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Kat1 is a highly selective inward-rectifying K(+) channel that opens for extended periods under conditions of extreme hyperpolarization. Over 200 point mutants in the pore region of the Kat1 K(+) channel were generated and examined in the yeast Saccharomyces cerevisiae and Xenopus oocytes to assess the effect of the mutations on ion selectivity. Su...
The Saccharomyces cerevisiae integral membrane protein Ssy1p functions with Ssy5p and Ptr3p to sense extracellular amino acids. Signal transduction leads to processing and nuclear localization of Stp1p and Stp2p, transcriptional activators of many amino acid transporter genes. Ssy1p is structurally related to amino acid permeases, but unable to tra...
Heat-shock response is a general mechanism through which cells cope with external, as well as internal, stresses. Although the heat-shock response is elicited under conditions of stress, proteins involved in this set of pathways are also necessary for basic maintenance of cellular constituents, which involves regulation of proper levels, folded sta...
Recent studies of Saccharomyces cerevisiae revealed sensors that detect extracellular amino acids (Ssy1p) or glucose (Snf3p and Rgt2p) and are evolutionarily related to the transporters of these nutrients. An intriguing question is whether the evolutionary transformation of transporters into nontransporting sensors reflects a homeostatic capability...
Amino acids in the environment of Saccharomyces cerevisiae can transcriptionally activate a third of the amino acid permease genes through a signal that originates from the interaction between the extracellular amino acids and an integral plasma membrane protein, Ssy1p. Two plasma membrane-associated proteins, Ptr3p and Ssy5p, participate in the se...
Saccharomyces cerevisiae senses extracellular amino acids using two members of the family of amino acid transporters, Gap1 or Ssy1; aspects of the latter are reviewed here. Despite resemblance with bona fide transporters, Ssy1 appears unable to facilitate transport. Exposure of yeast to amino acids results in Ssy1-dependent transcriptional inductio...
Saccharomyces cerevisiae senses extracellular amino acids using two members of the family of amino acid transporters, Gap1 or Ssy1; aspects of the latter are reviewed here. Despite resemblance with bona fide transporters, Ssy1 appears unable to facilitate transport. Exposure of yeast to amino acids results in Ssy1-dependent transcriptional inductio...
Sensing of extracellular amino acids results in transcriptional induction of amino acid permease genes in yeast. Ssy1, a membrane
protein resembling amino acid permeases, is required for signaling but is apparently unable to transport amino acids and is
thus believed to be a sensor. By using a novel genetic screen in which potassium uptake was made...
Hsp90 complexes contain a class of co-chaperones characterized by a tetratricopeptide repeat (TPR) domain, which mediates binding to a carboxyl-terminal EEVD region in Hsp90. Among Hsp90 TPR co-chaperones in Saccharomyces cerevisiae, only Cns1 is essential. The amino terminus of Cns1, which harbors the TPR domain, is sufficient for viability when o...
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 co-chaperones: 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...
In recent years major progress has been achieved in the understanding of transport processes in higher plants. The boom in
the field of molecular plant physiology led to the identification and characterization of membrane transporters with transport
activities for potassium, calcium, sugars, nitrate, ammonium, sulphate, phosphate, amino acids, pept...
In recent years major progress has been achieved in the understanding of transport processes in higher plants. The boom in the field of molecular plant physiology led to the identification and characterization of membrane transporters with transport activities for potassium, calcium, sugars, nitrate, ammonium, sulphate, phosphate, amino acids, pept...
Ionic currents related to the major potassium uptake systems in Saccharomyces cerevisiae were examined by whole cell patch-clamping, under K+ replete conditions. Those currents have the following properties. They (1) are inward under all conditions investigated, (2) arise instantaneously with appropriate voltage steps, (3) depend solely upon the mo...
Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery.
Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including
glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60v-src. In a...
The heat shock response is a highly conserved mechanism that allows cells to withstand a variety of stress conditions. Activation
of this response is characterized by increased synthesis of heat shock proteins (HSPs), which protect cellular proteins from
stress-induced denaturation. Heat shock transcription factors (HSFs) are required for increased...
CyP-40 cyclophilins are found in association with molecular chaperone Hsp90.steroid receptor complexes. The amino-terminal portion of these cyclophilins harbors the characteristic peptidyl-prolyl isomerase (PPIase) domain, whereas three copies of the tetratricopeptide (TPR) motif, a structure shown to be involved in protein-protein interactions, an...
