Upstream induction sequence, the cis-acting element required for response to the allantoin pathway inducer and enhancement of operation of the nitrogen-regulated upstream activation sequence in Saccharomyces cerevisiae

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...

Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 79%

Gln3p Nuclear Localization and Interaction with Ure2p inSaccharomyces cerevisiae

Kulkarni

Abulhamd

Rai

et al. 2001

“…Thus, we tried to identify polyamine-preferential transporters and found that DUR3 and SAM3 are polyaminepreferential transporters. DUR3 and SAM3 were originally reported as transporters for urea and S-adenosylmethionine (19,33). However, normally urea and S-adenosylmethionine do not exist at high levels outside cells.…”

Section: Discussionmentioning

confidence: 99%

“…Plasmids-For construction of YEpDUR3, the gene for the DUR3 open reading frame and its upstream region (19) was amplified by PCR from yeast X2180-1A (MATa SUC2 mal0 gal2 CUP1) genomic DNA as a template using primers DUR3F (5Ј-CGCGGATCCTTAGCCAAGACCAAAGGT-TCT-3Ј) and DUR3R (5Ј-CGGGGTACCACGACAGAGAT-GCAAAAAATG-3Ј). The resulting DNA fragment was digested with BamHI and KpnI and inserted into the same restriction sites of the plasmid YEp352 (20).…”

Section: Methodsmentioning

confidence: 99%

Polyamine Uptake by DUR3 and SAM3 in Saccharomyces cerevisiae

Uemura

Kashiwagi

Igarashi

2007

It has been reported that GAP1 and AGP2 catalyze the uptake of polyamines together with amino acids in Saccharomyces cerevisiae. We have looked for polyamine-preferential uptake proteins in S. cerevisiae. DUR3 catalyzed the uptake of polyamines together with urea, and SAM3 was found to catalyze the uptake of polyamines together with S-adenosylmethionine, glutamic acid, and lysine. Polyamine uptake was greatly decreased in both DUR3-and SAM3-deficient cells. The K m values for putrescine and spermidine of DUR3 were 479 and 21.2 M, respectively, and those of SAM3 were 433 and 20.7 M, respectively. Polyamine stimulation of cell growth of a polyamine requiring mutant, which is deficient in ornithine decarboxylase, was not influenced by the disruption of GAP1 and AGP2, but it was diminished by the disruption of DUR3 and SAM3. Furthermore, the polyamine stimulation of cell growth of a polyamine-requiring mutant was completely inhibited by the disruption of both DUR3 and SAM3. The results indicate that DUR3 and SAM3 are major polyamine uptake proteins in yeast. We previously reported that polyamine transport protein kinase 2 regulates polyamine transport. It was found that DUR3 (but not SAM3) was activated by phosphorylation of Thr 250 , Ser 251 , and Thr 684 by polyamine transport protein kinase 2.Polyamines (putrescine, spermidine, and spermine) in cells, which are essential for cell growth, are regulated by biosynthesis, degradation, and transport (1-4). With regard to polyamine transport, the properties of four polyamine transport systems were characterized in Escherichia coli (5-8). They include spermidine-preferential and putrescine-specific uptake systems as well as PotE (involved in the excretion of putrescine by a putrescine-ornithine antiporter activity) and CadB (involved in the excretion of cadaverine by a cadaverine-lysine antiporter activity). The former two transport systems function at neutral pH (2), whereas the latter two transport systems at acidic pH (9). In Saccharomyces cerevisiae, we identified four genes that encode polyamine excretion proteins TPO1-TPO4, mainly located on the plasma membrane (10 -12). We also found that UGA4 (located on vacuoles) can catalyze the uptake of ␥-aminobutyric acid and putrescine (13), and TPO5 (located on Golgi or post-Golgi secretory vesicles) can catalyze the excretion of polyamines (14). Furthermore, we reported that GAP1, located on the plasma membrane, can catalyze the uptake of putrescine and spermidine together with the uptake of amino acids (15). Although it has been reported that AGP2 can selectively catalyze the uptake of spermidine (16), there is also a report that AGP2 functions as an amino acid permease (17). In this study, we looked for proteins that can preferentially catalyze the uptake of polyamines in S. cerevisiae. We found that DUR3 can catalyze the uptake of polyamines together with urea, and SAM3 (which belongs to the family of amino acid polyamine-organocation transporters (18)) can catalyze the uptake of putrescine and spermidine together wit...

“…The yeast allantoicase gene DAL2 codes for a 345-amino acid protein (1)(2)(3). Expression of the allantoin pathway enzymes is (i) induced by allophanate, (ii) sensitive to nitrogen catabolite repression, and (iii) responsive to mutation of the DAL80 and DAL81 loci, which have been shown to regulate the allantoin degradation system (4,5).…”

mentioning

confidence: 99%

Crystal Structure of Yeast Allantoicase Reveals a Repeated Jelly Roll Motif

Leulliot

Quevillon‐Chéruel

Sorel

et al. 2004

Allantoicase (EC 3.5.3.4) catalyzes the conversion of allantoate into ureidoglycolate and urea, one of the final steps in the degradation of purines to urea. The mechanism of most enzymes involved in this pathway, which has been known for a long time, is unknown. In this paper we describe the three-dimensional crystal structure of the yeast allantoicase determined at a resolution of 2.6 Å by single anomalous diffraction. This constitutes the first structure for an enzyme of this pathway. The structure reveals a repeated jelly roll ␤-sheet motif, also present in proteins of unrelated biochemical function. Allantoicase has a hexameric arrangement in the crystal (dimer of trimers). Analysis of the protein sequence against the structural data reveals the presence of two totally conserved surface patches, one on each jelly roll motif. The hexameric packing concentrates these patches into conserved pockets that probably constitute the active site.