GOLGI AND MULTIVESICULAR BODIES IN PROTEIN STORAGE VACUOLE FORMATION

蛋白质储存液泡形成中的高尔基体和多胞体

• 批准号: 7354979
• 负责人: ANDREW L. STAEHELIN
• 金额: $ 0.94万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-26 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7354979
• 关键词: proteins

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。拟南芥胚胎中的蛋白质储存液泡 (PSV) 含有两种类型的储存蛋白质：2S 白蛋白和 12S 球蛋白，以及植酸球蛋白。 2S 白蛋白和 12S 球蛋白均作为前体在 ER 中合成，输出至高尔基体，并在前蛋白加工后在 PSV 中积累。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。尚待确定的是储存蛋白、加工酶和植酸如何分类以及它们如何到达 PSV。多泡体 (MVB) 已被证明在豆类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、拟南芥中的免疫标记技术以及甘蓝型油菜中的亚细胞分级分离技术，研究了拟南芥PSV形成过程中的高尔基体堆栈、高尔基体衍生囊泡和MVB。 2S 和 12 储存蛋白均在高尔基池边缘形成高度浓缩的聚集体。然而，2S 蛋白似乎在最顺式池的芽中凝结，而 12S 蛋白则在顺式池和内侧池的芽中凝结。在高尔基体堆叠附近可以看到三种类型的囊泡：具有电子致密核心和外部半透明层的 130 nm 囊泡、30-40 nm 无涂层囊泡和 30-40 nm 网格蛋白涂层囊泡。使用针对两个 2S 前肽和 12S 储存蛋白产生的肽抗体揭示了致密核心囊泡含有 2S 蛋白前体。在网格蛋白包被的囊泡上观察到 Beta-VPE 和天冬氨酸蛋白酶。亚细胞分级研究证实了免疫标记揭示的分布模式。 MVB 的直径范围为 150 nm 至 350 nm，包含约 25 nm 的内部囊泡。在 MVB 的内腔中，我们检测到了储存蛋白前体的聚集体、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从 MVB 开始。此外，在 TGN、MVB 和 PSV 腔中检测到 AtELP，一种含有 N 末端前肽的液泡蛋白受体。在 MVB 中，在限制膜和内部囊泡上均观察到 AtELP 标记，这与受体在 MVB 中被靶向降解的观点一致。最后，AtELP 在 PSV 管腔中检测到，但在 PSV 膜上未检测到，这表明当 MVB 与 PSV 融合时，AtELP 已完全从 MVB 限制膜上去除。