Deletion of TRK1 and TRK2 abolishes high-affinity K+ uptake in Saccharomyces cerevisiae, resulting in the inability to grow on typical synthetic growth medium unless it is supplemented with very high concentrations of potassium. Selection for spontaneous suppressors that restored growth of trk1delta trk2delta cells on K+-limiting medium led to the...
A number of recent ion channel investigations have involved heterologous expression in Saccharomyces cerevisiae. With this system, the advantages of yeast genetic analysis are now applicable toward the study of ion channels. In addition, the microbial aspect of the system allows experiments to be designed on a scale much larger than can be accommod...
Deletion of the potassium transporter genes TRK1 and TRK2 impairs potassium uptake in Saccharomyces cerevisiae, resulting in a greatly increased requirement for the ion and the inability to grow on low pH medium. Selection for mutations that restored growth of trk1Delta trk2Delta cells on low pH (3.0) medium led to the isolation of a dominant suppr...
Among the highly conserved sites in K+ channel pores, the tyrosine-glycine sequence is believed to play an important role in selectivity. Here we describe a novel approach in which comprehensive mutagenesis of the YG sites of the voltage-gated K+ channel, Kat1, is combined with phenotypic screening in Saccharomyces cerevisiae and electrophysiologic...
We show that cells deleted for SNF3, HXT1, HXT2, HXT3, HXT4, HXT6, and HXT7 do not take up glucose and cannot grow on media containing glucose as a sole carbon source. The expression of Hxt1, Hxt2, Hxt3, Hxt6, or Gal2 in these cells resulted in glucose transport and allowed growth on glucose media. In contrast, the expression of Snf3 failed to conf...
Selection for the ability of Saccharomyces cerevisiae cells to take up histidinol, the biosynthetic precursor to histidine, results in dominant mutations at HOL1. The DNA sequence of HOL1 was determined, and it predicts a 65-kDa protein related to the major facilitator family (drug resistance subfamily) of putative transport proteins. Two classes o...
Cpr6 and Cpr7, the Saccharomyces cerevisiae homologs of cyclophilin-40 (CyP-40), were shown to form complexes with Hsp90, a protein chaperone that functions in several
signal transduction pathways. Deletion of CPR7 caused severe growth defects when combined with mutations that decrease the amount of Hsp90 or Sti1, another component of
the Hsp90 cha...
We report the analysis of two Saccharomyces cerevisiae cyclophilins, Cpr6 and Cpr7, identified by their ability to interact in vivo with the transcriptional regulator Rpd3. Both cyclophilins have an extended carboxy-terminal region containing a three-unit tetratricopeptide repeat (TPR) motif and share significant amino acid identity with the mammal...
K+ channels play diverse roles in mediating K+ transport and in modulating the membrane potential in higher plant cells during growth and development. Some of the diversity in K+ channel functions may arise from the regulated expression of multiple genes encoding different K+ channel polypeptides. Here we report the isolation of a novel Arabidopsis...
The Arabidopsis thaliana KAT1 cDNA encodes a voltage-gated inward-rectifying K+ channel. A KAT1 genomic DNA clone was isolated and sequenced, and a 5' promoter and coding sequences containing eight introns were identified. Reporter gene analysis of transgenic plants containing the KAT1 promoter fused to bacterial beta-glucuronidase showed robust be...
The regulatory step Start in the cell cycle of the budding yeast Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal). To study this inhibition, mutations were identified that alter the sensitivity of yeast cells to Nal. Nal-sensitive mutations were sought because the inhibitory effects of Nal on wild-type cells are only transient, and wil...
Transport-deficient strains of the yeast Saccharomyces cerevisiae have recently proven useful for cloning, by functional complementation, of cDNAs encoding heterologous membrane transporters: specifically, H(+)-amino acid symporters and K+ channels from the higher plant Arabidopsis thaliana. The present study uses whole-cell patch-clamp experiments...
The TRK2 gene in Saccharomyces cerevisiae encodes a membrane protein involved in potassium transport and is expressed at extremely low levels. Dominant cis-acting mutations (TRK2D), selected by their ability to confer TRK2-dependent growth on low-potassium medium, identified an upstream repressor element (URS1-TRK2) in the TRK2 promoter. The URS1-T...
Two suppressors of the growth deficiency of a potassium transport mutant of Saccharomyces cerevisiae were isolated from a mouse cDNA expression library. These suppressors, SKD1 and SKD2 (suppressor of K+ transport growth defect), were cDNAs encoding members of a family of ATPases involved in membrane fusion (N-ethylmaleimide-sensitive fusion protei...
