TorC1 and nitrogen catabolite repression control of integrated GABA shunt and retrograde pathway gene expression.

Despite our detailed understanding of how the lower GABA shunt and retrograde genes are regulated, there is a paucity of validated information concerning control of GAD1, the glutamate decarboxylase gene which catalyzes the first reaction of the GABA shunt. Further, integration of glutamate degradation via the GABA shunt has not been investigated. Here, we show that while GAD1 shares a response to rapamycin-inhibition of the TorC1 kinase, it does so independently of the Gln3 and Gat1 NCR-sensitive transcriptional activators that mediate transcription of the lower GABA shunt genes. We also show that GABA shunt gene expression increases dramatically in response to nickel ions. The α-ketoglutarate needed for the GABA shunt to cycle, thereby producing reduced pyridine nucleotides, derives from the retrograde pathway as shown by a similar high increase in the retrograde reporter, CIT2 when nickel is present in the medium. These observations demonstrate high integration of the GABA shunt, retrograde, peroxisomal glyoxylate cycle, and ß-oxidation pathways.

How to think about and do successful research What you probable did not learn when you first entered the laboratory.

There is a logic to doing successful research, but graduate students and indeed postdoctoral fellows and young independent investigators often learn it apprentice style, by experience. The purpose of this essay is to provide the product of that experience and advice that I have found useful to young researchers as they begin their training and careers.

Industry and academia-a perfect match.

My career developed very differently from those of most academic researchers. After school, I worked for 6 years in industries that employed yeast to manufacture ethanol and beer. At university, I was trained as a microbiologist with very little training in molecular biology. I retrained in 1987 in molecular yeast genetics and focused on genetic engineering of industrial yeasts to minimize the production of spoilage compounds in wine and ethyl carbamate, a carcinogen, in wine. The malolactic yeast ML01 and the urea-degrading yeast were the first genetically enhanced yeasts that obtained US FDA approval for commercial applications. Apart from applied research, I was fascinated by classic molecular yeast genetic studies using sophisticated techniques such as transcriptomics, proteomics, and metabolomics. Doing research at the University of British Columbia was stimulating and exciting, we established a core microarray and metabolomics facility that was used by many scientists at UBC and hospitals in Vancouver. I also established a state-of-the-art Wine Library that was used to study aging of wines produced in British Columbia. Finally, I have been fortunate to know and collaborate with leading yeast scientists who motivated me.

From blood to stress-my life investigating cell differentiation.

This short retrospective covers more than 50 years of research. I spent most of it doing yeast genetics and genetic engineering. It has been my great privilege to be part of the international group of yeast genetics researchers. With many of them named in this retrospective, I am connected in lifelong friendships and the same is true for my students and collaborators. The question which we wanted to ask is "How does the genome of the cell and cell differentiation adapt to changing and stressful environmental conditions?" The two examples we studied were sporulation and pseudohyphal growth. Both forms of differentiation are triggered by the stress of starvation. In the pathway of regulation of pseudohyphal growth, a yeast NADPH oxidase (discovered by our group) plays a major role.

Carl Singer (1945-2013) - Life on a Plate: yeast genetics meetings and micromanipulators.

Micromanipulators, more than any other instrument, opened the early doors to developing the powerful genetics of yeast that underlies much of the molecular work today. The ability to separate the spores of a tetrad and analyze their phenotypes generated the genetic maps and biology upon which subsequent cloning, sequencing, cutting edge molecular and cell biology depended. This work describes the development of those micromanipulators from garage to barn to factory and the developer of the sophisticated instruments we use today. For more than 30 years Carl Singer and his family were staunch and generous supporters of the International Conferences on Yeast Genetics and Molecular Biology meetings both in Europe and America. Carl Singer's displays at meetings became a traditional fixture and engaged the appetites of many students and advanced researchers to employ a technique that many perceived as too complicated or difficult, but which he made simple and easy to learn. His experiences also document a sketch of the international yeast meetings, their venues and how they developed through the years.

What do the pictures say-snapshots of a career.

What follows are snapshots of my career in chicken eyes, yeast and Rhodospirillum rubrum, castor beans, Escherichia coli and finally yeast again. In contrast, only a few of the failures that realistically make up a career are included. It is a tale of the generosity and influences of those who shaped what I am and what I learned in a wonderful profession. The science described is only that which I was lucky enough to do or was performed in my laboratory by those who really deserve the credit for any success that I've enjoyed. Not mentioned for lack of space are the critical contributions of many impressive investigators in the field of nitrogen-responsive regulation for no scientific investigation occurs in isolation.

Constitutive and nitrogen catabolite repression-sensitive production of Gat1 isoforms.

