Functional Domain Mapping and Subcellular Distribution of Dal82p in Saccharomyces cerevisiae

Scott, Stephanie; Dorrington, Rosemary A.; Svetlov, Vladimir; Beeser, Alexander; Distler, Mackenzie; Cooper, Terrance G.

doi:10.1074/jbc.275.10.7198

Cited by 13 publications

(19 citation statements)

References 51 publications

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“…(i) LexA-Dal82p mediates Gln3p-dependent lacZ expression under experimental conditions similar to those reported here (33,44). Furthermore, there is independent genetic evidence supporting the contention that Dal82p and Gln3p interact with one another, i.e.…”

Section: Discussionsupporting

confidence: 57%

“…6A) with NLS-lexA-URE2 pAA15, lexA operator -lacZ pHS18-34, and a GLN3-B42 plasmid (33,44) as bait, reporter, and prey, respectively. To our initial surprise, transformants of wild type cells, containing only the bait and reporter plasmids (pAA15 plus pHS18-34) produce high level β-galactosidase (Fig.…”

Section: Two-hybrid Assays With Ure2p As Baitmentioning

confidence: 99%

“…Samples were prepared for fluorescence microscopy as described earlier (9,15,33). Images were irradiated with a combination of white and/or epi-fluorescent light, viewed using the 63× objective of a Zeiss Axiophot microscope equipped with a GFP filter set and collected with a Zeiss Axiocam digital camera using AxioVision 2.0.5.3 software.…”

Section: Fluorescence Microscopymentioning

confidence: 99%

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Gln3p Nuclear Localization and Interaction with Ure2p inSaccharomyces cerevisiae

Kulkarni

Abulhamd

Rai

et al. 2001

Journal of Biological Chemistry

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Gln3p is one of two well characterized GATA family transcriptional activation factors whose function is regulated by the nitrogen supply of the cell. When nitrogen is limiting, Gln3p and Gat1p are concentrated in the nucleus where they bind GATA sequences upstream of nitrogen catabolite repression (NCR)-sensitive genes and activate their transcription. Conversely, in excess nitrogen, these GATA sequences are unoccupied by Gln3p and Gat1p because these transcription activators are excluded from the nucleus. Ure2p binds to Gln3p and Gat1p and is required for NCR-sensitive transcription to be repressed and for nuclear exclusion of these transcription factors. Here we show the following. (i) Gln3p residues 344-365 are required for nuclear localization. (ii) Replacing Ser-344, Ser-347, and Ser-355 with alanines has minimal effects on GFP-Gln3p localization. However, replacing Gln3p Ser-344, Ser-347, and Ser-355 with aspartates results in significant loss of its ability to be concentrated in the nucleus. (iii) N and C termini of the Gln3p region required for it to complex with Ure2p and be excluded from the nucleus are between residues 1-103 and 301-365, respectively. (iv) N and C termini of the Ure2p region required for it to interact with Gln3p are situated between residues 101-151 and 330-346, respectively. (v) Loss of Ure2p residues participating in either dimer or prion formation diminishes its ability to carry out NCR-sensitive regulation of Gln3p activity.Saccharomyces cerevisiae is increasingly used as a model to identify the functions of mammalian proteins as well as how their production and activities are regulated and integrated. One of the gene families shared by S. cerevisiae and higher eukaryotes is the GATA family of DNA-binding proteins. In animal cells, GATA family proteins were originally shown to be responsible for regulating globin gene expression (1). However, they are now known to regulate a diverse set of developmental functions (2, 3). In yeast, the GATA family proteins Gln3p, Gat1p/Nil1p, Dal80p, Deh1p/Gzf3p have been studied as the main regulators of nitrogen catabolic gene expression (4-6), and recently their regulatory functions also been shown to be far more diverse (7,8). Gln3p and Gat1p are transcriptional activators, whereas Dal80p and Deh1p repress transcription by competing with these activators for binding to their target GATA sequences (4)(5)(6)(9)(10)(11) HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptGln3p and Gat1p function is also dramatically regulated by the nitrogen supply of the cell. When nitrogen is limiting, Gln3p and Gat1p are concentrated in the nucleus, bind to the GATA sequences of their target promoters, and activate transcription (8,9,(12)(13)(14)(15). On the other hand, when nitrogen is in excess, Gln3p and Gat1p are excluded from the nucleus, and nitrogen catabolite repression (NCR) 1 -sensitive gene expression is repressed. Nuclear exclusion of Gln3p and Gat1p requires Ure2p, the first NCR regulator identified (16...

