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.
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