Browsing by Subject "Saccharomyces cerevisiae"
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Item Insights into the Metabolic Regulation by GATOR1 in Response to Amino Acid Signaling(2017-07-27) Chen, Jun; Liu, Yi; Tu, Benjamin; Phillips, Margaret A.; Goodman, Joel M.The GATOR1/SEACIT complex consisting of Iml1-Npr2-Npr3 inhibits Target of Rapamycin Complex 1 (TORC1) in response to amino acid insufficiency. In glucose medium, yeast mutants lacking the function of this complex grow poorly in the absence of amino acid supplementation, despite hallmarks of increased TORC1 signaling. Such mutants perceive they are amino acid-replete and thus repress metabolic activities that are important for achieving this state. I find that npr2∆ mutants have defective mitochondrial TCA cycle activity and retrograde response. Supplementation of glutamine, and especially aspartate, which are nitrogen-containing forms of TCA cycle intermediates, rescue growth of npr2∆ mutants. These amino acids are then consumed in biosynthetic pathways that require nitrogen to support proliferative metabolism. Our findings reveal that negative regulators of TORC1 such as GATOR1/SEACIT regulate the cataplerotic synthesis of these amino acids from the TCA cycle in tune with the amino acid and nitrogen status of cells.Item Insights into the Metabolic Regulation of Growth and Proliferation in Saccharomyces Cerevisiae(2013-10-04) Cai, Ling; Liu, Yi; DeBerardinis, Ralph J.; Li, Bing; Ross, Elliott M.Cells needs to gauge their capacity to grow based on nutrient availability, and adopt different metabolic strategies for optimal growth and survival. We have investigated the molecular mechanism of how growth decisions are made based on metabolic status and how metabolic enzymes are regulated by nutrient availability. In the first part of this study, we report that acetyl-CoA is the downstream metabolite of carbon sources that represents a critical metabolic signal for growth and proliferation. Upon entry into growth, intracellular acetyl-CoA levels increase substantially and consequently induce the Gcn5p/SAGA-catalyzed acetylation of histones at genes important vi for growth, thereby enabling their rapid transcription and commitment to growth. Acetyl-CoA functions as a carbon-source rheostat that signals the initiation of the cellular growth program by promoting the acetylation of histones specifically at growth genes. In the second part of the study, we report the dynamic modification of ribosome biogenesis transcription factor Ifh1p regulated by different metabolic cues. Ribosome biogenesis requires an enormous commitment of energy and resources in growing cells. We show that Ifh1p is dynamically acetylated and phosphorylated as a function of the growth state of cells. Ifh1p is acetylated at numerous sites in its N-terminal region by Gcn5p and deacetylated by NAD+-dependent deacetylases of the sirtuin family. Acetylation of Ifh1p is responsive to intracellular acetyl-CoA levels and serves to regulate the stability of Ifh1p. The phosphorylation of Ifh1p is mediated by Protein Kinase A and is dependent on TORC1 signaling. Instead of modulating overall rates of RP gene transcription or growth, these nutrient-responsive modifications of Ifh1p play a more prominent role in the regulation of cellular replicative lifespan. Finally, we report the different roles of acetyl-CoA synthetases Acs1p and Acs2p in yeast metabolism. While Acs2p is important for rapid growth in glucose medium, Acs1p has unique roles in more challenging growth conditions. It is important for yeast metabolic cycling and is recruited to foci structure near mitochondria that might be involved in shuttling acetyl-CoA into the mitochondria during hypoxia.Item An Isolated Clasp TOG Domain Suppresses Microtubule Catastrophe and Promotes Rescue(2019-04-12) Majumdar, Shreoshi; Zhang, Xuewu; Rice, Luke M.; Yu, Hongtao; Tu, BenjaminMicrotubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the CLASP family that reduce catastrophe and/or increase rescue also contain arrayed TOGs. How CLASP TOGs contribute to activity is poorly understood. Using S. cerevisiae Stu1 as a model CLASP, I report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. To begin with, I introduce microtubules, their dynamics and regulatory proteins in Chapter 1. In Chapter 2, I discuss how the two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. I also explore the structure of TOG2 and how it reveals a CLASP-specific residue that likely dictates distinctive tubulin-binding properties. Next, in Chapter 