The vacuoles of yeast and plant cells share many structural and functional features. In both, specific targeting determinants must be present on soluble proteins for them to be sorted from the bulk secretory flow to the vacuole. Neither the precise nature of these determinants nor the identity of receptor(s) responsible for sorting are known for yeast or plants. We have biochemically purified an approximate 80 kD protein from peas with the features expected of such a receptor: (1) It is present in clathrin- coated vesicles that are known to participate in transport of proteins from the Golgi to the vacuole. (2) It binds with high affinity to a peptide that is known to contain sequences necessary and sufficient to target a cysteine protease to the vacuole, and with decreased affinity to peptides that are less efficient in directing such targeting. (3) It is a transmembrane protein having the peptide binding domain within its intralumenal amino terminal portion and approximately 4 kD of the carboxyl terminus exposed on the cytoplasmic surface. Identification of this protein is a novel finding and may be expected to lead to new insights into mechanisms for vacuolar sorting in both yeast and plant cells. As yeast continue to be the major genetic system for identifying gene products that participate in compartmentalization and sorting of proteins in the eukaryotic secretory pathway characterization of such a receptor is likely to lead to information that will be important for mammalian cell biologists. Only one receptor responsible for directing proteins to destinations from the intracellular trafficking pathway (as distinguished from plasma membrane receptors mediating endocytosis) has been characterized, the mannose-6-phosphate receptor. Studies of the VTR are therefore likely to provide additional insights into mechanisms involved in such trafficking. Work proposed here will determine the structure, function, and biological significance of this candidate Vacuolar Targeting Receptor (VTR). We will use the purified protein to establish the structural requirements for binding to different peptides, and will correlate these results with the ability of the same peptide sequences to direct vacuolar sorting in vivo. Antibodies to the VTR will allow its intracellular distribution and trafficking to be characterized. A cDNA clone of the VTR will provide its primary structure and will allow experiments to knock out expression of VTR protein in plant cells, thereby establishing its function.
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