Gruis Darren, Schulze Jan, Jung Rudolf
Pioneer Hi-Bred International, A DuPont Company, Johnston, Iowa 50131-1004, USA.
Plant Cell. 2004 Jan;16(1):270-90. doi: 10.1105/tpc.016378. Epub 2003 Dec 19.
The role(s) of specific proteases in seed protein processing is only vaguely understood; indeed, the overall role of processing in stable protein deposition has been the subject of more speculation than direct investigation. Seed-type members of the vacuolar processing enzyme (VPE) family were hypothesized to perform a unique function in seed protein processing, but we demonstrated previously that Asn-specific protein processing in developing Arabidopsis seeds occurs independently of this VPE activity. Here, we describe the unexpected expression of vegetative-type VPEs in developing seeds and test the role(s) of all VPEs in seed storage protein accumulation by systematically stacking knockout mutant alleles of all four members (alphaVPE, betaVPE, gammaVPE, and deltaVPE) of the VPE gene family in Arabidopsis. The complete removal of VPE function in the alphavpe betavpe gammavpe deltavpe quadruple mutant resulted in a total shift of storage protein accumulation from wild-type processed polypeptides to a finite number of prominent alternatively processed polypeptides cleaved at sites other than the conserved Asn residues targeted by VPE. Although alternatively proteolyzed legumin-type globulin polypeptides largely accumulated as intrasubunit disulfide-linked polypeptides with apparent molecular masses similar to those of VPE-processed legumin polypeptides, they showed markedly altered solubility and protein assembly characteristics. Instead of forming 11S hexamers, alternatively processed legumin polypeptides were deposited primarily as 9S complexes. However, despite the impact on seed protein processing, plants devoid of all known functional VPE genes appeared unchanged with regard to protein content in mature seeds, relative mobilization rates of protein reserves during germination, and vegetative growth. These findings indicate that VPE-mediated Asn-specific proteolytic processing, and the physiochemical property changes attributed to this specific processing step, are not required for the successful deposition and mobilization of seed storage protein in the protein storage vacuoles of Arabidopsis seeds.
特定蛋白酶在种子蛋白质加工过程中的作用目前还了解得很模糊；实际上，加工过程在稳定蛋白质沉积中的整体作用更多是推测的主题，而非直接研究的对象。液泡加工酶（VPE）家族的种子类型成员曾被假设在种子蛋白质加工中发挥独特功能，但我们之前已证明，拟南芥发育种子中特定于天冬酰胺的蛋白质加工独立于这种VPE活性发生。在这里，我们描述了营养型VPEs在发育种子中的意外表达，并通过系统地叠加拟南芥VPE基因家族所有四个成员（αVPE、βVPE、γVPE和δVPE）的敲除突变等位基因，来测试所有VPEs在种子储存蛋白积累中的作用。在αvpeβvpeγvpeδvpe四重突变体中VPE功能的完全去除，导致储存蛋白积累从野生型加工多肽完全转变为有限数量的突出的经不同方式加工的多肽，这些多肽在VPE靶向的保守天冬酰胺残基以外的位点被切割。尽管经不同方式蛋白水解的豆球蛋白型球蛋白多肽在很大程度上以亚基内二硫键连接的多肽形式积累，其表观分子量与VPE加工的豆球蛋白多肽相似，但它们显示出明显改变的溶解性和蛋白质组装特性。经不同方式加工的豆球蛋白多肽不是形成11S六聚体，而是主要以9S复合物的形式沉积。然而，尽管对种子蛋白质加工有影响，但缺乏所有已知功能VPE基因的植物在成熟种子中的蛋白质含量、萌发期间蛋白质储备的相对动员率和营养生长方面似乎没有变化。这些发现表明，VPE介导的特定于天冬酰胺的蛋白水解加工以及归因于这一特定加工步骤的理化性质变化，对于拟南芥种子蛋白质储存液泡中种子储存蛋白的成功沉积和动员并非必需。
