Amino acid sensing by Ssy1.

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 induction of several genes, in particular some encoding amino acid transporters. Amino acids differ strongly in their potency, leucine being the most potent one known. Using a selection system in which potassium uptake was made dependent on amino acid signalling, our laboratory has obtained and described gain-of-function mutations in SSY1. Some alleles conferred inducer-independent signalling; others increased apparent affinity for inducers. These results revealed that amino acid transport is not required for signalling and support the notion that sensing by Ssy1 occurs via its direct interaction with extracellular amino acids. Current work includes development of quantitative assays of sensing. We use the finding by Per Ljungdahl's laboratory that the signal transduction from Ssy1 involves proteolytic removal of an inhibitory part of the transcriptional activator Stp1. Protein-A Z-domain fused to the C-terminus of Stp1 and Western analysis using antibody against horseradish peroxidase allow quantification of sensing.

Transcriptional regulation of the Saccharomyces cerevisiae amino acid permease gene BAP2.

Uptake of branched-chain amino acids by Saccharomyces cerevisiae from media containing a preferred nitrogen source is mediated by the permeases encoded by BAP2, BAP3, and VAP1/TAT1. The transcriptional activity of the BAP2 promoter is affected by a number of genes, including SSY1, which encodes an amino acid permease homologue that is necessary for transcription of BAP2. Other genes that control BAP2 encode known (Leu3p, Tup1p) and putative (Stp1p, Stp2p) transcription factors. We present evidence that the zinc-finger proteins Stp1p and Stp2p bind directly to the BAP2 promoter. Binding of Stplp to the BAP2 promoter in vivo and in vitro indicates that the STP gene family indeed encodes transcription factors. The presence of a Leu3p binding site in the BAP2 promoter is required for full promoter activity on synthetic complete medium. The capacity of Leu3p to activate BAP2 transcription correlates with conditions that affect the level of alpha-isopropyl malate. The effect of a tup1 deletion on BAP2 transcription depends on SSY1. In an ssy1 strain, the phenotype of tup1 conforms to the well-established role of Tup1p as part of a repressor complex, but in the SSY1 strain deletion of TUP1 causes a decrease in transcription, indicating that Tup1p may also have an activating role at the BAP2 promoter. Our results thus suggest a complex interplay between several transcription factors in the expression of BAP2.

Stp1p, Stp2p and Abf1p are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae.

Expression of the BAP3 gene of Saccharomyces cerevisiae, encoding a branched chain amino acid permease, is induced in response to the availability of several naturally occurring amino acids in the medium. This induction is mediated via an upstream activating sequence (called UAS(aa)) in the BAP3 promoter, and dependent on Stp1p, a nuclear protein with zinc finger domains, suggesting that Stp1p is a transcription factor involved in BAP3 expression. In this paper, we show that Stp2p, a protein with considerable similarity to Stp1p, is also involved in the induction of BAP3 expression. To gain more insight into the roles of STP1 and STP2, we have overexpressed both Stp1p and Stp2p in yeast cells. Gel shift assays with the UAS(aa)as a probe show that the UAS(aa)can form two major complexes. One complex is dependent on Stp2p overexpression and the other is formed independently of STP1 or STP2, suggesting that the UAS(aa)is also bound by another factor. Here we show that the other factor is Abf1p, which binds specifically to the UAS(aa)of BAP3.

Phenotypic characteristics associated with the acquisition of HSV-specific CD8 T-lymphocyte-mediated cytolytic function in vitro.

Class I MHC-restricted, HSV-1-specific CD8(+) cytolytic T lymphocyte (CTL) function is rarely detected in lymphocytes isolated directly from the lymph node draining the site of infection. However, culture in vitro for 24 to 72 h in the absence of exogenous antigen results in the development of easily detectable levels of HSV-1-specific CTL effectors. The inability to detect virus-specific CTL in HSV-1-infected mice is not well understood. However, since the in vitro culture of HSV-1-immune lymphocytes results in the transition to CTL function, studies of the changes occurring to the CD8(+) T cell subpopulation may provide important insights into the development of virus-specific CTL. Therefore, the phenotypic changes taking place in the CD8(+) population of T cells from draining popliteal lymph nodes of HSV-1-infected C57BL/6 (B6) mice were investigated, focusing on changes in the expression of cell surface markers associated with T lymphocyte activation. The results demonstrate an increase in the percentage of CD8(+) T cells expressing the activation markers CD44 and CD25 in parallel with the acquisition of HSV-specific CTL effector function. Cytolytic function was found exclusively within the CD8(+) CD44(hi) CD25(hi) fraction of cells in culture, but, surprisingly, was not detectable in CD8(+) CD44(hi) CD25(lo) T cells. This suggested that the acquisition of high levels of the high-affinity IL-2 receptor was closely linked to cytolytic function and may define an important developmental stage in the transition from noncytolytic to cytolytic effector cell. In support of this, CD8(+) CD25(hi) T cells isolated from the regional lymph node exhibited direct ex vivo cytolytic function, indicating that cytolytic effector cells were present in the lymph node, but must emigrate rapidly after attaining this level of differentiation.

The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae.

