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...
Calcium and other alkaline earth cations change the electrostatic potential adjacent to negatively charged bilayer membranes both by accumulating in the aqueous diffuse double layer adjacent to the membrane and by adsorbing to the phospholipids. The effects of these cations on the electrostatic potential are described adequately by the Gouy-Chapman-Stern theory. We report the results of experiments with ethane-bis-trimethylammonium, a cation that has been termed "dimethonium" or "ethamethonium" in analogy with hexamethonium (hexane-1,6-bis-trimethylammonium) and decamethonium (decane-1,10-bis-trimethylammonium). We examined the effect of dimethonium on the zeta potential of multilamellar vesicles formed from the negative lipid phosphatidylserine (PS) and from 5:1 phosphatidylcholine/phosphatidylserine mixtures in solutions containing 0.1, 0.01 and 0.001 M sodium, cesium, or tetramethylammonium chloride. We also examined the effect of dimethonium on the conductance of planar PS bilayer membranes and the 31P NMR signal from sonicated PS vesicles formed in 0.1 M NaCl. We found no evidence that dimethonium adsorbs specifically to bilayer membranes. All the results, except for those obtained with vesicles of low charge density formed in a solution with a high salt concentration, are consistent with the predictions of the Gouy-Chapman theory. We conclude that dimethonium, which does not have the pharmacological effects of hexamethonium and decamethonium, is a useful divalent cation for physiologists interested in investigating electrostatic potentials adjacent to biological membranes.
Topoisomerase inhibitors comprise an important group of agents that is used in cancer treatment. Because the development of resistance to cancer chemotherapeutic agents represents a major limitation of cancer chemotherapy, we investigated the mechanism of resistance by murine P388 leukemia to camptothecin (topoisomerase I inhibitor) or amsacrine (topoisomerase II inhibitor). The resistant cells contained reduced levels of topoisomerase activity and messenger RNA. The topoisomerase gene of these cells was rearranged (only in one allele) and hypermethylated. These topoisomerase gene alterations probably resulted in reduced transcription and, thus, enzyme production, which was correlated with resistance to the topoisomerase inhibitor.
, 1988) were expressed in Saccharomyces cerevisiae under the control of the inducible GAL] promoter. As determined by indirect immunofluorescence, T7 RNA polymerase lacking the NLS remained mostly in the cytoplasm, whereas the protein containing the NLS localized to the nucleus. T7 RNA polymerase containing a mutated NLS remained mostly cytoplasmic. Hybrid proteins containing the NLS near the amino terminus were enzymatically active in the yeast cell, initiating transcription selectively at a T7 promoter placed in yeast chromosomal or plasmid DNA and stopping at a specific T7 terminator. At limiting enzyme concentrations, 5 to 10 times as much target RNA was produced when the polymerase contained the NLS, presumably because more enzyme reached the nucleus. Although substantial amounts of intact mRNA accumulated, no translation of target mRNAs in yeast cells was detected.The role of specific amino acid sequences in targeting proteins to the cell nucleus has recently been demonstrated both in higher eucaryotes (7,15,16,19; for reviews, see references 6, 28, and 32) and in Saccharomyces cerevisiae (12,25,26,33,34; for a review, see reference 32). These sequences have been termed nuclear localization signals (NLS). Although an absolute consensus NLS has not yet been defined, a number of NLS have been studied in some detail. The NLS most widely studied is a seven-amino-acid oligopeptide (Pro-Lys-Lys-128-Lys-Arg-Lys-Val) (the position of the second lysine in the simian virus 40 [SV40] T antigen is denoted) that is both necessary and sufficient to direct SV40 large T antigen to the nucleus in mammalian cells. This oligopeptide is able to direct a number of otherwise cytoplasmic proteins to the nucleus when fused to these proteins either by genetic engineering followed by expression in vivo (15, 26) or by chemical cross-linking in vitro (11,20,27). Mutation of the second lysine (Lys-128) of this sequence to asparagine or threonine was reported to render this signal nonfunctional (16,19).Bacteriophage T7 gene 1 encodes an active, single-chain RNA polymerase of approximately 100 kilodaltons (kDa) (2). T7 RNA polymerase is highly selective for a promoter sequence which is long enough that it is unlikely to be found in DNA unrelated to T7 DNA (9). It has previously been shown that T7 RNA polymerase can direct transcription from a T7 promoter in yeast nuclear DNA (3). Relatively high levels of T7 RNA polymerase were apparently needed (1 to 4% of total cell protein), and the enzyme was thought to enter the nucleus passively.The subcellular location of T7 RNA polymerase has been examined in monkey kidney (Vero) cells by using microinjection and transient expression in conjunction with immunofluorescence (8). Natural T7 RNA polymerase or derivatives that lacked an NLS remained largely cytoplasmic, but * Corresponding author.derivatives that contained the NLS of SV40 T antigen localized to the nucleus. As assayed in vitro, derivatives of T7 RNA polymerase that contained the SV40 NLS near the amino terminus were enzymatically a...
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