GOLGI AND MULTIVESICULAR BODIES IN PROTEIN STORAGE VACUOLE FORMATION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。拟南芥胚中的蛋白储存液泡（psv）包含2S白蛋白和12S球蛋白两种类型的储存蛋白，以及植酸球状体。2S白蛋白和12S球蛋白都在内质网中作为前体合成，输出到高尔基体，并在原蛋白加工后积累在psv中。储存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。目前尚不清楚储存蛋白、加工酶和植酸是如何分类的，以及它们是如何到达psv的。多泡体（Multivesicular bodies, MVBs）在豆科植物胚胎中含有储存蛋白，但它们在PSV组装中的确切作用尚不清楚。我们通过高压冷冻/冷冻替代样品的电子断层扫描和免疫标记技术，以及甘蓝型油菜的亚细胞分离技术，研究了拟南芥PSV形成过程中的高尔基堆、高尔基衍生囊泡和MVBs。2S和12储存蛋白在高尔基池边缘形成高度凝聚的聚集体。然而，虽然2S蛋白似乎在顺式池的芽中凝结，但12S蛋白在顺式池和内侧池的芽中凝结。在高尔基堆附近可以看到三种类型的囊泡：130 nm的囊泡，具有电子密集的核心和外层半透明层，30-40 nm的无包被囊泡，30-40 nm的包被囊泡。密集的核心囊泡含有2S蛋白前体，这是利用针对两个2S前肽的肽抗体和12S储存蛋白发现的。β - vpe和天冬氨酸蛋白酶在网格蛋白包被的囊泡上可见。亚细胞分离研究证实了免疫标记所揭示的分布模式。MVBs的直径从150纳米到350纳米不等，内部包含约25纳米的囊泡。在MVBs的管腔中，我们检测到储存蛋白前体的聚集，大量成熟形式的2S和12S蛋白，β - vpe和天冬氨酸蛋白酶。这表明密集的囊泡和网格蛋白包被的囊泡融合到MVBs中，并且储存蛋白的加工开始于MVBs。此外，在psv的TGN、MVBs和管腔中检测到含有n端前肽的空泡蛋白受体AtELP。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中被降解的观点一致。最后，在PSV管腔中检测到AtELP，但未在PSV膜上检测到，这表明当MVBs与PSV融合时，AtELP已完全从MVB限制膜上移除。

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.