Amino acid transporters of the yeast plasma membrane (permeases) belong to a family of integral membrane proteins with pronounced structural similarity. We present evidence that a member of this family, encoded by the open reading frame (ORF) YDR160w (SSY1), is required for the expression of a set of transporter genes. Thus, deletion of the SSY1 gene causes loss of leucine-inducible transcription of the amino acid permease genes BAP2, TAT1 and BAP3 (ORF YDR046c) and the peptide transporter, PTR2. D-leucine can generate the signal without entering the cell. We propose that Ssy1p is situated in the plasma membrane and is involved in sensing leucine in the medium.

STP1, a gene involved in pre-tRNA processing in yeast, is important for amino-acid uptake and transcription of the permease gene BAP2.

The bap1 mutant of Saccharomyces cerevisiae was previously isolated by its reduced uptake of branched-chain amino acids. In the present study, the corresponding wild-type gene was cloned and partial sequencing and subsequent genetic analysis revealed identity to STP1, a gene involved in tRNA maturation. The decrease in amino-acid uptake caused by stp1 mutations is independent of GCN4. It was previously found that the BAP2 promoter can be activated by the presence of amino acids, notably leucine, in the medium. We found that this activation depends on STP1. As a simple hypothesis we propose that Stp1p is a transcription factor which activates BAP2, and probably other amino-acid permease genes.

Amino acids induce expression of BAP2, a branched-chain amino acid permease gene in Saccharomyces cerevisiae.

Branched-chain amino acid uptake in Saccharomyces cerevisiae is mediated by at least three transport systems: the general amino acid permease Gap1p, the branched-chain amino acid permease Bap2p, and one or more so far unknown permeases. Regulation of the transcription of BAP2 is mainly subject to the presence of certain amino acids in the medium. The level of transcription is low during growth on a minimal medium with proline as the sole nitrogen source. As assayed with a lacZ fusion, the level of transcription is slightly higher (3-fold) on a minimal medium with ammonium ions as a nitrogen source, and transcription is induced about 20-fold by addition of leucine (0.2 mM). As little as 10 microM leucine causes a fivefold induction. Addition of (L)-leucine to minimal proline medium, on the other hand, has no effect on BAP2 transcription. The two known permeases for transport of branched-chain amino acids, Gap1p and Bap2p, are thus not active at the same time. The BAP2 promoter contains one or two putative Gcn4p binding sites and one putative Leu3p binding site. None of the three is needed for induction by leucine. Induction of BAP2 transcription by leucine is accompanied by an increase in branched-chain amino acid uptake. This elevation is interpreted to be partly the result of an increased level of the Bap2p permease in the plasma membrane, because deletion of BAP2 slightly decreases the induction of uptake. There is still a leucine-inducible increase in branched-chain amino acid uptake in a delta gap1 delta bap2 strain, indicating that BAP2 shares leucine induction with at least one remaining branched-chain amino acid-transporting permease.

BAP2, a gene encoding a permease for branched-chain amino acids in Saccharomyces cerevisiae.

To select the gene coding for an isoleucine permease, an isoleucine dependent strain (ilv1 cha1) was transformed with a yeast genomic multicopy library, and colonies growing at a low isoleucine concentration were selected. Partial sequencing of the responsible plasmid insert revealed the presence of a previously sequenced 609 codon open reading frame of chromosome II with homology to known permeases. Deletion, extra dosage and C-terminal truncation of this gene were constructed in a strain lacking the general amino acid permease, and amino acid uptake was measured during growth in synthetic complete medium. The following observations prompted us to name the gene BAP2 (branched-chain amino acid permease). Deletion of BAP2 reduced uptake of leucine, isoleucine and valine by 25-50%, while the uptake of 8 other L-alpha-amino acids was unaltered or slightly increased. Introduction of BAP2 on a centromere-based vector, leading to a gene dosage of two or slightly more, caused a 50% increase in leucine uptake and a smaller increase for isoleucine and valine. However, when the 29 C-terminal codons of the plasmid-borne copy of BAP2 were substituted, the cells more than doubled the uptake of leucine, isoleucine and valine, while no or little increase in uptake was observed for the other 8 amino acids.

A peptide from Tetrahymena disrupts subunit organization of E. coli RNA polymerase.

Incubation of the E. coli RNA polymerase with a polypeptide factor from the protozoan Tetrahymena reduces the affinity of the holoenzyme for DNA. SDS-polyacrylamide gel electrophoresis of the peptide-treated RNA polymerase showed that the band pattern of the polymerase subunits was strongly altered. The three large subunits, beta', beta and sigma, disappear and a high number of rapidly migrating bands appeared. However, a brief heat treatment of the samples almost restored the original RNA polymerase subunit composition, and in addition a high molecular weight protein band approximately 240 kDa appeared. It is suggested that the Tetrahymena peptide specifically binds to the RNA polymerase and changes the structures of the large subunits.

Purification and partial characterization of a transcription-inhibitory peptide from Tetrahymena.

