Clathrin-coated vesicles are known to be involved in the transport ofproteins from the Golgi to the vacuole in plant cells. The mechanisms by which proteins are directed into this pathway are not known. Here we identify an integral membrane protein of -80 kDa, extracted from clathrin-coated vesicles of developing pea (Pisum saivum L.) cotyledons, that bound at neutral pH to an affinity column prepared with the N-terminal targeting determinant of the vacuolar thiol protease, proaleurain, and eluted when the pH was lowered to 4. The protein was not retained on a control column prepared with the N-terminal sequence of a homologous, secreted thiol protease, endopeptidase B. The 80-kDa protein also accumulated in a membrane fraction that is less dense than clathrin-coated vesicles. In vitro studies demonstrated a binding constant of 37 nM between the =80-kDa protein and the proaleurain targeting determinant. A peptide with a vacuolar targeting determinant from prosporamin weakly competed for binding to the "80-kDa protein, while a peptide carrying a single amino acid substitution known to abolish prosporamin vacuolar targeting had no measurable binding affinity for the protein. The binding protein is a glycoprotein with a transmembrane orientation in which the C terminus is exposed to the cytoplasm. The binding domain is located in the N-terminal luminal portion of the protein. These properties of the binding protein are consistent with the function of a receptor that would select proteins in the trans-Golgi for sorting to clathrin-coated vesicles and delivery to the vacuole.Vacuoles are acidic compartments occupying up to 80% of the volume of mature plant cells. In addition to functioning in the maintenance ofturgor and as a depository for such solutes as amino acids, sugars, organic acids, and mineral salts, the vacuoles also contain hydrolytic enzymes and, at certain stages of plant development, may serve as sites of accumulation of storage proteins (1). The accumulation of hydrolytic enzymes has been used as evidence to consider that the vacuole is a lytic compartment in plants analogous to the lysosome of the mammalian cell (2). Because of the presence of hydrolytic enzymes and the accumulation of reserve proteins, the vacuole/protein body is a useful system in which to study protein targeting in plants (3, 4). As in the mammalian lysosome system, soluble vacuolar proteins are synthesized in the endoplasmic reticulum and progress through the Golgi apparatus and clathrin-coated vesicles (CCVs) (5, 6) prior to accumulating in the vacuole. Although some soluble vacuolar proteins are glycosylated, N-linked oligosaccharides have no role in sorting glycoproteins to the vacuole (3, 4, 7). There is an accumulating body of information indicating that targeting of soluble proteins to the vacuole is mediated by determinants that reside in the polypeptide (3, 4, 7). To date, N-terminal (8-11) and C-terminal targeting determinant sequences (12, 13) have been identified; internal peptide sequences also appear to be in...

The processes by which soluble proteins are sorted from the secretory pathway to the vacuolar compartments in plant cells are poorly understood. In contrast to receptormediated sorting of lysosomal proteins in mammalian cells, where the sorting determinant is a Man-6-P residue added to Asn-linked oligosaccharides (Kornfeld, 1992), plant vacuolar sorting is determined by sequences within the polypeptides themselves (Bednarek and Raikhel, 1991;Matsuoka and Nakamura, 1991;Neuhaus et al., 1991;Saalbach et al., 1991;Holwerda et al., 1992). A similar strategy is used in yeast, where the tetrapeptide QRPL within the . '

Altlloutgh the iildllctioll of en-ynie sylntlhesis has heen extensi-ely studiecl in luicrol)ial systenms in recent years (21,31) NN'ith the intact seedlings, induction was initiated by the addition of appropriate amounts of substrate solutioni (KNO3) to the Pyre.: dish; nonsubstrate solutions for controls were added in the same manner.Cotyledons were removed from this tissue at various time intervals for assav. In the excised tissue studY, cotyledons were removed and immlaersed in 25 ml of applropriate medium (substrate, inhibitors, or control) contained in Erlennmever flasks. Samples for assay were taken as described.

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