3, I study how, contrary to the expectation that TOGs must work in arrays, the isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function. Finally, in Chapter 4, I will summarize my work and provide insight into future directions.Item Metabolic Regulation at Sub-Organelle Length Scales: Inter-Organelle Contacts and Lipid Droplets(2021-09-29) Rogers, Sean W.; Radhakrishnan, Arun; Henne, W. Mike; Liou, Jen; Rosen, Michael K.For cells to properly respond to environmental changes, cellular interiors must be exquisitely organized both spatially and temporally. In particular, metabolism must be spatially coordinated so metabolites are appropriately shunted into either storage or growth. Despite our understanding of how membrane-bound organelles organize metabolic processes, little is known about how metabolic regulation occurs at sub-organelle length scales. At these length scales, physical interactions between the endoplasmic reticulum (ER) and other organelles at ER-membrane-contact-sites (ER-MCSs) are now recognized as sub-organelle hubs for the regulation of metabolic processes. Our work uses the nucleus-vacuole-junction (NVJ) in S. cerevisiae (yeast) as a model ER-MCS to further an understanding about potential general functions of ER-MCSs. We have noted that the NVJ, a physical connection between the nuclear-ER and the vacuole, is a hub for lipid metabolic enzymes and regulators. When yeast are exposed to low glucose conditions, the NVJ recruits several metabolic proteins, including the enzyme Hmg1. Hmg1 catalyzes the conversion of HMG-CoA to mevalonate and is the rate-limiting enzyme in sterol biogenesis. We noted that Hmg1 is less catalytically active when Nvj1, the protein that recruits Hmg1 to the NVJ, is genetically ablated, or when Nvj1 lacks a minimal motif required to recruit Hmg1. Hmg1 NVJ partitioning is accompanied by its assembly into high molecular weight species, which may underlie its increase in enzymatic efficiency. Indeed, artificial tetramerization of Hmg1 overcomes the deficiencies of an Nvj1 knock-out. During Hmg1 partitioning, mevalonate is preferentially shunted into synthesis of sterol-esters (SEs), which are storage lipids found in large cytoplasmic organelles, lipid droplets (LDs). Coordinately, glucose starvation promotes the degradation of triglycerides (TAGs), the other major lipid species contained in LDs. We found that the SE/TAG imbalance in LDs during glucose starvation leads to a phase separation of SEs from a liquid to liquidcrystalline state. Upon SE phase separation, the proteome of LDs is considerably changed. Collectively, our studies of the NVJ have identified a novel function for an ER-MCS and connected it to a lipid metabolic circuit that controls the proteome of LDs.Item Metabolic Regulation of Quiescence Entry and Exit in Saccharomyces cerevisiae(2014-12-03) Shi, Lei; Kohler, Jennifer J.; Yu, Hongtao; Cobb, Melanie H.; Tu, BenjaminUnicellular microorganisms often enter a state called quiescence when they encounter harsh environmental conditions. They stop growth and proliferation until conditions improve. In quiescence, the budding yeast slows down transcription three- to five-fold, while its translation rate drops to 0.3% of that in growth phase. Importantly, yeast quiescent cells have remarkably higher stress resistance than growing cells. Once conditions improve, they readily re-enter the cell cycle. Though quiescence is an important phase of cell life, its understanding has been limited. Previously, Allen et al. reported the isolation of quiescent yeast cells from stationary phase culture by cell density fractionation (Allen et al., 2006). Cells of high density, which they termed quiescent cells, were more stress-resistant and had more growth potential when conditions improved. However, it remained unknown how quiescent cells became dense and what mechanism allowed cells to enter quiescence. I report the intracellular glycogen and trehalose accumulation leads to increased density of quiescent yeast cells. Glycogen and trehalose are two carbon reserves yeast cells accumulate during entry into quiescence. Cells unable to produce glycogen and trehalose exhibit no density change during the entry of quiescence. Furthermore, yeast cells lacking trehalose dramatically slowed down the adaptation and growth in fresh nutrients. Thus, trehalose is a key determinant of the quiescent state and possibly fuels rapid cell cycle progression in the presence of fresh nutrients. When conditions improve, quiescent yeast cells readily re-enter growth. The proper regulation