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

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.

Download Full-text

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 ◽

10.1093/genetics/166.4.1727 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1727-1739 ◽

Cited By ~ 1

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.24.22.9771-9785.2004 ◽

2004 ◽

Vol 24 (22) ◽

pp. 9771-9785 ◽

Cited By ~ 58

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.

Download Full-text

The Regulatory Mechanism of Water Activities on Aflatoxins Biosynthesis and Conidia Development, and Transcription Factor AtfB Is Involved in This Regulation

Toxins ◽

10.3390/toxins13060431 ◽

2021 ◽

Vol 13 (6) ◽

pp. 431

Author(s):

Longxue Ma ◽

Xu Li ◽

Xiaoyun Ma ◽

Qiang Yu ◽

Xiaohua Yu ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Environmental Factors ◽

Health Risks ◽

Sexual Development ◽

Regulatory Mechanism ◽

Food Storage ◽

Economic Losses ◽

Transcriptional Level ◽

Conidia Production

Peanuts are frequently infected by Aspergillus strains and then contaminated by aflatoxins (AF), which brings out economic losses and health risks. AF production is affected by diverse environmental factors, especially water activity (aw). In this study, A. flavus was inoculated into peanuts with different aw (0.90, 0.95, and 0.99). Both AFB1 yield and conidia production showed the highest level in aw 0.90 treatment. Transcriptional level analyses indicated that AF biosynthesis genes, especially the middle- and later-stage genes, were significantly up-regulated in aw 0.90 than aw 0.95 and 0.99. AtfB could be the pivotal regulator response to aw variations, and could further regulate downstream genes, especially AF biosynthesis genes. The expressions of conidia genes and relevant regulators were also more up-regulated at aw 0.90 than aw 0.95 and 0.99, suggesting that the relative lower aw could increase A. flavus conidia development. Furthermore, transcription factors involved in sexual development and nitrogen metabolism were also modulated by different aw. This research partly clarified the regulatory mechanism of aw on AF biosynthesis and A. flavus development and it would supply some advice for AF prevention in food storage.

Download Full-text

TFEB controls retromer expression in response to nutrient availability

The Journal of Cell Biology ◽

10.1083/jcb.201903006 ◽

2019 ◽

Vol 218 (12) ◽

pp. 3954-3966 ◽

Cited By ~ 8

Author(s):

Rachel Curnock ◽

Alessia Calcagni ◽

Andrea Ballabio ◽

Peter J. Cullen

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Amino Acid ◽

Cell Surface ◽

Nutrient Uptake ◽

Nutrient Availability ◽

Amino Acid Starvation ◽

Endosomal Recycling ◽

Transcription Factor Eb ◽

Glutamine Transporters

Endosomal recycling maintains the cell surface abundance of nutrient transporters for nutrient uptake, but how the cell integrates nutrient availability with recycling is less well understood. Here, in studying the recycling of human glutamine transporters ASCT2 (SLC1A5), LAT1 (SLC7A5), SNAT1 (SLC38A1), and SNAT2 (SLC38A2), we establish that following amino acid restriction, the adaptive delivery of SNAT2 to the cell surface relies on retromer, a master conductor of endosomal recycling. Upon complete amino acid starvation or selective glutamine depletion, we establish that retromer expression is upregulated by transcription factor EB (TFEB) and other members of the MiTF/TFE family of transcription factors through association with CLEAR elements in the promoters of the retromer genes VPS35 and VPS26A. TFEB regulation of retromer expression therefore supports adaptive nutrient acquisition through endosomal recycling.

Download Full-text

Genome-wide analysis, transcription factor network approach and gene expression profile of GH3 genes over early somatic embryogenesis in Coffea spp

BMC Genomics ◽

10.1186/s12864-019-6176-1 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 1

Author(s):

Renan Terassi Pinto ◽

Natália Chagas Freitas ◽

Wesley Pires Flausino Máximo ◽

Thiago Bergamo Cardoso ◽

Débora de Oliveira Prudente ◽

...

