We have characterized the requirements to inhibit the function of the plant vacuolar sorting receptor BP80 in vivo and gained insight into the crucial role of receptor recycling between the prevacuolar compartment and the Golgi apparatus. The drug wortmannin interferes with the BP80-mediated route to the vacuole and induces hypersecretion of a soluble BP80-ligand. Wortmannin does not prevent receptor-ligand binding itself but causes BP80 levels to be limiting. Consequently, overexpression of BP80 partially restores vacuolar cargo transport. To simulate receptor traffic, we tested a truncated BP80 derivative in which the entire lumenal domain of BP80 has been replaced by the green fluorescent protein (GFP). The resulting chimeric protein (GFP-BP80) accumulates in the prevacuolar compartment as expected, but a soluble GFP fragment can also be detected in purified vacuoles. Interestingly, GFP-BP80 coexpression interferes with the correct sorting of a BP80-ligand and causes hypersecretion that is reversible by expressing a 10-fold excess of full-length BP80. This suggests that GFP-BP80 competes with endogenous BP80 mainly at the retrograde transport route that rescues receptors from the prevacuolar compartment. Treatment with wortmannin causes further leakage of GFP-BP80 from the prevacuolar compartment to the vacuoles, whereas BP80-ligands are secreted. We propose that recycling of the vacuolar sorting receptor from the prevacuolar compartment to the Golgi apparatus is an essential process that is saturable and wortmannin sensitive.

Protein sorting to plant vacuoles is known to be dependent on a considerable variety of protein motifs recognized by a family of sorting receptors. This can involve either traffic from the endoplasmic reticulum (ER) through the Golgi apparatus or direct ER-to-vacuole transport. Barley aspartic protease (Phytepsin) was shown previously to reach the vacuole via trafficking through the Golgi apparatus. Here we show that Phytepsin normally exits the ER in a COPII-mediated manner, because the Phytepsin precursor accumulates in the ER upon specific inhibition of the formation of COPII vesicles in vivo. Phytepsin differs from its yeast and mammalian counterparts by the presence of a saposin-like plant-specific insert (PSI). Deletion of this domain comprising 104 amino acids causes efficient secretion of the truncated molecule (Phytepsin ⌬ PSI) without affecting the enzymatic activity of the enzyme. Interestingly, deletion of the PSI also changes the way in which Phytepsin exits the ER. Inhibition of COPII vesicle formation causes accumulation of the Phytepsin precursor in the ER but has no effect on the secretion of Phytepsin ⌬ PSI. This suggests either that vacuolar sorting commences at the ER export step and involves recruitment into COPII vesicles or that the PSI domain carries two signals, one for COPII-dependent export from the ER and one for vacuolar delivery from the Golgi. The relevance of these observations with respect to the bulk flow model of secretory protein synthesis is discussed. INTRODUCTIONPlant cells contain at least two functionally distinct vacuolar compartments: the central lytic vacuole, which is related to the mammalian lysosome, and the so-called storage vacuoles, which appear to be unique to the plant kingdom (Marty, 1999). Whereas the lytic vacuole is typical for vegetative cells, storage vacuoles are found mostly in reserve tissues of seeds. Exceptions to this are the vegetative storage vacuoles that are formed during stress conditions and that could be related to a neutral vacuolar compartment recently discovered in tobacco protoplasts (Di Sansebastiano et al., 1998). It was first believed that the various vacuolar compartments share a common origin but change appearance and contents according to physiological conditions or tissue type. However, the simultaneous presence of storage vacuoles and lytic vacuoles within the same cell, as determined using specific membrane markers (Hoh et al., 1995;Paris et al., 1996;Jauh et al., 1998), argues against this notion. In addition, the great variety of vacuolar sorting signals described in plants (Matsuoka and Neuhaus, 1999) also suggests the possibility that the various types of vacuoles have different origins and are supported by different protein transport pathways (Chrispeels and Herman, 2000).The first evidence for distinct transport routes arose from the use of pharmacological agents that exhibit a differential effect on the vacuolar sorting of a variety of cargo molecules (Gomez and Chrispeels, 1993;Matsuoka et al., 1995). Furthermore, the to...

No abstract

In this study, we demonstrate that the folding and assembly of IgG in transgenic tobacco plants is orchestrated by BiP (binding protein), an endoplasmic reticulum resident chaperone. Expression of BiP and calreticulin was examined in transgenic tobacco plants that express immunoglobulin chains, either singly or in combination to form IgG antibody. BiP mRNA expression was lowest in wild-type nontransformed plants and those that expressed immunoglobulin light chain alone. Higher mRNA levels were detected in plants expressing fully assembled immunoglobulin (light and heavy chains), and the most abundant levels of RNA transcript were found in those plants that expressed immunoglobulin heavy chain alone. Estimation of total BiP demonstrated a similar pattern, with the highest levels detected in plants expressing immunoglobulin heavy chain alone. Immunoprecipitation studies demonstrated that BiP was associated with immunoglobulin chains extracted from protoplast lysates, but not from secreted fluids. Again, most BiP was coprecipitated from plants expressing heavy chain only and those that produced full length IgG. The binding of BiP to Ig heavy chains was ATP-sensitive. Co-expression of heavy and light chain resulted in IgG assembly and displacement of BiP from the heavy chain as the amount of light chain increased. Although calreticulin mRNA and total protein levels varied in a similar manner to those of BiP in the transgenic plants, there was no evidence for association between calreticulin and Ig chains, by coimmunoprecipitation. The results indicate that BiP, but not calreticulin, takes part in immunoglobulin folding and assembly in transgenic plants.Keywords: BiP; IgG; transgenic plants; immunoglobulin assembly; chaperones.A wide variety of functional recombinant antibody molecules have been expressed successfully in transgenic plants, ranging from small monomeric fragments [1-3] to full length IgG [2,4,5] as well as more complex multimeric secretory antibodies [6]. The synthesis, folding and assembly of complex mammalian proteins, such as full length immunoglobulins (Igs) in plants can be extremely efficient, resulting in expression levels of between 1 and 5% of total plant protein [4,6,7], that compare favourably with mammalian hybridoma cell culture. Protein folding and assembly within cells is a complex process with stringent quality control mechanisms (reviewed in [8]). It is largely regulated by enzymes and an array of molecular chaperones. In mammalian and plant cells, the best characterized chaperone is BiP (binding protein), a lumenal endoplasmic reticulum (ER) resident member of the heat shock protein 70 family of stress proteins [9]. BiP has been identified in various mammals [10][11][12][13], yeast [14,15] and plants [16,17]. By binding to newly synthesized polypeptides, BiP is thought to stabilize partially folded intermediates during folding and assist in the assembly of protein oligomers [18]. BiP also has other functions in protein translocation into the ER, prevention and dissolution of protein a...