scholarly journals Amino Acid Signaling in Yeast: Casein Kinase I and the Ssy5 Endoprotease Are Key Determinants of Endoproteolytic Activation of the Membrane-Bound Stp1 Transcription Factor

2004 ◽  
Vol 24 (22) ◽  
pp. 9771-9785 ◽  
Author(s):  
Fadi Abdel-Sater ◽  
Mohamed El Bakkoury ◽  
Antonio Urrestarazu ◽  
Stephan Vissers ◽  
Bruno André
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Amino Acid ◽  
Signaling Pathway ◽  
Serine Proteases ◽  
Casein Kinase ◽  
Casein Kinase I ◽  
Amino Acid Permease ◽  
Membrane Bound ◽  
Amino Acid Signaling

ABSTRACT Saccharomyces cerevisiae cells possess a plasma membrane sensor able to detect the presence of extracellular amino acids and then to activate a signaling pathway leading to transcriptional induction of multiple genes, e.g., AGP1, encoding an amino acid permease. This sensing function requires the permease-like Ssy1 and associated Ptr3 and Ssy5 proteins, all essential to activation, by endoproteolytic processing, of the membrane-bound Stp1 transcription factor. The SCFGrr1 ubiquitin-ligase complex is also essential to AGP1 induction, but its exact role in the amino acid signaling pathway remains unclear. Here we show that Stp1 undergoes casein kinase I-dependent phosphorylation. In the yck mutant lacking this kinase, Stp1 is not cleaved and AGP1 is not induced in response to amino acids. Furthermore, we provide evidence that Ssy5 is the endoprotease responsible for Stp1 processing. Ssy5 is significantly similar to serine proteases, its self-processing is a prerequisite for Stp1 cleavage, and its overexpression causes inducer-independent Stp1 cleavage and high-level AGP1 transcription. We further show that Stp1 processing also requires the SCFGrr1 complex but is insensitive to proteasome inhibition. However, Stp1 processing does not require SCFGrr1, Ssy1, or Ptr3 when Ssy5 is overproduced. Finally, we describe the properties of a particular ptr3 mutant that suggest that Ptr3 acts with Ssy1 in amino acid detection and signal initiation. We propose that Ssy1 and Ptr3 form the core components of the amino acid sensor. Upon detection of external amino acids, Ssy1-Ptr3 likely allows—in a manner dependent on SCFGrr1—the Ssy5 endoprotease to gain access to and to cleave Stp1, this requiring prior phosphorylation of Stp1 by casein kinase I.

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 The Prodomain of Ssy5 Protease Controls Receptor-Activated Proteolysis of Transcription Factor Stp1

2010 ◽  
Vol 30 (13) ◽  
pp. 3299-3309 ◽  
Author(s):  
Thorsten Pfirrmann ◽  
Stijn Heessen ◽  
Deike J. Omnus ◽  
Claes Andréasson ◽  
Per O. Ljungdahl
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Amino Acid ◽  
Signaling Pathway ◽  
Temperature Control ◽  
Regulatory Mechanism ◽  
Potent Inhibitor ◽  
Large N

ABSTRACT Extracellular amino acids induce the yeast SPS sensor to endoproteolytically cleave transcription factors Stp1 and Stp2 in a process termed receptor-activated proteolysis (RAP). Ssy5, the activating endoprotease, is synthesized with a large N-terminal prodomain and a C-terminal chymotrypsin-like catalytic (Cat) domain. During biogenesis, Ssy5 cleaves itself and the prodomain and Cat domain remain associated, forming an inactive primed protease. Here we show that the prodomain is a potent inhibitor of Cat domain activity and that its inactivation is a requisite for RAP. Accordingly, amino acid-induced signals trigger proteasome-dependent degradation of the prodomain. A mutation that stabilizes the prodomain prevents Stp1 processing, whereas destabilizing mutations lead to constitutive RAP-independent Stp1 processing. We fused a conditional degron to the prodomain to synthetically reprogram the amino acid-responsive SPS signaling pathway, placing it under temperature control. Our results define a regulatory mechanism that is novel for eukaryotic proteases functioning within cells.

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 Constitutive and Hyperresponsive Signaling by Mutant Forms of Saccharomyces cerevisiae Amino Acid Sensor Ssy1

2003 ◽  
Vol 2 (5) ◽  
pp. 922-929 ◽  
Author(s):  
Richard F. Gaber ◽  
Kim Ottow ◽  
Helge A. Andersen ◽  
Morten C. Kielland-Brandt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Direct Interaction ◽  
Acid Transport ◽  
Amino Acid Permease ◽  
Amino Acid Permeases ◽  
Mutant Forms ◽  
Amino Acid Sensor ◽  
Transcriptional Induction ◽  
Amino Acid Signaling

ABSTRACT 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 dependent on amino acid signaling, we obtained gain-of-function mutations in SSY1. Some alleles confer inducer-independent signaling; others increase the apparent affinity for inducers. The results reveal that amino acid transport is not required for signaling and support the notion that sensing by Ssy1 occurs via its direct interaction with extracellular amino acids.

