Coated Vesicles Are Involved in the Transport of Storage Proteins during Seed Development in Pisum sativum L

Abstract

During seed development, various storage proteins and hydrolases accumulate in specialized storage vacuoles, the protein bodies, via an elaborate intracellular transport system involving the rough endoplasmic reticulum, the Golgi apparatus, and transit vesicles. Clathrin-coated vesicles, similar to those which transport lysosomal proteins to lysosomes, an organelle analogous to the vacuole, in animal cells, could be involved in this intracellular transport mechanism. Clathrin-coated vesicles have been isolated from cotyledons of developing pea (Pisum sativum L.) seeds at the time of rapid protein accumulation and analyzed for the presence of protein body constituents. A 23,000 M, polypeptide, corresponding to pea lectin precursor, was found associated with the vesicles, as determined by immunoblotting. The lectin precursor was apparently sequestered within the vesicles, as the polypeptide was only susceptible to proteolysis if detergents were included in the digestion buffer. A number of glycosidase activities, including a-mannosidase, a-galactosidase, and f8-Nacetylhexosaminidase, were also associated with the vesicles. Thus, it appears that clathrin-coated vesicles are involved in the intracellular transport of storage proteins during seed development.

Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking inArabidopsis

“…CCVs were enriched according to Harley and Beevers (1989) with minor modifications as previously described (König et al, 2008b). To prevent protein degradation, Pefabloc (Carl Roth) and protease inhibitor cocktail (Carl Roth) were included in all buffers.…”

Mediation of Clathrin-Dependent Trafficking during Cytokinesis and Cell Expansion by Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE Proteins

“…N 2 ) and centrifuged (4 min at 100g). CCVs were purified by differential and density gradient centrifugation essentially as described (Depta and Robinson, 1986;Harley and Beevers, 1989). Briefly, the cell homogenate (H) underwent sequential differential centrifugation (4 min at 100g, 10 min at 1000g, and 25 min at 30,000g).…”

Sorting of proteins to vacuoles in plant cells

“…For vacuolar sorting, the destination would have an acidic pH that would cause dissociation of the receptor-ligand complex. As clathrin coated vesicles (CCVs) were known to function in traffic from Golgi to the lysosome/vacuole in mammalian and yeast cells, and as they similarly had been associated with transport of hydrolases to plant vacuoles [29], we developed an experimental strategy to search for a ssVSD-binding receptor in membranes purified from CCVs from developing peas [54].…”