络的一部分，我们表明拟南芥（Arabidopsis thaliana）叶绿体谷氨酰肽酶（CGEP）是一种同源寡聚基质丝氨酸（S9D）肽酶，具有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，但与基质 CLP 蛋白酶系统突变体表现出强烈的遗传相互作用，导致生长减慢。CGEP 的缺失上调了叶绿体蛋白伴侣机制和 70S 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 降解小于约 25 kD 的肽和蛋白质。CGEP 特异性切割 Glu C 端侧的底物，无论邻近残基如何，如使用与重组 CGEP 孵育的肽文库和质谱法所示。CGEP 在 E946 处显示出自催化 C 末端裂解，在体外和体内均去除了 15 个残基。在双子叶植物中鉴定出紧邻 E946 上游的保守基序 (A[S/T]GGG[N/G]PE _{946 )，但在单子叶植物中未鉴定到。}结构模型表明，C 端处理通过改善催化腔通道来增加底物尺寸上限。在cgep clpr2 - 1背景中与催化失活 CGEP-S781R 或具有未加工 C 末端的 CGEP 变体进行体内互补，以证明 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 末端原序列，表明它是植物最近的功能适应。