Institute of Botany, Christian-Albrechts-University of Kiel, Olshausenstraße 40, 24098 Kiel, Germany.
Solana Research, Eichenallee 9, Windeby, Germany.
J Exp Bot. 2019 Nov 18;70(21):6057-6069. doi: 10.1093/jxb/erz356.
Chloroplast protein degradation is known to occur both inside chloroplasts and in the vacuole. Genes encoding cysteine proteases have been found to be highly expressed during leaf senescence. However, it remains unclear where they participate in chloroplast protein degradation. In this study HvPAP14, which belongs to the C1A family of cysteine proteases, was identified in senescing barley (Hordeum vulgare L.) leaves by affinity enrichment using the mechanism-based probe DCG-04 targeting cysteine proteases and subsequent mass spectrometry. Biochemical analyses and expression of a HvPAP14:RFP fusion construct in barley protoplasts was used to identify the subcellular localization and putative substrates of HvPAP14. The HvPAP14:RFP fusion protein was detected in the endoplasmic reticulum and in vesicular bodies. Immunological studies showed that HvPAP14 was mainly located in chloroplasts, where it was found in tight association with thylakoid membranes. The recombinant enzyme was activated by low pH, in accordance with the detection of HvPAP14 in the thylakoid lumen. Overexpression of HvPAP14 in barley revealed that the protease can cleave LHCB proteins and PSBO as well as the large subunit of Rubisco. HvPAP14 is involved in the normal turnover of chloroplast proteins and may have a function in bulk protein degradation during leaf senescence.
质体蛋白降解既发生在质体内，也发生在液泡中。现已发现，编码半胱氨酸蛋白酶的基因在叶片衰老过程中高度表达。然而，它们在质体蛋白降解中参与的位置仍不清楚。本研究通过使用针对半胱氨酸蛋白酶的基于机制的探针 DCG-04 进行亲和富集，在衰老的大麦（Hordeum vulgare L.）叶片中鉴定到属于半胱氨酸蛋白酶 C1A 家族的 HvPAP14。通过生化分析和大麦原生质体中 HvPAP14:RFP 融合构建体的表达，鉴定了 HvPAP14 的亚细胞定位和可能的底物。HvPAP14:RFP 融合蛋白在内质网和小泡体中被检测到。免疫研究表明，HvPAP14 主要位于叶绿体中，在那里它与类囊体膜紧密结合。重组酶被低 pH 激活，这与在类囊体腔中检测到 HvPAP14 相一致。在大麦中过表达 HvPAP14 表明，该蛋白酶可以切割 LHCB 蛋白和 PSBO 以及 Rubisco 的大亚基。HvPAP14 参与质体蛋白的正常周转，并且在叶片衰老期间可能具有批量蛋白降解的功能。
