scholarly journals Constitutive Signal Transduction by Mutant Ssy5p and Ptr3p Components of the SPS Amino Acid Sensor System in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (6) ◽  
pp. 1116-1124 ◽  
Author(s):  
Peter Poulsen ◽  
Boqian Wu ◽  
Richard F. Gaber ◽  
Morten C. Kielland-Brandt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Transcription Factors ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Amino Acid Permease ◽  
Associated Proteins ◽  
Amino Acid Sensing ◽  
Median Effective Concentration ◽  
Mutant Cells

ABSTRACT 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 sensing, which results in cleavage of the transcription factors Stp1p and Stp2p, removing 10 kDa of the N terminus of each of them. This confers the transcription factors with the ability to gain access to the nucleus and activate transcription of amino acid permease genes. To extend our understanding of the role of Ptr3p and Ssy5p in this amino acid sensing process, we have isolated constitutive gain-of-function mutants in these two components by using a genetic screening in which potassium uptake is made dependent on amino acid signaling. Mutants which exhibit inducer-independent processing of Stp1p and activation of the amino acid permease gene AGP1 were obtained. For each component of the SPS complex, constitutive signaling by a mutant allele depended on the presence of wild-type alleles of the other two components. Despite the signaling in the absence of inducer, the processing of Stp1p was more complete in the presence of inducer. Dose response assays showed that the median effective concentration for Stp1p processing in the mutant cells was decreased; i.e., a lower inducer concentration is needed for signaling in the mutant cells. These results suggest that the three sensor components interact intimately in a complex rather than in separate reactions and support the notion that the three components function as a complex.

scholarly journals Different Ubiquitin Signals Act at the Golgi and Plasma Membrane to Direct GAP1 Trafficking

2008 ◽  
Vol 19 (7) ◽  
pp. 2962-2972 ◽  
Author(s):  
April L. Risinger ◽  
Chris A. Kaiser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Protein Sorting ◽  
High Capacity ◽  
Amino Acid Permease ◽  
Acid Addition ◽  
Amino Acid Levels ◽  
General Amino Acid Permease

The high capacity general amino acid permease, Gap1p, in Saccharomyces cerevisiae is distributed between the plasma membrane and internal compartments according to availability of amino acids. When internal amino acid levels are low, Gap1p is localized to the plasma membrane where it imports available amino acids from the medium. When sufficient amino acids are imported, Gap1p at the plasma membrane is endocytosed and newly synthesized Gap1p is delivered to the vacuole; both sorting steps require Gap1p ubiquitination. Although it has been suggested that identical trans-acting factors and Gap1p ubiquitin acceptor sites are involved in both processes, we define unique requirements for each of the ubiquitin-mediated sorting steps involved in delivery of Gap1p to the vacuole upon amino acid addition. Our finding that distinct ubiquitin-mediated sorting steps employ unique trans-acting factors, ubiquitination sites on Gap1p, and types of ubiquitination demonstrates a previously unrecognized level of specificity in ubiquitin-mediated protein sorting.

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

2007 ◽  
Vol 28 (2) ◽  
pp. 551-563 ◽  
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.

scholarly journals The External Amino Acid Signaling Pathway Promotes Activation of Stp1 and Uga35/Dal81 Transcription Factors for Induction of the AGP1 Gene in Saccharomyces cerevisiae

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1727-1739 ◽  
Author(s):  
Fadi Abdel-Sater ◽  
Ismaïl Iraqui ◽  
Antonio Urrestarazu ◽  
Bruno André
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Amino Acid ◽  
Signaling Pathway ◽  
Nitrogen Supply ◽  
Upstream Region ◽  
Gata Factor ◽  
Amino Acid Permease ◽  
Amino Acid Signaling

Abstract Yeast cells respond to the presence of amino acids in their environment by inducing transcription of several amino acid permease genes including AGP1, BAP2, and BAP3. The signaling pathway responsible for this induction involves Ssy1, a permease-like sensor of external amino acids, and culminates with proteolytic cleavage and translocation to the nucleus of the zinc-finger proteins Stp1 and Stp2, the lack of which abolishes induction of BAP2 and BAP3. Here we show that Stp1—but not Stp2—plays an important role in AGP1 induction, although significant induction of AGP1 by amino acids persists in stp1 and stp1 stp2 mutants. This residual induction depends on the Uga35/Dal81 transcription factor, indicating that the external amino acid signaling pathway activates not only Stp1 and Stp2, but also another Uga35/Dal81-dependent transcriptional circuit. Analysis of the AGP1 gene’s upstream region revealed that Stp1 and Uga35/Dal81 act synergistically through a 21-bp cis-acting sequence similar to the UASAA element previously found in the BAP2 and BAP3 upstream regions. Although cells growing under poor nitrogen-supply conditions display much higher induction of AGP1 expression than cells growing under good nitrogen-supply conditions, the UASAA itself is totally insensitive to nitrogen availability. Nitrogen-source control of AGP1 induction is mediated by the GATA factor Gln3, likely acting through adjacent 5′-GATA-3′ sequences, to amplify the positive effect of UASAA. Our data indicate that Stp1 may act in combination with distinct sets of transcription factors, according to the gene context, to promote induction of transcription in response to external amino acids. The data also suggest that Uga35/Dal81 is yet another transcription factor under the control of the external amino acid sensing pathway. Finally, the data show that the TOR pathway mediating global nitrogen control of transcription does not interfere with the external amino acid signaling pathway.

