Plant Biology Section, School of Integrative Plant Sciences (SIPS), Cornell University, Ithaca, NY, USA.
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia.
FEBS Lett. 2019 May;593(9):962-970. doi: 10.1002/1873-3468.13378. Epub 2019 Apr 20.
The prokaryotic N-degron pathway depends on the Clp chaperone-protease system and the ClpS adaptor for recognition of N-degron bearing substrates. Plant chloroplasts contain a diversified Clp protease, including the ClpS homolog ClpS1. Several candidate ClpS1 substrates have been identified, but the N-degron specificity is unclear. Here, we employed in vitro ClpS1 affinity assays using eight N-degron green fluorescence protein reporters containing either F, Y, L, W, I, or R in the N-terminal position. This demonstrated that ClpS1 has a restricted N-degron specificity, recognizing proteins bearing an N-terminal F or W, only weakly recognizing L, but not recognizing Y or I. This affinity is dependent on two conserved residues in the ClpS1 binding pocket and is sensitive to FR dipeptide competition, suggesting a unique chloroplast N-degron pathway.
原核 N 端肽降解途径依赖于 Clp 伴侣蛋白酶系统和 ClpS 接头来识别含有 N 端肽的底物。植物叶绿体含有多样化的 Clp 蛋白酶，包括 ClpS 同源物 ClpS1。已经鉴定出几种候选的 ClpS1 底物，但 N 端肽降解特异性尚不清楚。在这里，我们使用含有 F、Y、L、W、I 或 R 的 N 端肽绿色荧光蛋白报告基因的体外 ClpS1 亲和测定法，对 8 个 N 端肽降解蛋白进行了研究。结果表明，ClpS1 具有有限的 N 端肽降解特异性，仅能微弱识别 L，而不能识别 Y 或 I，只能识别 N 端含有 F 或 W 的蛋白。这种亲和力依赖于 ClpS1 结合口袋中的两个保守残基，并且对 FR 二肽竞争敏感，这表明存在一种独特的叶绿体 N 端肽降解途径。