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 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 Analysis of Htra Gene from Zebrafish (Danio Rerio)

2015 ◽  
Vol 10 (2) ◽  
Author(s):  
M. Murwantoko ◽  
Chio Oka ◽  
Masashi Kawaichi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Danio Rerio ◽  
Serine Proteases ◽  
Catalytic Domain ◽  
Fruit Fly ◽  
Wide Range ◽  
Igf Binding ◽  
Bacterial Homolog

HtrA which is characterized by the combination of a trypsin-like catalytic domain with at least one C-terminalPDZ domain is a highly conserved family of serine proteases found in a wide range of organisms. However theidentified HtrA family numbers varies among spesies, for example the number of mammalian, Eschericia coli,fruit fly-HtrA family are 4, 3 and 1 gene respectively. One gene is predicted exist in zebrafish. Since no completeinformation available on zebrafish HtrA, in this paper zebrafish HtrA (zHtrA) gene was analyzed. The zHtrA isbelonged to HtrA1 member and predicted encodes 478 amino acids with a signal peptide, a IGF binding domain,a Kazal-type inhibitor domain in the up stream of HtrA-bacterial homolog. At the amino acid sequence the zHtrA1showed the 69%, 69%, 68%, 54% and 54% with the rat HtrA1, mouse HtrA1, human HtrA1, human HtrA3 andmouse HtrA4 respectively. The zHtrA1 is firstly expressed at 60 hpf and mainly in the vertebral rudiments in thetail region.

scholarly journals Rhizobium leguminosarum Has a Second General Amino Acid Permease with Unusually Broad Substrate Specificity and High Similarity to Branched-Chain Amino Acid Transporters (Bra/LIV) of the ABC Family

2002 ◽  
Vol 184 (15) ◽  
pp. 4071-4080 ◽  
Author(s):  
A. H. F. Hosie ◽  
D. Allaway ◽  
C. S. Galloway ◽  
H. A. Dunsby ◽  
P. S. Poole
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rhizobium Leguminosarum ◽  
Binding Protein ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Amino Acid Permease ◽  
Branched Chain Amino Acid ◽  
General Amino Acid Permease ◽  
Branched Chain

ABSTRACT Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (BraRl). Characterization of the solute specificity of BraRl shows it to be the second general amino acid permease of R. leguminosarum. Although BraRl has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (l-glutamate, l-arginine, and l-histidine), in addition to neutral amino acids (l-alanine and l-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be α-amino acids. Consistent with this, BraRl is the first ABC transporter to be shown to transport γ-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by BraRl does not appear to be stereospecific as d amino acids cause significant inhibition of uptake of l-glutamate and l-leucine. Unlike all other solutes tested, l-alanine uptake is not dependent on solute binding protein BraCRl. Therefore, a second, unidentified solute binding protein may interact with the BraDEFGRl membrane complex during l-alanine uptake. Overall, the data indicate that BraRl is a general amino acid permease of the HAAT family. Furthermore, BraRl has the broadest solute specificity of any characterized bacterial amino acid transporter.

PREDICTION OF THE THREE-DIMENSIONAL STRUCTURE OF THE ENZYMATIC PART OF T-PA

1987 ◽  
Author(s):  
A Heckel ◽  
K M Hasselbach
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Serine Proteases ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
Salt Bridges ◽  
Three Dimensional Structure ◽  
Insertions And Deletions

Up to now the three-dimensional structure of t-PA or parts of this enzyme is unknown. Using computer graphical methods the spatial structure of the enzymatic part of t-PA is predicted on the hypothesis, the three-dimensional backbone structure of t-PA being similar to that of other serine proteases. The t-PA model was built up in three steps:1) Alignment of the t-PA sequence with other serine proteases. Comparison of enzyme structures available from Brookhaven Protein Data Bank proved elastase as a basis for modeling.2) Exchange of amino acids of elastase differing from the t-PA sequence. The replacement of amino acids was performed such that backbone atoms overlapp completely and side chains superpose as far as possible.3) Modeling of insertions and deletions. To determine the spatial arrangement of insertions and deletions parts of related enzymes such as chymotrypsin or trypsin were used whenever possible. Otherwise additional amino acid sequences were folded to a B-turn at the surface of the proteine, where all insertions or deletions are located. Finally the side chain torsion angles of amino acids were optimised to prevent close contacts of neigh bouring atoms and to improve hydrogen bonds and salt bridges.The resulting model was used to explain binding of arginine 560 of plasminogen to the active site of t-PA. Arginine 560 interacts with Asp 189, Gly 19 3, Ser 19 5 and Ser 214 of t-PA (chymotrypsin numbering). Furthermore interaction of chromo-genic substrate S 2288 with the active site of t-PA was studied. The need for D-configuration of the hydrophobic amino acid at the N-terminus of this tripeptide derivative could be easily explained.

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