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.

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.

BiP is found in association with calreticulin, both in the presence and absence of endoplasmic reticulum stress. Although the BiP-calreticulin complex can be disrupted by ATP, several properties suggest that the calreticulin associated with BiP is neither unfolded nor partially or improperly folded. (1) The complex is stable in vivo and does not dissociate during 8 hr of chase. (2) When present in the complex, calreticulin masks epitopes at the C terminus of BiP that are not masked when BiP is bound to an assembly-defective protein. And (3) overproduction of calreticulin does not lead to the recruitment of more BiP into complexes with calreticulin. The BiP-calreticulin complex can be disrupted by low pH but not by divalent cation chelators. When the endoplasmic reticulum retention signal of BiP is removed, complex formation with calreticulin still occurs, and this explains the poor secretion of the truncated molecule. Gel filtration experiments showed that BiP and calreticulin are present in distinct high molecular weight complexes in which both molecules interact with each other. The possible functions of this complex are discussed. INTRODUCTIONThe endoplasmic reticulum (ER) is the primary organelle of the endomembrane system and is responsible for the biosynthesis and maturation of proteins that are destined for secretion, for the plasma membrane, and for transport to various organelles of the endocytic and exocytic pathways (reviewed in Palade, 1975;Helenius et al., 1992; reviewed for plants in Vitale et al., 1993). In addition to nascent polypeptides and newly synthesized proteins, the ER lumen also contains a large and abundant family of soluble resident proteins known as the reticuloplasmins (Koch, 1987; reviewed for plants in Denecke, 1996). Several reticuloplasmins are now recognized as molecular chaperones (Gething and Sambrook, 1992;Hartl, 1996), which prevent the aggregation of unfolded and partially folded polypeptides, thus increasing the yield but not the rate of correct folding and assembly. Knowledge of the binding specificity of chaperones is rapidly expanding; however, mechanistic details of how the binding of the chaperone results in the unfolding of the protein followed by refolding and release from the chaperone remain to be established.Several reticuloplasmins have been identified in plants, including the binding protein BiP (Denecke et al., 1991;Fontes et al., 1991;Anderson et al., 1994), calreticulin (Chen et al., 1994;Dresselhaus et al., 1996), protein disulfide isomerase (PDI; Shorrosh and Dixon, 1991), and endoplasmin (Walther-Larsen et al., 1993;Denecke et al., 1993. Endoplasmin has homology with the cytosolic heat shock protein Hsp90 family, but no definitive function has been proposed. PDI catalyzes the formation of disulfide bonds, a process restricted to the ER lumen, hence the absence of cytosolic homologs of PDI.The best-characterized ER resident protein is BiP (Munro and Pelham, 1986). BiP belongs to the Hsp70 family of molecular chaperones. This family has members in every c...

BiP is found in association with calreticulin, both in the presence and absence of endoplasmic reticulum stress. Although the BiP-calreticulin complex can be disrupted by ATP, several properties suggest that the calreticulin associated with BiP is neither unfolded nor partially or improperly folded. (1) The complex is stable in vivo and does not dissociate during 8 hr of chase. (2) When present in the complex, calreticulin masks epitopes at the C terminus of BiP that are not masked when BiP is bound to an assembly-defective protein. And (3) overproduction of calreticulin does not lead to the recruitment of more BiP into complexes with calreticulin. The BiP-calreticulin complex can be disrupted by low pH but not by divalent cation chelators. When the endoplasmic reticulum retention signal of BiP is removed, complex formation with calreticulin still occurs, and this explains the poor secretion of the truncated molecule. Gel filtration experiments showed that BiP and calreticulin are present in distinct high molecular weight complexes in which both molecules interact with each other. The possible functions of this complex are discussed. INTRODUCTIONThe endoplasmic reticulum (ER) is the primary organelle of the endomembrane system and is responsible for the biosynthesis and maturation of proteins that are destined for secretion, for the plasma membrane, and for transport to various organelles of the endocytic and exocytic pathways (reviewed in Palade, 1975;Helenius et al., 1992; reviewed for plants in Vitale et al., 1993). In addition to nascent polypeptides and newly synthesized proteins, the ER lumen also contains a large and abundant family of soluble resident proteins known as the reticuloplasmins (Koch, 1987; reviewed for plants in Denecke, 1996). Several reticuloplasmins are now recognized as molecular chaperones (Gething and Sambrook, 1992;Hartl, 1996), which prevent the aggregation of unfolded and partially folded polypeptides, thus increasing the yield but not the rate of correct folding and assembly. Knowledge of the binding specificity of chaperones is rapidly expanding; however, mechanistic details of how the binding of the chaperone results in the unfolding of the protein followed by refolding and release from the chaperone remain to be established.Several reticuloplasmins have been identified in plants, including the binding protein BiP (Denecke et al., 1991;Fontes et al., 1991;Anderson et al., 1994), calreticulin (Chen et al., 1994;Dresselhaus et al., 1996), protein disulfide isomerase (PDI; Shorrosh and Dixon, 1991), and endoplasmin (Walther-Larsen et al., 1993;Denecke et al., 1993. Endoplasmin has homology with the cytosolic heat shock protein Hsp90 family, but no definitive function has been proposed. PDI catalyzes the formation of disulfide bonds, a process restricted to the ER lumen, hence the absence of cytosolic homologs of PDI.The best-characterized ER resident protein is BiP (Munro and Pelham, 1986). BiP belongs to the Hsp70 family of molecular chaperones. This family has members in every c...