The Gal3p-Gal80p-Gal4p Transcription Switch of Yeast: Gal3p Destabilizes the Gal80p-Gal4p Complex in Response to Galactose and ATP

Sil, Alok Kumar; Alam, Samina; Xin, Ping; Ma, Li; Morgan, Melissa A.; Lebo, C. M.; Woods, Michael P.; Hopper, James E.

doi:10.1128/mcb.19.11.7828

Cited by 69 publications

(59 citation statements)

References 88 publications

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“…4C). These results are in excellent agreement with previous biochemical experiments showing that in galactose media Gal80 remains physically associated with Gal4 at a second site (Sil et al 1999), again validating the FRET assay for detecting Gal4 interactions in vivo.…”

Section: The Gal4-tra1 Interaction Occurs Only In Galactose Mediumsupporting

confidence: 90%

In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer

Bhaumik¹,

Raha²,

Aiello³

et al. 2004

Genes Dev.

178

217

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Our understanding of eukaryotic transcriptional activation mechanisms has been hampered by an inability to identify the direct in vivo targets of activator proteins, primarily because of lack of appropriate experimental methods. To circumvent this problem, we have developed a fluorescence resonance energy transfer (FRET) assay to monitor interactions with transcriptional activation domains in living cells. We use this method to show that the Tra1 subunit of the SAGA (Spt/Ada/Gcn5/acetyltransferase) complex is the direct in vivo target of the yeast activator Gal4. Chromatin-immunoprecipitation experiments demonstrate that the Gal4-Tra1 interaction is required for recruitment of SAGA to the upstream activating sequence (UAS), and SAGA, in turn, recruits the Mediator complex to the UAS. The UAS-bound Mediator is required for recruitment of the general transcription factors to the core promoter. Thus, our results identify the in vivo target of an activator and show how the activator-target interaction leads to transcriptional stimulation. The FRET assay we describe is a general method that can be used to identify the in vivo targets of other activators. Transcription initiation by RNA polymerase II involves the assembly of general transcription factors (GTFs) on the core promoter to form a preinitiation complex (PIC). A variety of studies indicate that promoter-specific activator proteins (activators) work, at least in part, by increasing PIC formation (Orphanides et al. 1996;Roeder 1996;Ptashne and Gann 1997;. Activator-mediated stimulation of PIC assembly is believed to result from a direct interaction between the activation domain (AD) and one or more components of the transcription machinery, termed the "target." The unambiguous identification of the direct in vivo targets of activators has been a major challenge in the field.Transcriptional induction of genes involved in galactose utilization (GAL genes) has been a model experimental system for studying transcriptional activation mechanisms. The well-characterized acidic activator Gal4 is responsible for the transcriptional stimulation of GAL genes, such as GAL1, which contain Gal4-binding sites in their promoters (Johnston 1987;Johnston and Carlson 1992;Dudley et al. 1999). A variety of transcriptional components have been proposed to be the target of Gal4 including TBP (Melcher and Johnston 1995;Wu et al. 1996), TFIIB (Wu et al. 1996), Srb4 (Koh et al. 1998Park et al. 2000), Gal11 (Jeong et al. 2001), the Swi/Snf complex (Neely et al. 2002), and the SAGA (Spt/Ada/ Gcn5/acetyltransferase) complex (Bhaumik and Green 2001;Brown et al. 2001;Larschan and Winston 2001). These proposals are based on either in vitro protein interaction studies or inferences from various indirect in vivo experiments, and to date there is no definitive evidence that Gal4 directly interacts with any of these putative targets in vivo. The major obstacle has been the lack of an experimental strategy to measure direct interactions with transcriptional ADs in vivo.The development of spectra...

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Section: The Gal4-tra1 Interaction Occurs Only In Galactose Mediumsupporting

confidence: 90%

In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer

Bhaumik¹,

Raha²,

Aiello³

et al. 2004

Genes Dev.

178

217

View full text Add to dashboard Cite

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“…One model for how GlcNAc signaling may occur is based on galactose regulation in S. cerevisiae. Cells respond to elevated intracellular galactose, which acts together with ATP and Gal3 to relieve the inhibition of the Gal4 transcription factor by its negative regulator Gal80 (Sil et al, 1999). Another possibility is based on GlcNAc signaling in metazoan cells, in which elevated GlcNAc levels stimulate signal transduction via O-GlcNAc modification of proteins (Slawson and Hart, 2003;Zachara and Hart, 2006).…”

Section: Role Of Ngt1 In Hyphal Growth Stimulated By Glcnacmentioning

confidence: 99%

Identification of anN-Acetylglucosamine Transporter That Mediates Hyphal Induction inCandida albicans

