FUCTIONAL DIFFERENTIATION OF PLANT VACUOLES

从成熟的南瓜种子中纯化出积累大量贮藏蛋白前体的新型囊泡。这些小泡被命名为前体积累(PAC)小泡，平均直径为200至400 nm。它们包含一个电子致密的储存蛋白质核心，周围有一个电子半透明层，一些小泡也包含小的囊泡状结构。内质网内大量电子密度较高的储存蛋白聚集体发育成PAC小泡的电子密度较高的核心，然后离开内质网。这种独特的PAC小泡可能直接介导了不溶性蛋白质聚集体到蛋白质储存液泡的运输途径。我们还发现，在营养细胞中可以通过异位表达注定要被分割成PAC小泡的蛋白质来诱导PAC小泡，积累在PAC小泡中的非活性前体应该转化为…更多的是成熟形式。液泡处理酶(VPE)对植物液泡中各种种子蛋白质的成熟起重要作用。拟南芥有三种Vpe同源物：βVpe是种子特有的，αVpe和γVpe是营养器官特有的。我们发现营养VPE具有液泡加工活性，并定位于裂解的液泡中。随着衰老相关基因SAG2的表达水平的提高，两种营养VPEs在初生叶衰老过程中的表达水平也随之增加。营养VPE可能调节一些已知对这些处理有反应的功能性空泡蛋白的激活。为了研究蛋白质储存空泡中的VPE系统，我们分离了PAC小泡并表征了小泡的100 kDa成分(PVIOO)。PVIOO由一个疏水信号肽和以下三个结构域组成：一个11 kDa的Cysrich结构域和4个CxxxC基序(C，Cys)，一个34 kDa的Arg/Glu富含结构域，由6个同源重复组成，以及一个50 kDa的viclin样结构域。两个Cysrich多肽、三个富含Arg/Glu的多肽以及PV100的Asn-Gin键裂解产生了类viclin蛋白，所有这些蛋白的NH2末端都有一个焦谷氨酸。VPE负责裂解单一前体PVIOO的Asn-Gin键，以产生多个种子蛋白。很可能ASN-Gin伸展不仅为VPE提供了裂解位点，而且还产生了氨基肽酶抗性蛋白。富含半胱氨酸的多肽具有胰酶抑制作用，富含精氨酸/谷氨酸的多肽具有细胞毒活性。我们的发现表明，PVIOO在种子细胞的液泡中被转化为不同的功能蛋白。较少

Novel vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified from maturing pumpkin seeds. These vesicles were designated precursor-accumulating (PAC) vesicles and *ave diameters of 200 to 400 nm. They contain an electron-dense core of storage proteins surrounded by an electron-translucent layer, and some vesicles also contained small vesicle-like structures. Numerous electron-dense aggregates of storage proteins within the endoplasmic reticulum were found to be develop into the electron-dense cores of the PAC vesicles and then leave the endoplasmic reticulum. The unique PAC vesicles might mediate a transport pathway for insoluble aggregates of storage proteins directly to protein storage vacuoles. We also found that PAC vesicle can be induced in vegetative cells by ectopic expression of the protein that is destined to be compartmentalized into the PAC vesicles.Inactive precursors that are accumulated in the PAC vesicles should be converted int … More o mature forms. Vacuolar processing enzyme (VPE) has been shown to be responsible for maturation of various seed proteins in plant vacuoles. Arabidopsis has three VPE homologues; βVPE is specific to seeds and αVPE and γVPE are specific to vegetative organs. We found that the vegetative VPE has a vacuolar processing activity and is localized in the lytic vacuoles. The mRNA levels of both vegetative VPEs were increased in the primary leaves during senescence in parallel with the increase of the mRNA level of a senescence- associated gene (SAG2). The vegetative VPE might regulate the activation of some functional vacuolar proteins that are known to respond to these treatments.To investigate a VPE system in protein-storage vacuoles, we isolated the PAC vesicles and characterized a l00-kDa component (PVIOO) of the vesicles. PVIOO was composed of a hydrophobic signal peptide and the following three domains: an 11-kDa Cysrich domain with four CxxxC motifs (C, Cys), a 34-kDa Arg/Glu-rich domain composed of six homologous repeats, and a 50-kDa vicilin-like domain. Two Cysrich peptides, three Arg/Glu-rich peptides and the vicilin-like protein were produced by cleaving Asn-Gin bonds of PV100 and that all these proteins had a pyroglutamate at their NH2 terminus. VPE was responsible for cleaving Asn-Gin bonds of a single precursor, PVIOO, to produce multiple seed proteins. It is likely that the Asn-Gin stretches not only provide cleavage sites for VPE but also produce aminopeptidase-resistant proteins. Cys-rich peptide function as a trypsin inhibitor and Arg/Glu-rich peptides function as cytotoxic peptides. Our findings suggested that PVIOO is converted into different functional proteins in the vacuoles of seed cells. Less