Keyword(s):

Amino Acids ◽

Transcription Factor ◽

Somatic Embryogenesis ◽

Amino Acid ◽

Clonal Propagation ◽

Family Members ◽

Network Approach ◽

Embryogenic Potential ◽

Auxin Homeostasis ◽

Early Somatic Embryogenesis

Abstract Background Coffee production relies on plantations with varieties from Coffea arabica and Coffea canephora species. The first, the most representative in terms of coffee consumption, is mostly propagated by seeds, which leads to management problems regarding the plantations maintenance, harvest and processing of grains. Therefore, an efficient clonal propagation process is required for this species cultivation, which is possible by reaching a scalable and cost-effective somatic embryogenesis protocol. A key process on somatic embryogenesis induction is the auxin homeostasis performed by Gretchen Hagen 3 (GH3) proteins through amino acid conjugation. In this study, the GH3 family members were identified on C. canephora genome, and by performing analysis related to gene and protein structure and transcriptomic profile on embryogenic tissues, we point a GH3 gene as a potential regulator of auxin homeostasis during early somatic embryogenesis in C. arabica plants. Results We have searched within the published C. canephora genome and found 17 GH3 family members. We checked the conserved domains for GH3 proteins and clustered the members in three main groups according to phylogenetic relationships. We identified amino acids sets in four GH3 proteins that are related to acidic amino acid conjugation to auxin, and using a transcription factor (TF) network approach followed by RT-qPCR we analyzed their possible transcriptional regulators and expression profiles in cells with contrasting embryogenic potential in C. arabica. The CaGH3.15 expression pattern is the most correlated with embryogenic potential and with CaBBM, a C. arabica ortholog of a major somatic embryogenesis regulator. Conclusion Therefore, one out of the GH3 members may be influencing on coffee somatic embryogenesis by auxin conjugation with acidic amino acids, which leads to the phytohormone degradation. It is an indicative that this gene can serve as a molecular marker for coffee cells with embryogenic potential and needs to be further studied on how much determinant it is for this process. This work, together with future studies, can support the improvement of coffee clonal propagation through in vitro derived somatic embryos.

Download Full-text

Suppression of the mTORC1/STAT3/Notch1 pathway by activated AMPK prevents hepatic insulin resistance induced by excess amino acids

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00202.2013 ◽

2014 ◽

Vol 306 (2) ◽

pp. E197-E209 ◽

Cited By ~ 36

Author(s):

Hongliang Li ◽

Jiyeon Lee ◽

Chaoyong He ◽

Ming-Hui Zou ◽

Zhonglin Xie

Keyword(s):

Insulin Resistance ◽

Amino Acids ◽

Amino Acid ◽

Signaling Pathway ◽

High Protein Diet ◽

Chronic Administration ◽

Hepatic Insulin Resistance ◽

Notch1 Signaling ◽

Mtorc1 Signaling ◽

Notch1 Signaling Pathway

Nutrient overload is associated with the development of obesity, insulin resistance, and type 2 diabetes. However, the underlying mechanisms for developing insulin resistance in the presence of excess nutrients are incompletely understood. We investigated whether activation of AMP-activated protein kinase (AMPK) prevents the hepatic insulin resistance that is induced by the consumption of a high-protein diet (HPD) and the presence of excess amino acids. Exposure of HepG2 cells to excess amino acids reduced AMPK phosphorylation, upregulated Notch1 expression, and impaired the insulin-stimulated phosphorylation of Akt Ser473 and insulin receptor substrate-1 (IRS-1) Tyr612. Inhibition of Notch1 prevented amino acid-induced insulin resistance, which was accompanied by reduced expression of Rbp-Jk, hairy and enhancer of split-1, and forkhead box O1. Mechanistically, mTORC1 signaling was activated by excess amino acids, which then positively regulated Notch1 expression through the activation of the signal transducer and activator of transcription 3 (STAT3). Activation of AMPK by metformin inhibited mTORC1-STAT3 signaling, thereby preventing excess amino acid-impaired insulin signaling. Finally, HPD feeding suppressed AMPK activity, activated mTORC1/STAT3/Notch1 signaling, and induced insulin resistance. Chronic administration of either metformin or rapamycin inhibited the HPD-activated mTORC1/STAT3/Notch1 signaling pathway and prevented hepatic insulin resistance. We conclude that the upregulation of Notch1 expression by hyperactive mTORC1 signaling is an essential event in the development of hepatic insulin resistance in the presence of excess amino acids. Activation of AMPK prevents amino acid-induced insulin resistance through the suppression of the mTORC1/STAT3/Notch1 signaling pathway.