Nitrogen catabolite repression (NCR)-sensitive transcription is activated by Gln3 and Gat1. In nitrogen excess, Gln3 and Gat1 are cytoplasmic, and transcription is minimal. In poor nitrogen, Gln3 and Gat1 become nuclear and activate transcription. A long standing paradox has surrounded Gat1 production. Gat1 was first reported as an NCR-regulated activity mediating NCR-sensitive transcription in gln3 deletion strains. Upon cloning, GAT1 transcription was, as predicted, NCR-sensitive and Gln3- and Gat1-activated. In contrast, Western blots of Gat1-Myc(13) exhibited two constitutively produced species. Investigating this paradox, we demonstrate that wild type Gat1 isoforms (IsoA and IsoB) are initiated at Gat1 methionines 40, 95, and/or 102, but not at methionine 1. Their low level production is the same in rich and poor nitrogen conditions. When the Myc(13) tag is placed after Gat1 Ser-233, four N-terminal Gat1 isoforms (IsoC-F) are also initiated at methionines 40, 95, and/or 102. However, their production is highly NCR-sensitive, being greater in proline than glutamine medium. Surprisingly, all Gat1 isoforms produced in sufficient quantities to be confidently analyzed (IsoA, IsoC, and IsoD) require Gln3 and UASGATA promoter elements, both requirements typical of NCR-sensitive transcription. These data demonstrate that regulated Gat1 production is more complex than previously recognized, with wild type versus truncated Gat1 proteins failing to be regulated in parallel. This is the first reported instance of Gln3 UASGATA-dependent protein production failing to derepress in nitrogen poor conditions. A Gat1-lacZ ORF swap experiment indicated sequence(s) responsible for the nonparallel production are downstream of Gat1 leucine 61.

A domain in the transcription activator Gln3 specifically required for rapamycin responsiveness.

Nitrogen-responsive control of Gln3 localization is implemented through TorC1-dependent (rapamycin-responsive) and TorC1-independent (nitrogen catabolite repression-sensitive and methionine sulfoximine (Msx)-responsive) regulatory pathways. We previously demonstrated amino acid substitutions in a putative Gln3 α-helix(656-666), which are required for a two-hybrid Gln3-Tor1 interaction, also abolished rapamycin responsiveness of Gln3 localization and partially abrogated cytoplasmic Gln3 sequestration in cells cultured under nitrogen-repressive conditions. Here, we demonstrate these three characteristics are not inextricably linked together. A second distinct Gln3 region (Gln3(510-589)) is specifically required for rapamycin responsiveness of Gln3 localization, but not for cytoplasmic Gln3 sequestration under repressive growth conditions or relocation to the nucleus following Msx addition. Aspartate or alanine substitution mutations throughout this region uniformly abolish rapamycin responsiveness. Contained within this region is a sequence with a predicted propensity to form an α-helix(583-591), one side of which consists of three hydrophobic amino acids flanked by serine residues. Substitution of aspartate for even one of these serines abolishes rapamycin responsiveness and increases rapamycin resistance without affecting either of the other two Gln3 localization responses. In contrast, alanine substitutions decrease rapamycin resistance. Together, these data suggest that targets in the C-terminal portion of Gln3 required for the Gln3-Tor1 interaction, cytoplasmic Gln3 sequestration, and Gln3 responsiveness to Msx addition and growth in poor nitrogen sources are distinct from those needed for rapamycin responsiveness.

Premature termination of GAT1 transcription explains paradoxical negative correlation between nitrogen-responsive mRNA, but constitutive low-level protein production.

The first step in executing the genetic program of a cell is production of mRNA. In yeast, almost every gene is transcribed as multiple distinct isoforms, differing at their 5' and/or 3' termini. However, the implications and functional significance of the transcriptome-wide diversity of mRNA termini remains largely unexplored. In this paper, we show that the GAT1 gene, encoding a transcriptional activator of nitrogen-responsive catabolic genes, produces a variety of mRNAs differing in their 5' and 3' termini. Alternative transcription initiation leads to the constitutive, low level production of 2 full length proteins differing in their N-termini, whereas premature transcriptional termination generates a short, highly nitrogen catabolite repression- (NCR-) sensitive transcript that, as far as we can determine, is not translated under the growth conditions we used, but rather likely protects the cell from excess Gat1.

Five conditions commonly used to down-regulate tor complex 1 generate different physiological situations exhibiting distinct requirements and outcomes.

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc(13) localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc(13) localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal.

gln3 mutations dissociate responses to nitrogen limitation (nitrogen catabolite repression) and rapamycin inhibition of TorC1.