The protozoa Tetrahymena excretes a small peptide complex with an Mr of about 5,000. The peptide inhibits transcription by reducing the activity of the RNA polymerase. We have purified and partially characterized the peptide complex. It contains two peptide chains of apparent Mr 2,300 and 2,600, respectively. Magnesium ions in connection with the SH groups of cysteine play a role in holding together the two chains of the intact complex.

Peptides regulate the activity of RNA polymerases in Tetrahymena.

An exponentially multiplying population of the protozoa Tetrahymena has a highly variable transcription rate when cultivated in a complex broth medium. It is shown that peptides in the medium specifically stimulate transcription and that the cells in response synthesize a peptide with a molecular weight of 3000-4000. This peptide inhibits transcription in vivo in cells with high rates of transcription and in in vitro transcription systems. We have partially purified radioactively labelled inhibitor peptide and found that cells stimulated to high transcription rate selectively accumulate inhibitor peptide in the nuclei. In vitro experiments have shown that the inhibitor peptide can reduce the RNA polymerase activity in the nuclei. The presence of inhibitor seems to lower the affinity of RNA-polymerases towards DNA and to increase their release during incubation of isolated nuclei. On the other hand, stimulatory peptides will compete with the inhibitor, and the polymerase activity in isolated nuclei is determined--at least partially--by the ratio between stimulatory and inhibitory peptide factors. It is suggested that regulatory peptides also are involved in regulation of transcription in vivo.

Platelet-derived growth factor stimulates chemotaxis and nucleic acid synthesis in the protozoan Tetrahymena.

Platelet-derived growth factor (PDGF) is in concentrations of a few nanograms per ml a very active chemoattractant for the free-living ciliated protozoan Tetrahymena; at the same time it induces a rapid increase in incorporation of radioactive nucleic acid precursors into RNA and DNA. We find it remarkable that this lower eukaryote responds to platelet-derived growth factor in very much the same way as fibroblastic cells.

Inhibition of RNA synthesis in yeast protoplasts by a peptide factor from Tetrahymena cells.

Protoplasts of Schizosaccharomyces pombe, grown on a rich nutrient medium, were treated with a peptide factor isolated from cultures of the protozoan Tetrahymena pyriformis. The peptide factor is known to inhibit RNA synthesis in Tetrahymena. It has now been shown that the peptide factor also inhibits RNA synthesis in yeast protoplasts without affecting protein synthesis.

On the role of small peptides in the regulation of RNA synthesis in Tetrahymena pyriformis.

Tetrahymena cells secrete a factor which inhibits RNA synthesis in vivo and in vitro. The factor is a relatively small peptide with a molecular weight between 300 and 1500 Daltons. Other, non-specific peptides in the broth medium or added to a chemically defined medium have a stimulatory effect on RNA synthesis in vivo and such peptides also stimulate the in vitro synthesis of RNA in a r-chromatin preparation. On the basis of these results we conclude that such extracellular small peptides compete with a specific factor which is part of the intracellular regulatory mechanism controlling the rate of RNA synthesis. The consequence of such competition is a high overproduction of ribosomal RNA in cells inoculated on peptide-rich broth media.

Regulation of RNA synthesis in Tetrahymena pyriformis: secretion of regulatory factors.

The rate of ribosomal RNA synthesis varies greatly with the population density in both exponentially and synchronously growing populations of Tetrahymena pyriformis. Shortly after inoculation of the population - at relatively low cell densities - a gene-dose effect dominates the picture, and a doubling in the gene number is immediately followed by a doubling in the rate of RNA synthesis. However, also other mechanisms are controlling the rate of RNA synthesis. Generally one finds high rates of RNA synthesis in the lag phase of newly inoculated cells, decreasing rate of RNA synthesis during most of the exponential growth phase and very low rate of synthesis in stationary phase cells. We now have results which show that the repression of RNA synthesis in densely populated cultures is caused by a dialysable factor, which is secreted by the cells. If cells are inoculated on a medium which contains this factor the high initial rate of RNA synthesis normally observed is prevented, but the cells multiply and grow with normal generation time until normal stationary-phase population densities are reached.

Tetrahymena cells secrete a low-molecular weight factor which inhibits RNA synthesis in vivo and in vitro.

Medium which has been dialyzed against a dense population of Tetrahymena cells contains a factor which reduces the rate of RNA synthesis in other Tetrahymena cells inoculated on that medium. Furthermore, this extracellular factor inhibits the ribosomal RNA synthesis in vitro when added to an RNA synthesizing r-chromatin preparation.

Regulation of ribosomal RNA synthesis in Tetrahymena pyriformis.

Ribosomal RNA is synthesized at constant rate during most of the cell cycle in heat-shock synchronized populations of Tetrahymena pyriformis. Early in each macronuclear S-period the rate of synthesis increases abruptly, concomitant with replication of the genes coding for ribosomal RNA. The increase is prevented by inhibitors of DNA replication, added prior to the S-period. Similarly, in cultures synchronized by starvation/refeeding, inhibition of DNA replication, at the time when the rDNA is replicated, will prevent the normal increase in rate of RNA synthesis which follows refeeding. We conclude that inhibition of rDNA replication interferes with the synthesis of rRNA, and we suggest that with respect to rRNA synthesis a gene dosis effect is operating in fast-growing Tetrahymena cells.