Álvarez

Konopka

2007

MBoC

127

171

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The sugar N-acetylglucosamine (GlcNAc) plays an important role in nutrient sensing and cellular regulation in a wide range of organisms from bacteria to humans. In the fungal pathogen Candida albicans, GlcNAc induces a morphological transition from budding to hyphal growth. Proteomic comparison of plasma membrane proteins from buds and from hyphae induced by GlcNAc identified a novel hyphal protein (Ngt1) with similarity to the major facilitator superfamily of transporters. An Ngt1-GFP fusion was detected in the plasma membrane after induction with GlcNAc, but not other related sugars. Ngt1-GFP was also induced by macrophage phagocytosis, suggesting a role for the GlcNAc response in signaling entry into phagolysosomes. NGT1 is needed for efficient GlcNAc uptake and for the ability to induce hyphae at low GlcNAc concentrations. High concentrations of GlcNAc could bypass the need for NGT1 to induce hyphae, indicating that elevated intracellular levels of GlcNAc induce hyphal formation. Expression of NGT1 in Saccharomyces cerevisiae promoted GlcNAc uptake, indicating that Ngt1 acts directly as a GlcNAc transporter. Transport mediated by Ngt1 was specific, as other sugars could not compete for the uptake of GlcNAc. Thus, Ngt1 represents the first eukaryotic GlcNAc transporter to be discovered. The presence of NGT1 homologues in the genome sequences of a wide range of eukaryotes from yeast to mammals suggests that they may also function in the cellular processes regulated by GlcNAc, including those that underlie important diseases such as cancer and diabetes. INTRODUCTIONN-acetylglucosamine (GlcNAc) is an amino sugar that carries out important roles in a broad range of cells from bacteria to humans. One aspect of GlcNAc function is to mediate cellular signaling. In bacteria, GlcNAc induces components that are important for colonization of human hosts, including fimbrins that mediate adhesion to host cells (Sohanpal et al., 2004), multidrug exporter genes (Hirakawa et al., 2006) and Curli fibers that promote biofilm formation (Barnhart et al., 2006). In mammals, GlcNAc is a key sensor of nutrient status that is involved in insulin signaling, cell cycle control, and other essential processes. Nutritional effects that lead to increased GlcNAc levels result in its conversion to UDP-GlcNAc and subsequent attachment to proteins on serine or threonine residues in a dynamic manner that is analogous to modification of proteins by phosphorylation (Slawson and Hart, 2003;Zachara and Hart, 2006). In fact, the interplay between O-GlcNAc modification and phosphorylation of proteins regulates many critical transcription factors, such as c-myc and p53 (Chou and Hart, 2001;Yang et al., 2006). O-GlcNAc modification also regulates other processes including proteosome function (Zachara and Hart, 2004). In addition to these regulatory roles, GlcNAc contributes to the N-linked glycosylation, glycophosphatidylinositol (GPI) anchor addition to proteins and is polymerized into chitin, which forms part of the fungal cell wall and the exoskele...

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“…Also, the cellular uptake of the signal molecules (FK506 vs. galactose) could be different. Because there is evidence suggesting that the Gal80p-Gal3p interaction is capable of perturbing the Gal80p-Gal4p binding reaction (29), it is also possible that the surrogate systems do not fully recapitulate the elements of Gal3p induction function.…”

Section: Gal Gene Activation Correlates With Decreased Binding Betweenmentioning

confidence: 99%

Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein

Peng

Hopper

2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

106

127

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Galactose-inducible genes (GAL genes) in yeast Saccharomyces cerevisiae are efficiently transcribed only when the sequencespecific transcription activator Gal4p is activated. Activation of Gal4p requires the interaction between the Gal4p inhibitory protein Gal80p and the galactokinase paralog, Gal3p. It has been proposed that Gal3p binds to a Gal80p-Gal4p complex in the nucleus to activate Gal4p. Here, we present evidence that the Gal3p-Gal80p interaction occurs in the cytoplasm, and concurrently, Gal80p is removed from Gal4p at the GAL gene promoter. We also show that GAL gene expression can be activated by heterologous protein-protein interaction in the cytoplasm that is independent of galactose and Gal3p function. These results indicate that galactose-triggered Gal3p-Gal80p association in the cytoplasm activates Gal4p in the nucleus.

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The Gal3p-Gal80p-Gal4p Transcription Switch of Yeast: Gal3p Destabilizes the Gal80p-Gal4p Complex in Response to Galactose and ATP

Cited by 69 publications

References 88 publications

In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer

In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer

Identification of anN-Acetylglucosamine Transporter That Mediates Hyphal Induction inCandida albicans

Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein

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