The GATA family transcription activator, Gln3 responds to the nitrogen requirements and environmental resources of the cell. When rapidly utilized, "good" nitrogen sources, e.g., glutamine, are plentiful, Gln3 is completely sequestered in the cytoplasm, and the transcription it mediates is minimal. In contrast, during nitrogen-limiting conditions, Gln3 quickly relocates to the nucleus and activates transcription of genes required to scavenge alternative, "poor" nitrogen sources, e.g., proline. This physiological response has been designated nitrogen catabolite repression (NCR). Because rapamycin treatment also elicits nuclear Gln3 localization, TorC1 has been thought to be responsible for NCR-sensitive Gln3 regulation. However, accumulating evidence now suggests that GATA factor regulation may occur by two separate pathways, one TorC1-dependent and the other NCR-sensitive. Therefore, the present experiments were initiated to identify Gln3 amino acid substitutions capable of dissecting the individual contributions of these pathways to overall Gln3 regulation. The rationale was that different regulatory pathways might be expected to operate through distinct Gln3 sensor residues. We found that C-terminal truncations or amino acid substitutions in a 17-amino acid Gln3 peptide with a predicted propensity to fold into an α-helix partially abolished the ability of the cell to sequester Gln3 in the cytoplasm of glutamine-grown cells and eliminated the rapamycin response of Gln3 localization, but did not adversely affect its response to limiting nitrogen. However, overall wild type control of intracellular Gln3 localization requires the contributions of both individual regulatory systems. We also found that Gln3 possesses at least one Tor1-interacting site in addition to the one previously reported.

Alterations in the Ure2 αCap domain elicit different GATA factor responses to rapamycin treatment and nitrogen limitation.

Ure2 is a phosphoprotein and central negative regulator of nitrogen-responsive Gln3/Gat1 localization and their ability to activate transcription. This negative regulation is achieved by the formation of Ure2-Gln3 and -Gat1 complexes that are thought to sequester these GATA factors in the cytoplasm of cells cultured in excess nitrogen. Ure2 itself is a dimer the monomer of which consists of two core domains and a flexible protruding αcap. Here, we show that alterations in this αcap abolish rapamycin-elicited nuclear Gln3 and, to a more limited extent, Gat1 localization. In contrast, these alterations have little demonstrable effect on the Gln3 and Gat1 responses to nitrogen limitation. Using two-dimensional PAGE we resolved eight rather than the two previously reported Ure2 isoforms and demonstrated Ure2 dephosphorylation to be stimulus-specific, occurring after rapamycin treatment but only minimally if at all in nitrogen-limited cells. Alteration of the αcap significantly diminished the response of Ure2 dephosphorylation to the TorC1 inhibitor, rapamycin. Furthermore, in contrast to Gln3, rapamycin-elicited Ure2 dephosphorylation occurred independently of Sit4 and Pph21/22 (PP2A) as well as Siw14, Ptc1, and Ppz1. Together, our data suggest that distinct regions of Ure2 are associated with the receipt and/or implementation of signals calling for cessation of GATA factor sequestration in the cytoplasm. This in turn is more consistent with the existence of distinct pathways for TorC1- and nitrogen limitation-dependent control than it is with these stimuli representing sequential steps in a single regulatory pathway.

Nitrogen-responsive regulation of GATA protein family activators Gln3 and Gat1 occurs by two distinct pathways, one inhibited by rapamycin and the other by methionine sulfoximine.

Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin.

Effects of abolishing Whi2 on the proteome and nitrogen catabolite repression-sensitive protein production.

In yeast physiology, a commonly used reference condition for many experiments, including those involving nitrogen catabolite repression (NCR), is growth in synthetic complete (SC) medium. Four SC formulations, SCCSH,1990, SCCSH,1994, SCCSH,2005, and SCME, have been used interchangeably as the nitrogen-rich medium of choice [Cold Spring Harbor Yeast Course Manuals (SCCSH) and a formulation in the methods in enzymology (SCME)]. It has been tacitly presumed that all of these formulations support equivalent responses. However, a recent report concluded that (i) TorC1 activity is downregulated by the lower concentration of primarily leucine in SCME relative to SCCSH. (ii) The Whi2-Psr1/2 complex is responsible for this downregulation. TorC1 is a primary nitrogen-responsive regulator in yeast. Among its downstream targets is control of NCR-sensitive transcription activators Gln3 and Gat1. They in turn control production of catabolic transporters and enzymes needed to scavenge poor nitrogen sources (e.g., Proline) and activate autophagy (ATG14). One of the reporters used in Chen et al. was an NCR-sensitive DAL80-GFP promoter fusion. This intrigued us because we expected minimal if any DAL80 expression in SC medium. Therefore, we investigated the source of the Dal80-GFP production and the proteomes of wild-type and whi2Δ cells cultured in SCCSH and SCME. We found a massive and equivalent reorientation of amino acid biosynthetic proteins in both wild-type and whi2Δ cells even though both media contained high overall concentrations of amino acids. Gcn2 appears to play a significant regulatory role in this reorientation. NCR-sensitive DAL80 expression and overall NCR-sensitive protein production were only marginally affected by the whi2Δ. In contrast, the levels of 58 proteins changed by an absolute value of log2 between 3 and 8 when Whi2 was abolished relative to wild type. Surprisingly, with only two exceptions could those proteins be related in GO analyses, i.e., GO terms associated with carbohydrate metabolism and oxidative stress after shifting a whi2Δ from SCCSH to SCME for 6 h. What was conspicuously missing were proteins related by TorC1- and NCR-associated GO terms.

