We report the isolation of a cDNA (KAT1)

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related cochaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well de®ned, and so we examined in vivo the in¯uences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding af®nity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.

Inward-rectifying potassium channels located in the plasma membrane of higher plant and animal cells contribute to cellular homeostasis and excitability. The genes encoding this specific class of K+ channels have not been functionally identified. This report shows that a single messenger RNA transcript from the Arabidopsis thaliana KAT1 complementary DNA confers the functional expression of a hyperpolarization-activated K+ channel in Xenopus oocytes. The channels encoded by KAT1 are highly selective for K+ over other monovalent cations, are blocked by tetraethylammonium and barium, and have a single channel conductance of 28 +/- 7 picosiemens with 118 millimolar K+ in the bathing solution. These functional characteristics, typical of inward-rectifying K+ channels in eukaryotic cells, demonstrate that KAT1 encodes an inward-rectifying K+ channel.

In Saccharomyces cerevisiae, TRKI and TRK2 encode the high-and low-affinity K+ transporters, respectively. In cells containing a deletion of TRKI, transcription levels of TRK2 are extremely low and are limiting for growth in media containing low levels of K+ (Trk-phenotype). Recessive mutations in RPDI and RPD3 suppress the Trk-phenotype of tr*IA cells. We show here that 7pd3 mutations derepress TRK2, conferring an approximately fourfold increase in transcription. rpd3 mutations confer pleiotropic phenotypes, induding (i) mating defects, (ii) hypersensitivity to cycloheximide, (ill) inability to sporulate as homozygous diploids, and (iv) constitutive derepression of acid phosphatase. RPD3 was cloned and is predicted to encode a 48-kDa protein with no extensive imilarity to proteins contained in current data bases. Deletion of RPD3 is not lethal but confers phenotypes identical to those caused by spontaneous mutations. RPD3 is required for both full repression and fufl activation of transcription of target genes including PHOS, STE6, and TY2. RPD3 is the second gene required for this function, since RPDI is also required. The effects of mutations in RPDI and RPD3 are not additive, suggesting that these genes are involved in the same transcriptional regulatory function or pathway.Genetic selections and screenings for yeast mutants that exhibit increased expression of a structural gene have proven successful in the identification of transcription factors. The selection schemes described thus far have used as model systems structural genes required for growth in defined media. In the starting wild-type strain, transcription of the structural gene is limiting for growth and therefore mutations that increase its transcription can be selected.Some of these mutational studies have identified genes that encode proteins whose role in transcription is known or readily determined. Genetic mapping of the sitl and sit2 mutations demonstrated that they reside in the genes that encode the two largest subunits of RNA polymerase II (1). SPT15 (8, 47) was shown to encode TATA-binding factor TFIID (7, 13). SPTIJ and SPT12 (8) are allelic to structural genes HTAI and HTBI, which encode histone proteins H2A and H2B (6,15,29). It was recently shown that SPT2 (31) is allelic to SIN) (30,36), whose sequence reveals a protein with significant similarity to nonhistone chromatin component HMG1 (2, 12). Finally, GALI1 (SPT13) (9, 28, 39) has recently been shown to function by establishing or maintaining phosphorylation of transactivator GAL4 (24). The roles of a large number of other genes, identified through similar genetic selections, remain to be determined and are likely to define new functions required for transcriptional regulation.We have used as a model system transcription of TRK2, a yeast gene that encodes the low-affinity K+ transporter. In cells deleted for TRKI, the high-affinity K+ transporter gene, expression of TRK2 is limiting for growth on media containing low levels of K+ (Trk-phenotype; 11). We have isolated recessive mu...

We identified a 180-kilodalton plasma membrane protein in Saccharomyces cerevisiae required for high-affinity transport (uptake) of potassium. The gene that encodes this putative potassium transporter (TRK1) was cloned by its ability to relieve the potassium transport defect in trk1 cells. TRK1 encodes a protein 1,235 amino acids long that contains 12 potential membrane-spanning domains. Our results demonstrate the physical and functional independence of the yeast potassium and proton transport systems. TRK1 is nonessential in S. cerevisiae and maps to a locus unlinked to PMA1, the gene that encodes the plasma membrane ATPase. Haploid cells that contain a null allele of TRK1 (trk1 delta) rely on a low-affinity transporter for potassium uptake and, under certain conditions, exhibit energy-dependent loss of potassium, directly exposing the activity of a transporter responsible for the efflux of this ion.