Download Full-text

348 SENESCENCE OF DAYLILY (HEMEROCALLIS) IS ASSOCIATED WITH EXPRESSION OF A MADS-BOX GENE

HortScience ◽

10.21273/hortsci.29.5.480e ◽

1994 ◽

Vol 29 (5) ◽

pp. 480e-480

Author(s):

Nathan E. Lange ◽

Michael S. Reid ◽

Victoriano Valpuesta ◽

Consuelo Guerrero ◽

Miguel A. Botella

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Amino Acid ◽

Blot Hybridization ◽

Flower Senescence ◽

Northern Blot Hybridization ◽

Mads Box ◽

Homologous Genes ◽

Mads Box Gene ◽

Commercially Important

As in many commercially important flowers, especially the monocotyledonous geophytes, senescence of the ephemeral daylily flower (Hemerocallis) does not involve ethylene. By differentially screening a cDNA library constructed from mRNA extracted from daylily petals in the earliest stages of senescence, clones were isolated whose transcription is up-regulated coordinately with the onset of senescence. One of these clones, sen12, was found to be a transcription factor. The deduced amino acid sequence of sen12 contains a MADS-box and an associated K-box similar to transcription factors suggested to control floral morphogenesis in a variety of different species. Northern blot hybridization showed sen12 to be highly upregulated before and during visible flower senescence. The expression of homologous genes during senescence of other flowers will be reported.

Download Full-text

CmMYB#7, an R3 MYB transcription factor, acts as a negative regulator of anthocyanin biosynthesis in chrysanthemum

Journal of Experimental Botany ◽

10.1093/jxb/erz121 ◽

2019 ◽

Vol 70 (12) ◽

pp. 3111-3123 ◽

Cited By ~ 8

Author(s):

Lili Xiang ◽

Xiaofen Liu ◽

Heng Li ◽

Xueren Yin ◽

Donald Grierson ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Binding Site ◽

Transient Expression ◽

Regulatory Mechanism ◽

Anthocyanin Biosynthesis ◽

Expression Patterns ◽

Negative Regulator ◽

Myb Transcription Factor ◽

Anthocyanin Content

Abstract ‘Jimba’, a well-known white flowered chrysanthemum cultivar, occasionally and spontaneously produces red colored petals under natural cultivation, but there is little information about the molecular regulatory mechanism underlying this process. We analysed the expression patterns of 91 MYB transcription factors in ‘Jimba’ and ‘Turning red Jimba’ and identified an R3 MYB, CmMYB#7, whose expression was significantly decreased in ‘Turning red Jimba’ compared with ‘Jimba’, and confirmed it is a passive repressor of anthocyanin biosynthesis. CmMYB#7 competed with CmMYB6, which together with CmbHLH2 is an essential component of the anthocyanin activation complex, for interaction with CmbHLH2 through the bHLH binding site in the R3 MYB domain. This reduced binding of the CmMYB6–CmbHLH2 complex and inhibited its ability to activate CmDFR and CmUFGT promoters. Moreover, using transient expression assays we demonstrated that changes in the expression of CmMYB#7 accounted for alterations in anthocyanin content. Taken together, our findings illustrate that CmMYB#7 is a negative regulator of anthocyanin biosynthesis in chrysanthemum.

Download Full-text