Functional Analysis of the Precursor-Accumulating Vesicle

Plant seeds accumulate stored proteins as a source of amino acids for germination and subsequent seedling growth. Although most of the storage proteins are transported to protein storage vacuoles through the Golgi, at the same time, some ER-derived structures play a role in this process. PAC (precursor accumulation) vesicles are ER derived compartments found in mature seeds of pumpkins and castor beans. PAC vesicles have a core with high electron density and a periphery with low electron density. Some PAC vesicles also contain small vesicular structures.

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PAC Vesicles Store Protein Precursors in a Golgi-Independent Manner

• PAC vesicles accumulate the precursors of storage proteins, 2S albumin, and 11S globulin, to be transported to protein storage vacuoles. The precursor proteins form aggregates in the ER lumen, which develop into PAC vesicles. PAC vesicles are responsible for aggregating proteins into protein storage vacuoles in a Golgi-independent manner.

Fig.1. ER-derived compartments for protein storage.

• We help our customers study this process through the following methods. To characterize these vesicles, we perform an ultrastructural analysis and isolation of the plants at the middle stage of seed maturation. Subsequently, the isolated vesicles are examined by electron microscopy and immunoelectron microscopy.
• To examine the protein composition of the vesicles, we subject the pure vesicle preparation to SDS-PAGE and then either to staining with Coomassie blue or immunoblotting with specific antibodies against each of three major storage proteins.
• Finally, biochemical analysis of the isolated vesicles is performed to elucidate the mechanisms by which these proteins are targeted to protein-storage vacuoles.

PAC Vesicles Store Protein Precursors in a Gorgi-Dependent Manner

• In addition, some protein precursors that escape from the ER and reach the Golgi with the help of other transport vesicles are recycled to the PAC vesicle by PV72, which may recycle between the PAC vesicle and the Golgi complex.
• We provide ideas for analyzing this path mechanism. The type I integrated membrane protein PV72 on the vesicle membrane is identified by proteomic analysis of the isolated vesicles. Then, through transient expression, immunofluorescent analysis, and binding assay, we reach the conclusion that PV72 is specifically and transiently accumulated at the middle stage of seed maturation in association with the synthesis of storage proteins.

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