Characterization of Clathrin-Coated Vesicles

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It has been demonstrated that cotyledons from developing peas provide a rich source of clathrin coated vesicles (ccvs). It appears that, in similarity to mammalian ccvs, those from plants are composed of a clathrin cage consisting of heavy and light chains, adaptors, and a membrane vesicle component containing receptors to which cargo proteins are complexed. The ccvs isolated from cotyledons are primarily of trans-Golgi origin, and their adaptors are expected to differ from those of the better characterized mammalian ccvs which are principally of plasma membrane origin. It is planned to extend on the preliminary findings which have shown a different peptide composition of the adaptor fraction from plant ccvs. A potential receptor protein which interacts with the N-terminal targeting peptide of vacuolar proteins has been identified. This receptor will be characterized and its role in selecting protein body proteins, along with its interaction with ccv components, will be investigated. The information accumulated will provide crucial information to the understanding of vesicular transport of proteins in plants and will provide a basis for extended comparative studies with animal and other systems. %%% A key feature of all eukaryotic cells is that they are physically compartmentalized. That is, cytoplasm is not a uniform mixture of cellular components, but rather it is highly organized in such a way that specific functions, and the components that carry out those functions, are localized in discrete regions of the cell, or even external to the cell (e.g., secreted materials, cell walls, etc.). Some of these functionally distinct compartments are bounded by membranes. It follows that there must be mechanisms to ensure that specific proteins get to their particular subcellular locations. The coated vesicle system is part of this mechanism. Coated vesicles are shuttle vesicles, or transporters, of proteins contained within them. In mammalian systems, the role of coated vesicles in endocytosis (the transport of external proteins into the cell) and in intracellular movement of newly synthesized proteins from the Golgi apparatus to pre-lysosomal or pre-secretory compartments is well-studied. In plants, relatively little is known about coated vesicle function or structure, and some of the available information is the subject of controversy in the field. Coated vesicles are very likely to play significant roles in the transport of specific proteins to the vacuole, to seed storage bodies, and to the growing cell wall. As such, a detailed understanding of their structure and function will be critical to agricultural biotechnology. This project promises to yield solid, reliable information about a very important plant biology subject.