Distinct phosphatase requirements and GATA factor responses to nitrogen catabolite repression and rapamycin treatment in Saccharomyces cerevisiae.

In yeast, rapamycin (Rap)-inhibited TorC1, and the phosphatases it regulates (Sit4 and PP2A) are components of a conserved pathway regulating the response of eukaryotic cells to nutrient availability. TorC1 and intracellular nitrogen levels regulate the localization of Gln3 and Gat1, the activators of nitrogen catabolite repression (NCR)-sensitive genes whose products are required to utilize poor nitrogen sources. In nitrogen excess, Gln3 and Gat1 are cytoplasmic, and NCR-sensitive transcription is repressed. During nitrogen limitation or Rap treatment, Gln3 and Gat1 are nuclear, and transcription is derepressed. We previously demonstrated that the Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for nuclear Gln3 localization differ. We now show that Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for NCR-sensitive and Rap-induced nuclear Gat1 localization markedly differ from those of Gln3. Our data suggest that Gln3 and Gat1 localizations are controlled by two different regulatory pathways. Gln3 localization predominantly responds to intracellular nitrogen levels, as reflected by its stronger NCR-sensitivity, weaker response to Rap treatment, and strong response to methionine sulfoximine (Msx, a glutamine synthetase inhibitor). In contrast, Gat1 localization predominantly responds to TorC1 regulation as reflected by its weaker NCR sensitivity, stronger response to Rap, and immunity to the effects of Msx. Nuclear Gln3 localization in proline-grown (nitrogen limited) cells exhibits no requirement for Pph21/22-Tpd3/Cdc55, whereas nuclear Gat1 localization under these conditions is absolutely dependent on Pph21/22-Tpd3/Cdc55. Furthermore, the extent to which Pph21/22-Tpd3-Cdc55 is required for the TorC1 pathway (Rap) to induce nuclear Gat1 localization is regulated in parallel with Pph21/22-Tpd3-Cdc55-dependent Gln3 dephosphorylation and NCR-sensitive transcription, being highest in limiting nitrogen and lowest when nitrogen is in excess.

N- and C-terminal Gln3-Tor1 interaction sites: one acting negatively and the other positively to regulate nuclear Gln3 localization.

Gln3 activates Nitrogen Catabolite Repression, NCR-sensitive expression of the genes required for Saccharomyces cerevisiae to scavenge poor nitrogen sources from its environment. The global TorC1 kinase complex negatively regulates nuclear Gln3 localization, interacting with an α-helix in the C-terminal region of Gln3, Gln3656-666. In nitrogen replete conditions, Gln3 is sequestered in the cytoplasm, whereas when TorC1 is down-regulated, in nitrogen restrictive conditions, Gln3 migrates into the nucleus. In this work, we show that the C-terminal Gln3-Tor1 interaction site is required for wild type, rapamycin-elicited, Sit4-dependent nuclear Gln3 localization, but not for its dephosphorylation. In fact, truncated Gln31-384 can enter the nucleus in the absence of Sit4 in both repressive and derepressive growth conditions. However, Gln31-384 can only enter the nucleus if a newly discovered second positively-acting Gln3-Tor1 interaction site remains intact. Importantly, the N- and C-terminal Gln3-Tor1 interaction sites function both autonomously and collaboratively. The N-terminal Gln3-Tor1 interaction site, previously designated Gln3URS contains a predicted α-helix situated within an unstructured coiled-coil region. Eight of the thirteen serine/threonine residues in the Gln3URS are dephosphorylated 3-15-fold with three of them by 10-15-fold. Substituting phosphomimetic aspartate for serine/threonine residues in the Gln3 URS abolishes the N-terminal Gln3-Tor1 interaction, rapamycin-elicited nuclear Gln3 localization, and ½ of the derepressed levels of nuclear Gln3 localization. Cytoplasmic Gln3 sequestration in repressive conditions, however, remains intact. These findings further deconvolve the mechanisms that achieve nitrogen-responsive transcription factor regulation downstream of TorC1.