To acquire information on the relationships between structural maturation of proteins in the endoplasmic reticulum (ER) and their transport along the secretory pathway, we have analyzed the destiny of an assembly-defective form of the trimeric vacuolar storage glycoprotein phaseolin. In leaves of transgenic tobacco, where assembly-competent phaseolin is correctly targeted t o the vacuole, defective phaseolin remains located in the ER or a closely related compartment where it represents a major ligand of the chaperone BiP. Defective phaseolin maintained susceptibility to endoglycosidase H and was slowly degraded by a process that is not inhibited by heat shock or brefeldin A, indicating that degradation does not involve transport along the secretory pathway. These results provide evidence for the presente of a quality control mechanism in the ER of plant cells that avoids intracellular trafficking of severely defective proteins and eventually leads t o their degradation.

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI 332 ) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded.Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI 332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI 332 . Cell fractionation experiments indicate that RI 332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitinationdependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI 332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI 332 , its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61.From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.

Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential.

To acquire information on the relationships between structural maturation of proteins in the endoplasmic reticulum (ER) and their transport along the secretory pathway, we have analyzed the destiny of an assembly-defective form of the trimeric vacuolar storage glycoprotein phaseolin. In leaves of transgenic tobacco, where assembly-competent phaseolin is correctly targeted to the vacuole, defective phaseolin remains located in the ER or a closely related compartment where it represents a major ligand of the chaperone BiP. Defective phaseolin maintained susceptibility to endoglycosidase H and was slowly degraded by a process that is not inhibited by heat shock or brefeldin A, indicating that degradation does not involve transport along the secretory pathway. These results provide evidence for the presence of a quality control mechanism in the ER of plant cells that avoids intracellular trafficking of severely defective proteins and eventually leads to their degradation.

Phaseolin, one of the major legume proteins for human nutrition, is a trimeric glycoprotein of the 7S class that accumulates in the protein storage vacuoles of common bean. Phaseolin is cotranslationally introduced into the lumen of the endoplasmic reticulum; from there, it is transported through the Golgi complex to the storage vacuoles. Phaseolin is also transported to the vacuole in vegetative tissues of transgenic plants. By transient and permanent expression in tobacco leaf cells, we show here that vacuolar sorting of phaseolin is saturable and that saturation leads to Golgimediated secretion from the cell. A mutated phaseolin, in which the four C-terminal residues (Ala, Phe, Val, and Tyr) were deleted, efficiently formed trimers but was secreted entirely outside of the cells in transgenic tobacco leaves, indicating that the deleted sequence contains information necessary for interactions with the saturable vacuolar sorting machinery. In the apoplast, the secreted phaseolin remained intact; this is similar to what occurs to wild-type phaseolin in bean storage vacuoles, whereas in vegetative vacuoles of transgenic plants, the storage protein is fragmented. INTRODUCTIONPhaseolin is the major storage protein of common bean. Phaseolin is a member of the 7S vicilin class and one of the most important legume proteins for human nutrition; a number of efforts have been made to improve its nutritional value (Hoffman et al., 1988; Dyer et al., 1995). The structure, genetic makeup, cotranslational and post-translational modifications, and intracellular transport of phaseolin have been elucidated largely by numerous investigators (Bollini et al., 1982;Slightom et al., 1985;Sturm et al., 1987;Lawrence et al., 1994), but the mechanisms that allow correct intracellular targeting of phaseolin and the other 7S storage proteins have not been fully characterized.Phaseolin is a homotrimeric soluble protein that accumulates in the protein storage vacuoles of cotyledonary cells. Its synthesis, maturation, and intracellular targeting are mediated by the secretory pathway, which delivers proteins into the endoplasmic reticulum (ER) and from there to the cell surface or the vacuoles (Okita and Rogers, 1996). The Golgi complex as well as other intermediate compartments mediate this traffic.Protein constructs that have a transient signal peptide for cotranslational insertion into the ER, but no other specific sorting signal, are secreted from plant cells (Denecke et al., 1990;Hunt and Chrispeels, 1991). Soluble proteins destined for the different vacuoles are sorted from the proteins destined for the apoplast, probably at the exit of the Golgi complex (Ahmed et al., 1997;Paris et al., 1997). Sorting occurs because vacuolar proteins have structures, often identified as short stretches of amino acids in propeptides, that are not present in apoplastic proteins. The signals are variable, and different vacuolar sorting mechanisms must exist (Matsuoka et al., 1995;Kirsch et al., 1996). A putative integral membrane receptor that recognize...