scholarly journals Amino Acid Signaling in Saccharomyces cerevisiae: a Permease-Like Sensor of External Amino Acids and F-Box Protein Grr1p Are Required for Transcriptional Induction of the AGP1 Gene, Which Encodes a Broad-Specificity Amino Acid Permease

1999 ◽  
Vol 19 (2) ◽  
pp. 989-1001 ◽  
Author(s):  
Ismaïl Iraqui ◽  
Stephan Vissers ◽  
Florent Bernard ◽  
Johan-Owen de Craene ◽  
Eckhard Boles ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Glucose Sensors ◽  
Amino Acid Permease ◽  
Transporter Family ◽  
F Box Protein ◽  
Transcriptional Induction ◽  
Amino Acid Signaling ◽  
Broad Specificity

ABSTRACT The SSY1 gene of Saccharomyces cerevisiaeencodes a member of a large family of amino acid permeases. Compared to the 17 other proteins of this family, however, Ssy1p displays unusual structural features reminiscent of those distinguishing the Snf3p and Rgt2p glucose sensors from the other proteins of the sugar transporter family. We show here that SSY1 is required for transcriptional induction, in response to multiple amino acids, of theAGP1 gene encoding a low-affinity, broad-specificity amino acid permease. Total noninduction of the AGP1 gene in thessy1Δ mutant is not due to impaired incorporation of inducing amino acids. Conversely, AGP1 is strongly induced by tryptophan in a mutant strain largely deficient in tryptophan uptake, but it remains unexpressed in a mutant that accumulates high levels of tryptophan endogenously. Induction of AGP1requires Uga35p(Dal81p/DurLp), a transcription factor of the Cys6-Zn2 family previously shown to participate in several nitrogen induction pathways. Induction of AGP1by amino acids also requires Grr1p, the F-box protein of the SCFGrr1 ubiquitin-protein ligase complex also required for transduction of the glucose signal generated by the Snf3p and Rgt2p glucose sensors. Systematic analysis of amino acid permease genes showed that Ssy1p is involved in transcriptional induction of at least five genes in addition to AGP1. Our results show that the amino acid permease homologue Ssy1p is a sensor of external amino acids, coupling availability of amino acids to transcriptional events. The essential role of Grr1p in this amino acid signaling pathway lends further support to the hypothesis that this protein participates in integrating nutrient availability with the cell cycle.

scholarly journals Genetic and Biochemical Analysis of the Yeast Plasma Membrane Ssy1p-Ptr3p-Ssy5p Sensor of Extracellular Amino Acids

2001 ◽  
Vol 21 (3) ◽  
pp. 814-826 ◽  
Author(s):  
Hanna Forsberg ◽  
Per O. Ljungdahl
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Membrane System ◽  
Dominant Negative ◽  
Amino Acid Permease ◽  
Yeast Plasma Membrane ◽  
Sensing System ◽  
Amino Acid Availability ◽  
Cell Extracts

ABSTRACT Ssy1p and Ptr3p are known components of a yeast plasma membrane system that functions to sense the presence of amino acids in the extracellular environment. In response to amino acids, this sensing system initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and transport proteins, including multiple, genetically distinct amino acid permeases. We have found that SSY5 encodes a third component of this amino acid sensing system. Mutations inSSY5 manifest phenotypes that are indistinguishable from those resulting from either single ssy1 andptr3 mutations or ssy5 ssy1 and ssy5 ptr3 double mutations. Although Ssy5p is predicted to be a soluble protein, it exhibits properties indicating that it is a peripherally associated plasma membrane protein. Each of the three sensor components, Ssy1p, Ptr3p, and Ssy5p, adopts conformations and modifications that are dependent upon the availability of amino acids and on the presence of the other two components. These results suggest that these components function as part of a sensor complex localized to the plasma membrane. Consistent with a sensor complex, the overexpression of SSY1 or the unique N-terminal extension of this amino acid permease homologue inactivates the amino acid sensor in a dominant-negative manner. Each of the components of the Ssy1p-Ptr3p-Ssy5p (SPS) signaling system undergoes rapid physical changes, reflected in altered electrophoretic mobility, when leucine is added to cells grown in media lacking amino acids. Furthermore, the levels of each SPS sensor component present in whole-cell extracts diminish upon leucine addition. The rapid physical alterations and reduced levels of sensor components are consistent with their being downregulated in response to amino acid availability. These results reveal the dynamic nature of the amino acid-initiated signals transduced by the SPS sensor.

