FK506 and rapamycin are related immunosuppressive compounds that block helper T cell activation by interfering with signal transduction. In vitro, both drugs bind and inhibit the FK506-binding protein (FKBP) proline rotamase. Saccharomyces cerevisiae cells treated with rapamycin irreversibly arrested in the G1 phase of the cell cycle. An FKBP-rapamycin complex is concluded to be the toxic agent because (i) strains that lack FKBP proline rotamase, encoded by FPR1, were viable and fully resistant to rapamycin and (ii) FK506 antagonized rapamycin toxicity in vivo. Mutations that conferred rapamycin resistance altered conserved residues in FKBP that are critical for drug binding. Two genes other than FPR1, named TOR1 and TOR2, that participate in rapamycin toxicity were identified. Nonallelic noncomplementation between FPR1, TOR1, and TOR2 alleles suggests that the products of these genes may interact as subunits of a protein complex. Such a complex may mediate nuclear entry of signals required for progression through the cell cycle.

No abstract

Phosphinothricin (PPT) is a potent inhibitor of glutamine synthetase in plants and is used as a non‐selective herbicide. The bar gene which confers resistance in Streptomyces hygroscopicus to bialaphos, a tripeptide containing PPT, encodes a phosphinothricin acetyltransferase (PAT) (see accompanying paper). The bar gene was placed under control of the 35S promoter of the cauliflower mosaic virus and transferred to plant cells using Agrobacterium‐mediated transformation. PAT was used as a selectable marker in protoplast co‐cultivation. The chimeric bar gene was expressed in tobacco, potato and tomato plants. Transgenic plants showed complete resistance towards high doses of the commercial formulations of phosphinothricin and bialaphos. These data present a successful approach to obtain herbicide‐resistant plants by detoxification of the herbicide.

SummaryWith renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.

A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide‐resistant plants.

Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology.

FK 506 and cyclosporin A are potent immunosuppressive compounds that inhibit T-cell activation by interfering with signal transduction. In vitro, FK 506 binds and inhibits the activity of FK 506-binding protein (FKBP), a peptidylprolyl rotamase (cis-rans isomerase). Cyclosporin A acts similarly on a different proline rotamase, cyclophilin. Experiments described here demonstrate genetically that FKBP is a target for FK 506 in vivo. We have isolated the gene encoding the FKBP proline rotamase (FPRI) from Saccharomyces cerevisiae. The encoded yeast protein is highly homologous with bovine and human FKBP and shares no homology with cyclophilin. Disruption of FPRI and CPRI (encoding cyclophilin) individually or in combination is not lethal; thus, either enzymatic proline rotamerization is not essential for life or an unknown proline rotamase can substitute for the missing enzymes. Overexpression or disruption of FPRI confers resistance to growth inhibition by FK 506, suggesting that FKBP is a target for FK 506 in yeast. However, FKBP is only one of at least two targets because strains lacking FKBP are only partially resistant to FK 506. FK 506, a macrolide antibiotic produced by Streptomyces tsukubaensis (1, 2), is a potent immunosuppressive compound (3-5) that inhibits graft rejection after organ transplantation (6-8). Human clinical trials have indicated that FK 506 is effective against graft rejection at lower doses and with a different spectrum of side effects compared with the widely prescribed immunosuppresive drug cyclosporin A (CsA) (9, 10). In addition, both FK 506 and CsA have potential therapeutic indications in the treatment of immune and autoimmune disorders (12, 13).Although structurally unrelated, FK 506 and CsA appear to suppress the human immune system by similar means. In human T cells, antigen presentation to the T-cell receptor triggers a signal transduction cascade resulting in increased gene expression and T-cell activation (14, 15). FK 506 and CsA do not inhibit the first steps in this pathway (inositol 1,4,5-trisphosphate/diacylgylcerol production or Ca2" release from the endoplasmic reticulum) (5, 15-17) but rather block a subsequent step required for synthesis or activation of the transcription factors (NF-AT, AP-3, and NF-KB), which promote expression of lymphokine genes (such as interleukin 2) (14,(18)(19)(20)(21) (27). These findings suggest that proline rotamases play a role in T-cell signal transduction, perhaps by some posttranslational regulatory mechanism involving folding of key substrates. Consistent with this, a component of the Drosophila visual transduction system is a cyclophilin homolog (28, 29). Because cyclophilin and FKBP differ in substrate specificity in vitro (30), each may fold different proteins in vivo, only some of which need participate in signal transduction.To determine the role of proline rotamases in cellular physiology and to define a possible intracellular target of FK 506, we have taken a genetic approach with the unicellular eukaryotic yeast, Sacchar...

Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)‐anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide‐containing, mild base‐resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI‐anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI‐anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI‐anchored proteins.