Substrate specificity and gene expression of the amino-acid permeases in Saccharomyces cerevisiae

The activities of the proline-specific permease (PUT4) and the general amino acid permease (GAP1) of Saccharomyces cerevisiae vary 70- to 140-fold in response to the nitrogen source of the growth medium. The PUT4 and GAP1 permease activities are regulated by control of synthesis and control of activity. These permeases are irreversibly inactivated by addition of ammonia or glutamine, lowering the activity to that found during steady-state growth on these nitrogen sources. Mutants altered in the regulation of the PUT4 permease (Per-) have been isolated. The mutations in these strains are pleiotropic and affect many other permeases, but have no direct effect on various cytoplasmic enzymes involved in nitrogen assimilation. In strains having one class of mutations (per1), ammonia inactivation of the PUT4 and GAP1 permeases did not occur, whereas glutamate and glutamine inactivation did. Thus, there appear to be two independent inactivation systems, one responding to ammonia and one responding to glutamate (or a metabolite of glutamate). The mutations were found to be nuclear and recessive. The inactivation systems are constitutive and do not require transport of the effector molecules per se, apparently operating on the inside of the cytoplasmic membrane. The ammonia inactivation was found not to require a functional glutamate dehydrogenase (NADP). These mutants were used to show that ammonia exerts control of arginase synthesis largely by inducer exclusion. This may be the primary mode of nitrogen regulation for most nitrogen-regulated enzymes of S. cerevisiae.

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Activation of the SPS Amino Acid-Sensing Pathway in Saccharomyces cerevisiae Correlates with the Phosphorylation State of a Sensor Component, Ptr3

Molecular and Cellular Biology ◽

10.1128/mcb.00929-07 ◽

2007 ◽

Vol 28 (2) ◽

pp. 551-563 ◽

Cited By ~ 47

Author(s):

Zhengchang Liu ◽

Janet Thornton ◽

Mário Spírek ◽

Ronald A. Butow

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Nuclear Translocation ◽

Proteolytic Processing ◽

Casein Kinase I ◽

Positive Regulators ◽

Amino Acid Permeases ◽

Amino Acid Sensing ◽

Amino Acid Sensor

ABSTRACT Cells of the budding yeast Saccharomyces cerevisiae sense extracellular amino acids and activate expression of amino acid permeases through the SPS-sensing pathway, which consists of Ssy1, an amino acid sensor on the plasma membrane, and two downstream factors, Ptr3 and Ssy5. Upon activation of SPS signaling, two transcription factors, Stp1 and Stp2, undergo Ssy5-dependent proteolytic processing that enables their nuclear translocation. Here we show that Ptr3 is a phosphoprotein whose hyperphosphorylation is increased by external amino acids and is dependent on Ssy1 but not on Ssy5. A deletion mutation in GRR1, encoding a component of the SCFGrr1 E3 ubiquitin ligase, blocks amino acid-induced hyperphosphorylation of Ptr3. We found that two casein kinase I (CKI) proteins, Yck1 and Yck2, previously identified as positive regulators of SPS signaling, are required for hyperphosphorylation of Ptr3. Loss- and gain-of-function mutations in PTR3 result in decreased and increased Ptr3 hyperphosporylation, respectively. We found that a defect in PP2A phosphatase activity leads to the hyperphosphorylation of Ptr3 and constitutive activation of SPS signaling. Two-hybrid analysis revealed interactions between the N-terminal signal transduction domain of Ssy1 with Ptr3 and Yck1. Our findings reveal that CKI and PP2A phosphatase play antagonistic roles in SPS sensing by regulating Ptr3 phosphorylation.

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Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2723-2735.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2723-2735 ◽

Cited By ~ 1

Author(s):

C M Moehle ◽

A G Hinnebusch

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Ribosomal Protein ◽

Binding Sites ◽

Protein Gene ◽

Ribosomal Protein Gene ◽

Ribosomal Protein Genes ◽

Biosynthetic Genes ◽

Stringent Control

An amino acid limitation in bacteria elicits a global response, called stringent control, that leads to reduced synthesis of rRNA and ribosomal proteins and increased expression of amino acid biosynthetic operons. We have used the antimetabolite 3-amino-1,2,4-triazole to cause histidine limitation as a means to elicit the stringent response in the yeast Saccharomyces cerevisiae. Fusions of the yeast ribosomal protein genes RPL16A, CRY1, RPS16A, and RPL25 with the Escherichia coli lacZ gene were used to show that the expression of these genes is reduced by a factor of 2 to 5 during histidine-limited exponential growth and that this regulation occurs at the level of transcription. Stringent regulation of the four yeast ribosomal protein genes was shown to be associated with a nucleotide sequence, known as the UASrpg (upstream activating sequence for ribosomal protein genes), that binds the transcriptional regulatory protein RAP1. The RAP1 binding sites also appeared to mediate the greater ribosomal protein gene expression observed in cells growing exponentially than in cells in stationary phase. Although expression of the ribosomal protein genes was reduced in response to histidine limitation, the level of RAP1 DNA-binding activity in cell extracts was unaffected. Yeast strains bearing a mutation in any one of the genes GCN1 to GCN4 are defective in derepression of amino acid biosynthetic genes in 10 different pathways under conditions of histidine limitation. These Gcn- mutants showed wild-type regulation of ribosomal protein gene expression, which suggests that separate regulatory pathways exist in S. cerevisiae for the derepression of amino acid biosynthetic genes and the repression of ribosomal protein genes in response to amino acid starvation.