scholarly journals Amino Acids Regulate Retrieval of the Yeast General Amino Acid Permease from the Vacuolar Targeting Pathway

2006 ◽  
Vol 17 (7) ◽  
pp. 3031-3050 ◽  
Author(s):  
Marta Rubio-Texeira ◽  
Chris A. Kaiser
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Membrane Trafficking ◽  
Amino Acid Permease ◽  
A Genome ◽  
High Amino Acid ◽  
General Amino Acid Permease ◽  
Intracellular Sorting ◽  
Amino Acid Concentrations

Intracellular sorting of the general amino acid permease (Gap1p) in Saccharomyces cerevisiae depends on availability of amino acids such that at low amino acid concentrations Gap1p is sorted to the plasma membrane, whereas at high concentrations Gap1p is sorted to the vacuole. In a genome-wide screen for mutations that affect Gap1p sorting we identified deletions in a subset of components of the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular endosome (MVE). Gap1p-GFP is delivered to the vacuolar interior by the MVE pathway in wild-type cells, but when formation of the MVE is blocked by mutation, Gap1p-GFP efficiently cycles from this compartment to the plasma membrane, resulting in unusually high permease activity at the cell surface. Importantly, cycling of Gap1p-GFP to the plasma membrane is blocked by high amino acid concentrations, defining recycling from the endosome as a major step in Gap1p trafficking under physiological control. Mutations in LST4 and LST7 genes, previously identified for their role in Gap1p sorting, similarly block MVE to plasma membrane trafficking of Gap1p. However, mutations in other recycling complexes such as the retromer had no significant effect on the intracellular sorting of Gap1p, suggesting that Gap1p follows a genetically distinct pathway for recycling. We previously found that Gap1p sorting from the Golgi to the endosome requires ubiquitination of Gap1p by an Rsp5p ubiquitin ligase complex, but amino acid abundance does not appear to significantly alter the accumulation of polyubiquitinated Gap1p. Thus the role of ubiquitination appears to be a signal for delivery of Gap1p to the MVE, whereas amino acid abundance appears to control the cycling of Gap1p from the MVE to the plasma membrane.

scholarly journals Deletion of RTS1, Encoding a Regulatory Subunit of Protein Phosphatase 2A, Results in Constitutive Amino Acid Signaling via Increased Stp1p Processing

2006 ◽  
Vol 5 (1) ◽  
pp. 174-179 ◽  
Author(s):  
Nadine Eckert-Boulet ◽  
Katrin Larsson ◽  
Boqian Wu ◽  
Peter Poulsen ◽  
Birgitte Regenberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Regulatory Subunit ◽  
Mutant Library ◽  
Amino Acid Permease ◽  
Phosphatase 2A ◽  
Amino Acid Sensing ◽  
Amino Acid Signaling

ABSTRACT In Saccharomyces cerevisiae, extracellular amino acids are sensed at the plasma membrane by the SPS sensor, consisting of the transporter homologue Ssy1p, Ptr3p, and the endoprotease Ssy5p. Amino acid sensing results in proteolytic truncation of the transcription factors Stp1p and Stp2p, followed by their relocation from the cytoplasm to the nucleus, where they activate transcription of amino acid permease genes. We screened a transposon mutant library for constitutively signaling mutants, with the aim of identifying down-regulating components of the SPS-mediated pathway. Three isolated mutants were carrying a transposon in the RTS1 gene, which encodes a regulatory subunit of protein phosphatase 2A. We investigated the basal activity of the AGP1 and BAP2 promoters in rts1Δ cells and found increased transcription from these promoters, as well as increased Stp1p processing, even in the absence of amino acids. Based on our findings we propose that the phosphatase complex containing Rts1p keeps the SPS-mediated pathway down-regulated in the absence of extracellular amino acids by dephosphorylating a component of the pathway.

scholarly journals Changes in membrane proteins associated with inhibition of the general amino acid permease of yeast (Saccharomyces cerevisiae)

1981 ◽  
Vol 196 (2) ◽  
pp. 531-536 ◽  
Author(s):  
J R Woodward ◽  
H L Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Wild Type ◽  
Amino Acid Permease ◽  
Yeast Saccharomyces Cerevisiae ◽  
General Amino Acid Permease ◽  
Wild Type Yeast

The general amino acid permease (‘Gap’) system of the wild-type yeast (Saccharomyces cerevisiae) strain Y185 is inhibited by the uptake and accumulation of its substrate amino acids. Surprisingly, this inhibition persists even after ‘pools’ of amino acids, accumulated initially, have returned to normal sizes. Recovery from this inhibition depends on a supply of energy and involves the synthesis of a membrane protein component of the Gap system.

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