Barley cysteine protease PAP14 plays a role in degradation of chloroplast proteins
Barley cysteine protease PAP14 plays a role in degradation of chloroplast proteins
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DOI：
10.1093/jxb/erz356
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发表时间：
2019-11-01
影响因子：
6.9
通讯作者：
Krupinska, Karin
中科院分区：
文献类型：
--
作者：
Frank, Susann;Hollmann, Julien;Krupinska, Karin
已知叶绿体蛋白质降解发生在叶绿体内部和液泡中。已发现编码半胱氨酸蛋白酶的基因在叶子衰老过程中高度表达。然而，目前尚不清楚它们在何处参与叶绿体蛋白质降解。在这项研究中，HvPAP14 属于半胱氨酸蛋白酶 C1A 家族，通过使用基于机制的探针 DCG-04 靶向半胱氨酸蛋白酶的亲和富集和随后的质谱分析，在衰老大麦 (Hordeum vulgare L.) 叶子中进行了鉴定。 HvPAP14:RFP 融合构建体在大麦原生质体中的生化分析和表达用于鉴定 HvPAP14 的亚细胞定位和假定底物。在内质网和囊泡体中检测到 HvPAP14:RFP 融合蛋白。免疫学研究表明，HvPAP14 主要位于叶绿体中，与类囊体膜紧密结合。根据类囊体腔中 HvPAP14 的检测，重组酶在低 pH 条件下被激活。 HvPAP14 在大麦中的过度表达表明，该蛋白酶可以裂解 LHCB 蛋白和 PSBO 以及 Rubisco 大亚基。 HvPAP14 参与叶绿体蛋白的正常周转，并可能在叶片衰老过程中的大量蛋白降解中发挥作用。
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.