Trusova Svetlana V, Teplova Anastasia D, Golyshev Sergei A, Galiullina Raisa A, Morozova Ekaterina A, Chichkova Nina V, Vartapetian Andrey B
Department of Chemistry and Biochemistry of Nucleoproteins, Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia.
Front Plant Sci. 2019 Jul 18;10:873. doi: 10.3389/fpls.2019.00873. eCollection 2019.
Phytaspases belong to the family of plant subtilisin-like proteases and are distinct from other family members, as they have strict and rarely occurring aspartate cleavage specificity and unusual localization dynamics. After being secreted into the apoplast of healthy plant tissues, phytaspases are able to return back into cells that have been committed to cell death due to a variety of biotic and abiotic stresses. It was recently discovered that retrograde transport of phytaspases involves clathrin-mediated endocytosis. Here, consequences of phytaspase internalization were studied. Proteolytic activity of phytaspases in the apoplast and intracellular protein fractions obtained from leaves containing either endogenous phytaspase only or transiently producing phytaspase-EGFP protein (Phyt-EGFP) was determined. We demonstrated that triggering phytaspase internalization by antimycin A-induced oxidative stress is accompanied by re-distribution of phytaspase activity from the apoplast to the cell interior. Inhibition of clathrin-mediated endocytosis by co-production of the Hub protein prevented phytaspase internalization and phytaspase activity re-localization. Specificity of endocytic uptake of phytaspases was demonstrated by the co-production of an apoplast-targeted mRFP protein marker, which retained its apoplastic localization when phytaspase internalization was essentially complete. Overproduction of Phyt-EGFP, but not of the proteolytically inactive phytaspase mutant, caused moderate damage in young seedlings, whereas antimycin A treatment induced a pronounced loss of cell viability independent of the Phyt-EGFP overproduction. Interestingly, inhibition of clathrin-mediated endocytosis abrogated cell death symptoms in both cases. In contrast to stress-induced internalization of tobacco phytaspase, phytaspase-EGFP protein (Phyt-EGFP) was spontaneously internalized when transiently produced in leaves. The Phyt-EGFP uptake was dependent on clathrin-mediated endocytosis as well, the internalized protein being initially visualized within the membranous vesicles. At later time points, the EGFP tag was cleaved off from Phyt, though the elevated level of intracellular Phyt proteolytic activity persisted. Our data, therefore, point to clathrin-mediated endocytosis as a means to deliver proteolytically active phytaspases into plant cells. It would be interesting to learn whether or not phytaspases are unique among the large family of plant subtilisin-like proteases in their ability to utilize retrograde trafficking.
植物天冬氨酸特异性蛋白酶属于植物枯草杆菌蛋白酶样蛋白酶家族，与其他家族成员不同，因为它们具有严格且罕见的天冬氨酸切割特异性以及异常的定位动态。在分泌到健康植物组织的质外体后，植物天冬氨酸特异性蛋白酶能够回到由于各种生物和非生物胁迫而进入细胞死亡程序的细胞中。最近发现，植物天冬氨酸特异性蛋白酶的逆行运输涉及网格蛋白介导的内吞作用。在此，研究了植物天冬氨酸特异性蛋白酶内化的后果。测定了仅含有内源性植物天冬氨酸特异性蛋白酶或瞬时产生植物天冬氨酸特异性蛋白酶 - 绿色荧光蛋白（Phyt - EGFP）的叶片中质外体和细胞内蛋白质组分中植物天冬氨酸特异性蛋白酶的蛋白水解活性。我们证明，抗霉素A诱导的氧化应激触发植物天冬氨酸特异性蛋白酶内化，伴随着植物天冬氨酸特异性蛋白酶活性从质外体重新分布到细胞内部。通过共同产生Hub蛋白抑制网格蛋白介导的内吞作用可防止植物天冬氨酸特异性蛋白酶内化和植物天冬氨酸特异性蛋白酶活性重新定位。通过共同产生质外体靶向的mRFP蛋白标记物证明了植物天冬氨酸特异性蛋白酶内吞摄取的特异性，当植物天冬氨酸特异性蛋白酶内化基本完成时，该标记物保留其质外体定位。过量表达Phyt - EGFP，但不包括蛋白水解无活性的植物天冬氨酸特异性蛋白酶突变体，会对幼苗造成中度损伤，而抗霉素A处理会导致明显的细胞活力丧失，与Phyt - EGFP的过量表达无关。有趣的是，在这两种情况下，抑制网格蛋白介导的内吞作用都消除了细胞死亡症状。与烟草植物天冬氨酸特异性蛋白酶的应激诱导内化相反，当在叶片中瞬时产生时，植物天冬氨酸特异性蛋白酶 - 绿色荧光蛋白（Phyt - EGFP）会自发内化。Phyt - EGFP的摄取也依赖于网格蛋白介导的内吞作用，内化的蛋白最初在膜泡中可见。在随后的时间点，EGFP标签从Phyt上被切割下来，尽管细胞内Phyt蛋白水解活性水平仍然升高。因此，我们的数据表明网格蛋白介导的内吞作用是将具有蛋白水解活性的植物天冬氨酸特异性蛋白酶递送到植物细胞中的一种方式。了解植物天冬氨酸特异性蛋白酶在利用逆行运输方面是否在植物枯草杆菌蛋白酶样蛋白酶大家族中独一无二将是很有趣的。