Disruption of genes encoding endogenous transport proteins in Saccharomyces cerevisiae has facilitated the recent cloning, by functional expression, of cDNAs encoding K+ channels and amino acid transporters from the plant Arabidopsis thaliana [1-4]. In the present study, we demonstrate in whole-cell patch clamp experiments that the inability of trk...
Selectable markers integrated by the 'gamma' deletion method (Sikorski and Hieter, 1989) can be efficiently replaced in vivo with other markers by transformation with homologous plasmids. Transformation frequencies in experiments designed to replace original selectable markers with an alternate marker were high and molecular analysis confirmed that...
The ability to express heterologous proteins in K+ uptake-defective strains of Saccharomyces cerevisiae can be exploited to identify cDNAs encoding heterologous K+ channels. Moreover, the ability of heterologous potassium channels like KAT1 and AKT1 to suppress completely the conditional negative growth phenotype of S. cerevisiae cells containing m...
In Saccharomyces cerevisiae, TRK1 and TRK2 are required for high- and low-affinity K+ transport. Among suppressors of the K+ transport defect in trk1 delta trk2 delta cells, we have identified members of the sugar transporter gene superfamily. One suppressor encodes the previously identified glucose transporter HXT1, and another encodes a new membe...
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 complementa...
In Saccharomyces cerevisiae, TRK1 and TRK2 are required for high- and low-affinity K+ transport. Among suppressors of the K+ transport defect in trk1 delta trk2 delta cells, we have identified members of the sugar transporter gene superfamily. One suppressor encodes the previously identified glucose transporter HXT1, and another encodes a new membe...
We report the isolation of a cDNA (KAT1) from Arabidopsis thaliana that encodes a probable K+ channel. KAT1 was cloned by its ability to suppress a K+ transport-defective phenotype in mutant Saccharomyces cerevisiae cells. This suppression is sensitive to known K+ channel blockers, including tetraethylammonium and Ba2+ ions. The KAT1 cDNA contains...
Two Saccharomyces cerevisiae genes previously unknown to be required for DNA synthesis have been identified by screening a collection of temperature-sensitive mutants. The effects of mutations in DNA43 and DNA52 on the rate of S phase DNA synthesis were detected by monitoring DNA synthesis in synchronous populations that were obtained by isopycnic...
This chapter reviews the molecular genetics of yeast ion transport. The H+-ATPase is the principal electrogenic ion pump in the plasma membranes of fungi. The electrochemical energy of this gradient drives the import of nutrients and other ions. Besides providing the energy for nutrient uptake, the proton pump also controls the intracellular pH. Th...
We show that the extent of transcriptional regulation of many, apparently unrelated, genes in Saccharomyces cerevisiae is
dependent on RPD1 (and RPD3 [M. Vidal and R. F. Gaber, Mol. Cell. Biol. 11:6317-6327, 1991]). Genes regulated by stimuli as
diverse as external signals (PHO5), cell differentiation processes (SPO11 and SPO13), cell type (RME1, F...
In Saccharomyces cerevisiae, TRK1 and TRK2 encode the high- and low-affinity K+ transporters, respectively. In cells containing
a deletion of TRK1, transcription levels of TRK2 are extremely low and are limiting for growth in media containing low levels
of K+ (Trk- phenotype). Recessive mutations in RPD1 and RPD3 suppress the TRK2, conferring an ap...
We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces
cerevisiae. TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with
a large hydrophilic region between M3 and M4. These structural features closely resemble thos...
We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces cerevisiae. TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with a large hydrophilic region between M3 and M4. These structural features closely resemble thos...
In Saccharomyces cerevisiae, high-affinity K+ uptake is dependent upon a 180-kDa plasma membrane protein encoded by TRK1 (c-TRK1) [Gaber et al., Mol. Cell. Biol. 8 (1988) 2848-2859)]. Although hybridization with a c-TRK1 probe revealed highly homologous sequences in the genomes of most Saccharomyces species, the TRK1 sequence in S. uvarum (u-TRK1)...
TRK1, the gene encoding the high affinity K+ transporter in Saccharomyces cerevisiae, is nonessential due to the existence of a functionally independent low affinity transporter. To identify the gene(s) encoding the low affinity K+ transporter, we screened trk1 delta cells for mutants (Kla-) that require higher concentrations of K+ in the medium to...
Saccharomyces cerevisiae cells containing a deletion of TRK1, the gene encoding the high affinity potassium transporter, retain only low affinity uptake of this ion and consequently lose the ability to grow in media containing low levels (0.2 mM) of potassium. Using a trk1 delta strain, we selected spontaneous Trk+ pseudorevertants that regained th...