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Role of IME1 expression in regulation of meiosis in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6103 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6103-6113 ◽

Cited By ~ 58

Author(s):

H E Smith ◽

S S Su ◽

L Neigeborn ◽

S E Driscoll ◽

A P Mitchell

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Specific Gene ◽

Alpha Cells ◽

Cell Type ◽

Yeast Saccharomyces Cerevisiae ◽

Specific Gene Expression ◽

Gal1 Promoter ◽

Acid Protein

Two signals are required for meiosis and spore formation in the yeast Saccharomyces cerevisiae: starvation and the MAT products a1 and alpha 2, which determine the a/alpha cell type. These signals lead to increased expression of the IME1 (inducer of meiosis) gene, which is required for sporulation and sporulation-specific gene expression. We report here the sequence of the IME1 gene and the consequences of IME1 expression from the GAL1 promoter. The deduced IME1 product is a 360-amino-acid protein with a tyrosine-rich C-terminal region. Expression of PGAL1-IME1 in vegetative a/alpha cells led to moderate accumulation of four early sporulation-specific transcripts (IME2, SPO11, SPO13, and HOP1); the transcripts accumulated 3- to 10-fold more after starvation. Two sporulation-specific transcripts normally expressed later (SPS1 and SPS2) did not accumulate until PGAL1-IME1 strains were starved, and the intact IME1 gene was not activated by PGAL1-IME1 expression. In a or alpha cells, which lack alpha 2 or a1, expression of PGAL1-IME1 led to the same pattern of IME2 and SPO13 expression as in a/alpha cells, as measured with ime2::lacZ and spo13::lacZ fusions. Thus, in wild-type strains, the increased expression of IME1 in starved a/alpha cells can account entirely for cell type control, but only partially for nutritional control, of early sporulation-specific gene expression. PGAL1-IME1 expression did not cause growing cells to sporulate but permitted efficient sporulation of amino acid-limited cells, which otherwise sporulated poorly. We suggest that IME1 acts primarily as a positive regulator of early sporulation-specific genes and that growth arrest is an independent prerequisite for execution of the sporulation program.

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Transcriptional and Translational Regulation of Gene Expression in the General Control of Amino-Acid Biosynthesis in Saccharomyces cerevisiae

Progress in Nucleic Acid Research and Molecular Biology ◽

10.1016/s0079-6603(08)60712-6 ◽

1990 ◽

pp. 195-240 ◽

Cited By ~ 33

Author(s):

Alan G. Hinnebusch

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Translational Regulation ◽

Regulation Of Gene Expression ◽

Amino Acid Biosynthesis ◽

General Control ◽

Acid Biosynthesis ◽

Transcriptional And Translational Regulation

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Two FK506 resistance-conferring genes in Saccharomyces cerevisiae, TAT1 and TAT2, encode amino acid permeases mediating tyrosine and tryptophan uptake.

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6597 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6597-6606 ◽

Cited By ~ 112

Author(s):

A Schmidt ◽

M N Hall ◽

A Koller

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

T Cell Activation ◽

Cell Activation ◽

Amino Acid Permease ◽

High Affinity ◽

New Genes ◽

Eukaryotic Microorganisms ◽

Amino Acid Permeases ◽

Tryptophan Uptake

The macrocyclic lactone FK506 exerts immunosuppressive effects on T lymphocytes by interfering with signal transduction leading to T-cell activation and also inhibits the growth of eukaryotic microorganisms, including Saccharomyces cerevisiae. We reported previously that an FK506-sensitive target in S. cerevisiae is required for amino acid import and that overexpression of two new genes, TAT1 and TAT2 (formerly called TAP1 and TAP2), confers resistance to the drug. Here we report that TAT1 and TAT2 encode novel members of the yeast amino acid permease family composed of integral membrane proteins that share 30 to 40% identity. TAT1 is the tyrosine high-affinity transporter, which also mediates low-affinity or low-capacity uptake of tryptophan. TAT2 is the tryptophan high-affinity transporter. FK506 does not reduce the levels of TAT1 and TAT2 transcripts, indicating that the inhibition of amino acid transport by the drug is posttranscriptional.

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