LEM 1 , an ATP - binding - cassette transporter , selectively modulates the biological potency of steroid hormones ( glucocorticoid receptor / yeast / PDR 5 / STSI ) ANASTASIA KRALLI , SEAN

The rat glucocorticoid receptor confers hormone-dependent transcriptional enhancement when expressed in yeast, thereby enabling the genetic identification of nonreceptor proteins that function in the hormone signaltransduction pathway. We isolated a yeast mutant, leml, with increased sensitivity to dexamethasone and triamcinolone acetonide; responsiveness to a third agonist, deoxycorticosterone, is unaffected. Cloning of wild-type LEM1 revealed a putative transport protein of the ATP-binding cassette family. Dexamethasone accumulation is increased in leml cells, suggesting that wild-type LEM1 decreases dexamethasone potency by exporting this ligand. LEMI appears to affect certain steroids and not others. We propose that transporters like LEM1 can selectively modulate the intracellular levels of steroid hormones. Differential activities of such transporters in mammalian cells might regulate hormone availability and thereby hormone signaling in a cell-type specific manner. Glucocorticoids function by binding to the glucocorticoid receptor (GR), an intracellular protein that, once activated by hormone, binds to specific DNA sequences and regulates the transcription of adjacent genes. The protein machinery with which the receptor interacts in the course of signaling and transcriptional regulation has been evolutionarily conserved, as the rat GR can confer hormone-dependent transcriptional activation when expressed in cells from heterologous species as diverse as fungi, insects, and plants (1-4). Interestingly, the biological efficacies and potencies of certain steroid ligands differ when mammalian GR is expressed in different species. Two strong agonists in mammalian cells, dexamethasone (dex) and triamcinolone acetonide (TA), are very weak agonists in Saccharomyces cerevisiae (5); in contrast, agonists like deacylcortivazol and RU28362 efficiently activate GR in all species tested. Thus, dex and TA are more potent GR agonists than deoxycorticosterone (DOC) in mammalian cells, whereas DOC is more potent than dex and TA in yeast. Similarly, dex-mesylate, an antagonist in mammalian cells, is a strong agonist in cultured Drosophila melanogaster cells (3). Clearly, cellular factors other than the receptor must serve as determinants of ligand responsiveness by GR. By this view, speciesor cell-type-specific differences in these factors would produce the distinct ligand response characteristics of mammalian, Drosophila, and S. cerevisiae cells. Factors that modulate ligand effects might interact either with GR or with the ligand itself. GR-interacting factors could affect ligand binding or alter the activity of the hormonebound receptor; factors that interact with the ligand could modify it, sequester it, or affect its intracellular accumulation. Ligand accumulation could be affected by proteins that alter its flux across the cell membrane. Steroids are small hydrophobic molecules commonly assumed to diffuse through the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. membrane. However, steroid transport may be an active process. Corticosterone uptake by isolated rat liver membrane vesicles is saturable and produces elevated intravesicular hormone levels relative to the medium (6). In mouse L929 fibroblasts, cortisol and dex are exported in a saturable, energydependent, temperature-sensitive process (7, 8). Thus, in a given cell type, accumulation of a specific ligand may depend on the nature and activities of selective transporters. The ability of GR to function in yeast provides an opportunity to genetically identify nonreceptor factors in the glucocorticoid signal-transduction pathway. To search for proteins that selectively modulate the effect of particular steroid ligands (here termed LEM for ligand effect modulator), we screened for yeast mutants that increase GR responsiveness to TA and dex without altering responsiveness to DOC. MATERIALS AND METHODS Plasmids. Plasmids pGlN795, pGlF620S, pTCA/N795, and pLCA/N795 express wild-type (N795) or a point mutant (F620S) GR, from the constitutive yeast GPD promoter (1, 5). Plasmids pH2/ER (9), pH2/MR (9), and YEphPR-B (10) express respectively, the estrogen receptor (ER), the mineralocorticoid receptor (MR), and the progesterone receptor (PR). Reporter plasmids pAs26x (1) and pUCAsERE (11) contain three glucocorticoid/progesterone response elements and one estrogen response element, respectively, upstream of a minimal yeast CYC1 promoter driving the Escherichia coli lacZ gene. Plasmid pTCA/LEM1 carries the LEM1 gene with 1200 bp of 5' and 400 bp of 3' sequences inserted into the polylinker of pRS314 (12). In pTCA/leml::LEU2, the LEM1 sequences from -360 bp to +3950 bp relative to the translation initiation site were replaced by the LEU2 gene. Yeast Strains and Genetic Methods. We used the following yeast strains: BJG26.1 contains a GR-responsive ,3-galactosidase (1-gal) reporter gene (5); YPH252 (a, LEM1) (12); YNK100 (a, leml-1), derived by crossing the original m17 isolate twice to YPH252. The LEM1 locus was disrupted in the haploid YPH252 and YPH250 and the diploid YPH274 (12) strains by the one-step replacement method (13) and by using the DNA insert of the pTCA/leml ::LEU2 plasmid. Mutagenesis and Genetic Screen. BJG26.1 cells containing pTCA/N795 were grown in 5 ml of selective medium at 30°C to OD600 = 0.7, harvested by centrifugation (5000 x g for 5 min), washed twice with phosphate-buffered saline (PBS; 137 mM NaCl/2.7 mM KCl/4.3 mM Na2HPO4/1.4 mM KH2PO4), resuspended in 1 ml of PBS, and incubated with 50 /l of ethyl methanesulfonate (EMS; Sigma) for 1 h at 25°C. The EMS was neutralized with five volumes of 5% sodium thiosulfate, and Abbrevations: GR, glucocorticoid receptor; ER, estrogen receptor; MR, mineralocorticoid receptor; PR, progesterone receptor; dex, dexamethasone; DOC, deoxycorticosterone; TA, triamcinolone acetonide; LEM, ligand effect modulator; ABC, ATP-binding cassette; Mdr, multidrug resistance; P-gal, 3-galactosidase.