Saccharomyces cerevisiae histidine auxotrophs are unable to use L-histidinol as a source of histidine even when they have
a functional histidinol dehydrogenase. Mutations in the hol1 gene permit growth of His- cells on histidinol by enhancing the
ability of cells to take up histidinol from the medium. Second-site mutations linked to HOL1-1 further...
Saccharomyces cerevisiae histidine auxotrophs are unable to use L-histidinol as a source of histidine even when they have a functional histidinol dehydrogenase. Mutations in the hol1 gene permit growth of His- cells on histidinol by enhancing the ability of cells to take up histidinol from the medium. Second-site mutations linked to HOL1-1 further...
In Saccharomyces cerevisiae, methylation of the principal membrane sterol at C-24 produces the C-28 methyl group specific to ergosterol and represents one of the few structural differences between ergosterol and cholesterol. C-28 in S. cerevisiae has been suggested to be essential for the sparking function (W. J. Pinto and W. R. Nes, J. Biol. Chem....
In Saccharomyces cerevisiae, methylation of the principal membrane sterol at C-24 produces the C-28 methyl group specific to ergosterol and represents one of the few structural differences between ergosterol and cholesterol. C-28 in S. cerevisiae has been suggested to be essential for the sparking function (W. J. Pinto and W. R. Nes, J. Biol. Chem....
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...
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...
A genetic approach has been used to establish the molecular basis of 4-base codon recognition by frameshift suppressor tRNA
containing an extra nucleotide in the anticodon. We have isolated all possible base substitution mutations at the position
4 (N) in the 3'-CCCN-5' anticodon of a Saccharomyces cerevisiae frameshift suppressor glycine tRNA enco...
A genetic approach has been used to establish the molecular basis of 4-base codon recognition by frameshift suppressor tRNA containing an extra nucleotide in the anticodon. We have isolated all possible base substitution mutations at the position 4 (N) in the 3'-CCCN-5' anticodon of a Saccharomyces cerevisiae frameshift suppressor glycine tRNA enco...
Five previously unmapped frameshift suppressor genes have been located on the yeast genetic map. In addition, we have further characterized the map positions of two suppressors whose approximate locations were determined in an earlier study. These results represent the completion of genetic mapping studies on all 25 of the known frameshift suppress...
Two classes of frameshift suppressors distributed at 22 different loci were identified in previous studies in the yeast Saccharomyces cerevisiae. These suppressors exhibited allele-specific suppression of +1 G:C insertion mutations in either glycine or proline codons, designated as group II and group III frameshift mutations, respectively. Genes co...
The SUF16 frameshift suppressor locus encodes a glycine tRNA. The SUF16-1 suppressor tRNA is inferred by DNA sequence analysis to contain the four-base anticodon sequence 3'-CCCG-5' in place of the wild-type anticodon 3'-CCG-5'. SUF16-1 mediates translation of the four-base messenger RNA (mRNA) sequence 5'-GGGU-3' but apparently fails to act at the...
ICR-induced frameshift mutations at the his4 locus in Saccharomyces cerevisiae have been classified into several groups on the basis of their reversion and suppression properties. One group of externally suppressible his4 mutations, designated Group II, have been shown to contain +1 G:C insertions in glycine codons and are suppressed by any one of...
The recent proliferation of studies on moveable genetic elements has
suggested that genome rearrangement can contribute to the underlying
mechanisms involved in cell differentiation1. Evidence to
support this notion is rapidly accumulating in both prokaryotic and
eukaryotic organisms2-5. So far, however, relatively few
eukaryotes have lent themselv...
Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. Previous evidence indicated that suppressors of one group (Group II: SUF1, SUF3, SUF4, SUF5 and SUF6) represent mutations in the s...
The recessive gene, mound (mnd), allows the appearance of globose masses of compacted hyphae. Dikaryons of Schizophyllum commune that are heteroallelic for mnd [(mosaic dikaryons: (mnd + mnd(+))] have been successfully dedikaryotized in cholate-containing medium in order to recover the component nuclear types directly. The relative proportion of th...
A series of hemi-compatible dikaryon x monokaryon (di-mon) matings was designed to determine whether there was any genetic interaction between the dikaryotic nuclei. One of the nuclei in each dikaryon was known to carry a recessive gene (mnd) that promoted the development of an abnormal growth form, mound. Dikaryons containing both mnd( +) and mnd...
