Complementary DNAs derived from a mouse hybridoma messenger RNA were used to transform tobacco leaf segments followed by regeneration of mature plants. Plants expressing single gamma or kappa immunoglobulin chains were crossed to yield progeny in which both chains were expressed simultaneously. A functional antibody accumulated to 1.3% of total leaf protein in plants expressing full-length cDNAs containing leader sequences. Specific binding of the antigen recognized by these antibodies was similar to the hybridoma-derived antibody. Transformants having gamma- or kappa-chain cDNAs without leader sequences gave poor expression of the proteins. The increased abundance of both gamma- and kappa-chains in transformants expressing assembled gamma-kappa complexes was not reflected in increased mRNA levels. The results demonstrate that production of immunoglobulins and assembly of functional antibodies occurs very efficiently in tobacco. Assembly of subunits by sexual cross might be a generally applicable method for expression of heterologous multimers in plants.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytosplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBPI) Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target ofFKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.The macrolide drug rapamycin exhibits immunosuppressive as well as antineoplastic and antiproliferative properties (reviewed in reference 52). Despite the structural similarity between rapamycin and FK506, FK506 (as well as the cyclic undecapeptide cyclosporin A [CsA]) abrogates early events in T-cell activation by specifically blocking transcription of interleukin-2 (IL-2) (47, 70; reviewed in references 62 and 64), whereas rapamycin blocks subsequent lymphokine receptor-mediated processes (16,18).The blockade of T-cell signal transduction results from the interaction of these agents with specific intracellular receptors (or immunophilins). CsA binds to a class of proteins called cyclophilins (reviewed in reference 73), whereas the primary targets for both rapamycin and FK506 are the FKBPs (for FK506-binding proteins) (28,67,69). One FKBP subtype (FKBP12) has been purified from a variety of organisms and, like the cyclophilins, shown to be an enzyme with peptidyl-prolyl cis-trans isomerase (PPIase) activity (28,67). It is well established, however, that although ligand binding specifically inhibits enzymatic activity in vitro, this loss of function is not required for immunosuppression (6,24,29,30,37,45,74 interacting with other downstream cellular proteins. Thus, the immunophilins act as chaperones for these drugs, delivering them to another site of action in the cell.Both the cyclophilin-CsA and FKBP12-FK506 complexes bind to a specific protein phosphatase (calcineurin) which is hypothesized to control the activity of IL-2 gene-specific transcriptional activators (12, 24, 45, 55; reviewed in reference 63). In contrast, the downstream cellular targets for the rapamycin-sensitive signaling pathway have not been genetically characterized, although rapamycin has been shown recently to block the phosphorylation and activation of 70-kDa S6 (pp7OS6K) and p34cdc2 kinases in animal cells (8,11,51).Since rapamycin is a potent antifungal agent, we have used the power of yeast genetics to rapidly dissect the rapamycin-sensitive pathway, with the hope that a parallel pathway exists in mammalian cells. We and others previously identified and characterized the gene encoding a yeast homolog of human FKBP12 (hFKB12) (29,30,37,39,74). Deletion of this gene (which we call RBP1, for rapamycinbinding protein; also known as FPRI and FKB1 [30,37,74]) results in a recessive rapamycin-resistant phenotype, and expression of human FKBP12 in an rbpl deletion mutant restores rapamycin sensitivity (37).In this study, we hav...

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the response to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, -13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBPI (for rapamycin-binding protein). The RBPI protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.Agents that inhibit T-cell activation include cyclosporin A (CsA) (19) and the recently discovered macrolide FK506 (31, 36). CsA was originally discovered as an antifungal agent, and FK506 was identified as an inhibitor of interleukin-2 (IL-2) production (20). Despite the structural dissimilarity between these two immunosuppressive drugs, recent reports suggest that the targets for both agents, cyclophilin and FK506-binding protein (FKBP), respectively, are peptidylprolyl cis-trans isomerases (PPlases), enzymes that promote protein folding in vitro (12,15,34,35,37,40,41). Although the endogenous function of PPlases is not known, the fact that the immunosuppressive action of CsA and FK506 is linked to inhibition of PPIase activity suggests that they may be required in the regulation of intracellular signaling events leading to T-cell activation (8, 12, 37).Rapamycin, a macrolide antifungal agent with structural similarity to FK506 (32, 42), also exhibits immunosuppressive (3, 26, 38) as well as antineoplastic (9, 18) properties. Rapamycin and FK506 act as reciprocal antagonists in vivo (murine T cell activation [6]) and compete for binding to FKBP (15). Given these similarities, the mechanism of action of rapamycin remains enigmatic because, whereas FK506 (like CsA) acts to inhibit IL-2 transcription, rapamycin has no effe...

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