GOLGI AND MULTIVESICULAR BODIES IN PROTEIN STORAGE VACUOLE FORMATION

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。拟南芥胚胎中的蛋白质储存泡（PSV）含有两种类型的储存蛋白，2S白蛋白和12 S球蛋白，以及植酸球状体。2S白蛋白和12 S球蛋白在ER中作为前体合成，输出到高尔基体，并在前蛋白加工后积累在PSV中。贮藏蛋白的加工涉及半胱氨酸蛋白酶β-VPE（液泡蛋白酶）和至少一种天冬氨酸蛋白酶。还有待确定的是如何储存蛋白质，加工酶和植酸是分类的，以及它们如何到达PSV。多泡体（MVB）已被证明含有豆类胚胎中的存储蛋白，但其在PSV装配的确切作用尚不清楚。我们已经研究了高尔基体堆栈，高尔基体衍生的囊泡，和MVBs在拟南芥PSV形成的高压冷冻/冷冻替代样品的电子断层扫描，并通过免疫标记技术在拟南芥，并通过亚细胞分馏技术在甘蓝型油菜。2S和12贮藏蛋白都在高尔基池边缘形成高度浓缩的聚集体。然而，而2S蛋白似乎凝聚在芽的顺式最池，12 S蛋白这样做，在芽的顺式和内侧池。三种类型的囊泡被认为是在附近的高尔基体堆栈：130 nm的囊泡与电子致密的核心和外部半透明层，30-40 nm的非涂层囊泡，和30-40 nm网格蛋白涂层囊泡。致密的核心囊泡含有2S蛋白前体，如通过使用针对两个2S前肽和12 S储存蛋白的肽抗体所揭示的。β-VPE和天冬氨酸蛋白酶被视为网格蛋白包被的囊泡。亚细胞分级分离研究证实了免疫标记揭示的分布模式。MVB直径范围为150 nm至350 nm，含有约25 nm的内部囊泡。在MVB的内腔中，我们已经检测到贮存蛋白前体的聚集体、大量的2S和12 S蛋白的成熟形式、β-VPE和天冬氨酸蛋白酶。这表明致密囊泡和网格蛋白包被的囊泡融合成MVB，并且储存蛋白的加工开始于MVB。此外，AtELP，空泡蛋白含有N-末端前肽的受体，检测到在TGN中，在MVB中，并在管腔的PSV。在MVB中，在限制膜和内部囊泡上都观察到AtELP标记，这与受体在MVB中靶向降解的想法一致。最后，AtELP在PSV腔中检测到，但不在PSV膜上，这表明当MVB与PSV融合时，AtELP已完全从MVB限制膜上去除。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein storage vacuoles (PSVs) in the Arabidopsis embryo contain two types of storage proteins, the 2S albumins and the 12S globulins, as well as phytic acid globoids. Both 2S albumins and 12S globulins are synthesized as precursors in the ER, exported to the Golgi apparatus, and accumulated in PSVs after processing of the proproteins. Processing of the storage proteins involves the cysteine-protease beta-VPE (vacuolar protein enzyme) and at least one aspartic protease. Yet to be determined is how the storage proteins, processing enzymes, and phytic acid are sorted and how they get to the PSVs. Multivesicular bodies (MVBs) have been shown to contain storage proteins in legume embryos but their exact role in PSV assembly is not known. We have studied Golgi stacks, Golgi-derived vesicles, and MVBs during PSV formation in Arabidopsis by means of electron tomography of high-pressure frozen/freeze substituted samples and by immunolabeling techniques in Arabidopsis, and by subcellular fractionation techniques in Brassica napus. The 2S and 12 storage proteins both form highly condensed aggregates within the Golgi cisterna margins. However, whereas the 2S proteins appear to condense in buds of the cis-most cisterna, the 12S proteins do so in buds of cis and medial cisternae. Three types of vesicles are seen in the vicinity of Golgi stacks: 130 nm vesicles with an electron-dense core and an outer translucent layer, 30-40 nm non-coated vesicles, and 30-40 nm clathrin-coated vesicles. The dense core vesicles contain 2S protein precursors, as revealed by the use of peptide antibodies raised against two 2S propeptides, and 12S storage proteins. Beta-VPE and an aspartic protease were seen over clathrin-coated vesicles. Subcellular fractionation studies confirm the distribution pattern revealed by immunolabeling. MVBs range from 150 nm to 350 nm in diameter and contain internal vesicles of around 25 nm. In the lumen of MVBs, we have detected aggregates of storage protein precursors, high amounts of the mature forms of the 2S and 12S proteins, beta-VPE, and the aspartic protease. This suggests that dense vesicles and clathrin-coated vesicles fuse into MVBs, and that the processing of storage proteins begins in the MVBs. In addition, AtELP, a receptor for vacuolar proteins containing N-terminal propeptides, was detected in the TGN, in MVBs, and in the lumen of PSVs. In MVBs, AtELP labeling was observed over both the limiting membrane and internal vesicles, consistent with the idea that the receptor is targeted for degradation in the MVB. Finally, AtELP was detected in the lumen of PSV but not over the PSV membrane, suggesting that by the time MVBs fuse with PSVs, AtELP has been completely removed from the MVB limiting membrane.