In Vivo Trapping of Proteins Interacting with the Chloroplast CLPC1 Chaperone: Potential Substrates and Adaptors

The chloroplast stromal CLP protease
system is essential for growth
and development. It consists of a proteolytic CLP core complex that
likely dynamically interacts with oligomeric rings of CLPC1, CLPC2,
or CLPD AAA+ chaperones. These ATP-dependent chaperones
are predicted to bind and unfold CLP protease substrates, frequently
aided by adaptors (recognins), and feed them into the proteolytic
CLP core for degradation. To identify new substrates and possibly
also new adaptors for the chloroplast CLP protease system, we generated
an in vivo CLPC1 substrate trap with a C-terminal STREPII affinity
tag in Arabidopsis thaliana by mutating critical
glutamate residues (E374A and E718A) in the two Walker B domains of
CLPC1 required for the hydrolysis of ATP (CLPC1-TRAP). On the basis
of homology to nonplant CLPB/C chaperones, it is predicted that interacting
substrates are unable to be released; that is, they are trapped. When
expressed in the wild type, this CLPC1-TRAP induced a dominant visible
phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered. Affinity purification
of the CLPC1-TRAP resulted in a dozen proteins highly enriched compared
with affinity-purified CLPC1 with a C-terminal STREPII affinity tag
(CLPC1-WT). These enriched proteins likely represent CLP protease
substrates or new adaptors. Several of these trapped proteins overaccumulated
in clp mutants or were found as interactors for the
adaptor CLPS1, supporting their functional relationship to CLP function.
Importantly, the affinity purification of this CLPC1-TRAP also showed
high enrichment of all CLPP, CLPR, and CLPT subunits, indicating the
stabilization of the CLPC to CLP core interaction and providing direct
support for their physical and functional interaction.

叶绿体基质CLP蛋白酶系统（chloroplast stromal CLP protease system）对于植物的生长与发育不可或缺。该系统由蛋白水解型CLP核心复合物（CLP core complex）组成，可与CLPC1、CLPC2或CLPD的AAA+分子伴侣（AAA+ chaperones）寡聚环发生动态相互作用。此类ATP依赖型分子伴侣能够结合并解折叠CLP蛋白酶底物，该过程通常由适配蛋白（识别蛋白）辅助完成，并将底物递送至蛋白水解型CLP核心复合物中进行降解。
为鉴定叶绿体CLP蛋白酶系统的新型底物及潜在新型适配蛋白，我们在拟南芥（Arabidopsis thaliana）中构建了带有C端STREPII亲和标签（STREPII affinity tag）的体内CLPC1底物捕获株系（CLPC1-TRAP）：通过突变CLPC1两个ATP水解必需的Walker B结构域（Walker B domains）内的关键谷氨酸残基（E374A与E718A）实现该构建。基于与非植物CLPB/C分子伴侣的同源性，可预测与之结合的底物将无法被释放，即被捕获。
当在野生型植株中表达该CLPC1-TRAP时，会诱导出显性可见表型；而在clpc1-1无效突变体中，无法获得能够表达CLPC1-TRAP的存活突变株。对CLPC1-TRAP进行亲和纯化后，得到了十余种相较于带有C端STREPII亲和标签的野生型CLPC1（CLPC1-WT）亲和纯化产物显著富集的蛋白质。这些富集蛋白大概率为CLP蛋白酶底物或新型适配蛋白。其中部分捕获蛋白在clp突变体中存在过量积累现象，或可作为适配蛋白CLPS1的互作蛋白，这佐证了它们与CLP功能的相关性。
值得注意的是，对该CLPC1-TRAP的亲和纯化还显示所有CLPP、CLPR和CLPT亚基均得到高度富集，这表明CLPC与CLP核心复合物的相互作用得以稳定，并为二者的物理与功能互作提供了直接实验证据。