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Section: Discussionsupporting

confidence: 57%

Section: Two-hybrid Assays With Ure2p As Baitmentioning

confidence: 99%

Section: Fluorescence Microscopymentioning

confidence: 99%

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Gln3p Nuclear Localization and Interaction with Ure2p inSaccharomyces cerevisiae

Kulkarni

Abulhamd

Rai

et al. 2001

Journal of Biological Chemistry

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“…The resulting PCR products were cloned into pEGKG and completely sequenced; the constructs encoded the Yvh1p C-terminal 159 (pAB116) and 74 (pAB117) aa. To construct EGFP (enhanced green fluorescent protein) fusion proteins, pAB7 and pAB18 were digested with NdeI and the NdeI fragments were cloned into pNVS2, a pRS316-based, galactose-inducible EGFP fusion vector (37).…”

Section: Strains and Plasmidsmentioning

confidence: 99%

The Dual-Specificity Protein Phosphatase Yvh1p Regulates Sporulation, Growth, and Glycogen Accumulation Independently of Catalytic Activity in Saccharomyces cerevisiae via the Cyclic AMP-Dependent Protein Kinase Cascade

Beeser

Cooper

2000

J Bacteriol

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Yvh1p, a dual-specific protein phosphatase induced specifically by nitrogen starvation, regulates cell growth as well as initiation and completion of sporulation. We demonstrate that yvh1 disruption mutants are also unable to accumulate glycogen in stationary phase. A catalytically inactive variant of yvh1 (C117S) and a DNA fragment encoding only the Yvh1p C-terminal 159 amino acids (which completely lacks the phosphatase domain) complement all three phenotypes as well as the wild-type allele; no complementation occurs with a fragment encoding only the C-terminal 74 amino acids. These observations argue that phosphatase activity is not required for the Yvh1p functions we measured. Mutations which decrease endogenous cyclic AMP (cAMP) levels partially suppress the sporulation and glycogen accumulation defects. In addition, reporter gene expression supported by a DRR2 promoter fragment, containing two stress response elements known to respond to cAMP-protein kinase A, decreases in a yvh1 disruption mutant. Therefore, our results identify three cellular processes that both require Yvh1p and respond to alterations in cAMP, and they lead us to suggest that Yvh1p may be a participant in and/or a contributor to regulation of the cAMP-dependent protein kinase cascade. The fact that decreasing the levels of cAMP alleviates the need for Yvh1p function supports this suggestion.

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“…(Row 3) Enlargements of the B and C frames of row 2. (Row 4) GYC86 transformed with pNVS82, containing GAL1,10-EGFP-DAL82, prepared as described earlier (22). The slide in frame 4A was illuminated with a small amount of white as well as fluorescent light; in frame 4B only fluorescent light was used.…”

Section: Cooper Unpublished Data)mentioning

confidence: 99%

Green Fluorescent Protein-Dal80p Illuminates up to 16 Distinct Foci That Colocalize with and Exhibit the Same Behavior as Chromosomal DNA Proceeding through the Cell Cycle of Saccharomyces cerevisiae

et al. 2001

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Functional Domain Mapping and Subcellular Distribution of Dal82p in Saccharomyces cerevisiae

Cited by 13 publications

References 51 publications

Gln3p Nuclear Localization and Interaction with Ure2p inSaccharomyces cerevisiae

Gln3p Nuclear Localization and Interaction with Ure2p inSaccharomyces cerevisiae

The Dual-Specificity Protein Phosphatase Yvh1p Regulates Sporulation, Growth, and Glycogen Accumulation Independently of Catalytic Activity in Saccharomyces cerevisiae via the Cyclic AMP-Dependent Protein Kinase Cascade

Green Fluorescent Protein-Dal80p Illuminates up to 16 Distinct Foci That Colocalize with and Exhibit the Same Behavior as Chromosomal DNA Proceeding through the Cell Cycle of Saccharomyces cerevisiae

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