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...

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including beta-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.

Candida albicans is not inhibited by a number of drugs known to affect fungal cells. The basis for this resistance in most cases is unknown but has been attributed to the general impermeability of the fungal cell envelope. A gene (BENr) formerly shown to be responsible for the resistance of C. albicans to benomyl and methotrexate was shown in the present study to confer resistance to four other inhibitory compounds: cycloheximide, benztriazoles, 4-nitroquinoline-N-oxide, and sulfometuron methyl. Analysis of the protein database revealed an apparent similarity of the C. albicans gene to membrane protein genes encoding antibiotic resistance in prokaryotes and eukaryotes and a high degree of identity to a recently cloned gene encoding cycloheximide resistance in Candida maltosa. We propose that BENr encodes a protein that operates in a fashion similar, but not identical, to that described for other multiple-drug resistance proteins.

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By using orthogonal-field alternating gel electrophoresis (OFAGE), field-inversion gel electrophoresis (FIGE), and contour-clamped homogeneous field gel electrophoresis (CHEF), we have clearly resolved 11 chromosomal bands from various Candida albicans strains. OFAGE resolves the smaller chromosomes better, while FIGE, which under our conditions causes the chromosomes to run in the reverse order of OFAGE, is more effective in separating the larger chromosomes. CHEF separates all chromosomes under some conditions, but these conditions do not often resolve homologs. The strains examined are highly polymorphic for chromosome size. Fourteen cloned Candida genes, isolated on the basis of conferral of new properties to or complementation of auxotrophic deficiencies in Saccharomyces cerevisiae, and three sequences of unknown function have been hybridized to Southern transfers of CHEF, FIGE, and OFAGE gels. Four sets of resolvable bands have been shown to be homologous chromosomes. On the basis of these data, we suggest that C. albicans has seven chromosomes. Genes have been assigned to the seven chromosomes. Two chromosomes identified genetically have been located on the electrophoretic karyotype.

INTRODUCTIONPhenotypic variability involving morphology, synthesis of secondary metabo lites, or virulence is not uncommon in filamentous fungi. Efforts to explain this phenomenon have stimulated interest in extrachromal genetic elements such as mycoviruses, plasmids, or transposons as modulators of fungal gene expression. DNA plasmids are not prevalent in filamentous fungi, and the incidence of transposable elements has only recently been reported (62). In contrast, mycoviruses and related double-stranded (ds) RNA genetic elements are found associated with fungi at a very high frequency. Consequently, the . contribution of these genetic elements to phenotypic variability has come under increased scrutiny.The first fungal viruses were isolated from mushrooms in 1962 (57), followed six years later by the discovery of viruses in Penicilliumfuniculosum