of quiescence exit in response to the environment is of vital importance to balance cell growth and quiescence. In budding yeast, CLN3 is one of the G1 cyclins that govern cell cycle entry and transition from G1 to S phase. CLN3 is the first activated G1 cyclin that subsequently induces the other G1 cyclins. Notably, CLN3 deletion slows down yeast cell cycle entry. Thus, studying CLN3 expression in response to nutrients may reveal the key mechanism of quiescence exit. I report acetyl-CoA induces immediate CLN3 transcription in quiescent yeast cells. Acetate derived surge of acetyl-CoA promotes extensive histone H3 acetylation at CLN3 promoter mediated by the SAGA complex, a histone H3 acetyltransferase. The acetylated histones loosen the chromatin at CLN3 promoter and facilitate rapid CLN3 transcription. Thus, acetyl-CoA is sensed by the SAGA complex to induce CLN3 transcription that promotes quiescence exit and regrowth. Altogether, my studies have revealed insights into how yeast cells enter and exit quiescence metabolically. In addition to yeast cell quiescence regulation, my studies may also aid in understanding the key mechanisms of quiescence regulation in higher eukaryotic systems.Item [News](1988-06-10) Fenley, BobItem Novel Insights into the Regulation of Autophagy in Saccharomyces Cerevisiae(2011-12-15) Wu, Xi; Tu, BenjaminAutophagy is an evolutionarily conserved pathway for the degradation of intracellular contents. How autophagy is regulated, especially upon changes in metabolic and nutritional state, remains poorly understood. In Saccharomyces cerevisiae, autophagy is normally triggered by nutrient starvation. However, by using a prototrophic strain, I discovered that autophagy can be strongly induced upon switch from a rich medium (YPL) to a minimal medium (SL) without nutrient starvation. This new autophagy-inducing condition was termed SL-induced autophagy. Growth measurement confirmed that SL-induced autophagy was important for cellular homeostasis and growth following medium switch. A genetic screen uncovered IML1, NPR2, NPR3 and PBP1, which are all required for SL-induced autophagy, but not for nitrogen-starvation-induced autophagy. Iml1p, Npr2p and Npr3p function in the same complex and regulate autophagosome formation. During SL-induced autophagy, Iml1p can localize to the pre-autophagosomal structures, consistent with the role of the Iml1p complex in autophagosome formation. Moreover, a conserved domain in Iml1p was identified to be required for SL-induced autophagy as well as complex formation. I discovered that sulfur containing amino acids, but not non-sulfur containing amino acids, can specifically inhibit SL-induced autophagy. I further demonstrated that cysteine is a key metabolite that inhibits SL-induced autophagy by regulating cellular processes related to cysteine metabolism. Cysteine does not suppress SL-induced autophagy by regulating oxidative stress, protein urmylation and thiolation of cytosolic tRNAs. Future studies will be required to reveal the exact mechanism through which cysteine inhibits SL-induced autophagy. I also discovered that autophagy can be significantly induced upon depletion of a Fe-S cluster containing protein, Rli1p, and other factors that are also involved in rRNA processing and translation initiation. Interestingly, IML1, NPR2, NPR3 and PBP1 are also important for Rli1p-depletion-induced mitophagy. These results strongly suggest the mechanistic link between SL-induced autophagy and ribosome biogenesis or translation regulation. Collectively, my studies have demonstrated the existence of additional mechanisms that regulate autophagy in response to relatively more subtle changes in metabolic and nutritional state.Item On Sulfur Sensing in Saccharomyces cerevisiae(December 2021) Johnson, Zane Miller; Nijhawan, Deepak; De Martino, George; Yu, Hongtao; Tu, BenjaminThe unique chemistry available to sulfur compared to oxygen, such as the ability to exist in numerous oxidation states and greater nucleophilicity, makes many of the biochemical reactions requisite for cellular life possible. As a result of this critical importance, organisms have developed several mechanisms for sensing and maintaining levels of sulfur-containing metabolites. In the yeast Saccharomyces cerevisiae, regulation of sulfur metabolism can be distilled down to the actions of two proteins; the F-box protein Met30, and the transcriptional coactivator Met4. Met30 belongs to the family of SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligases, and negatively regulates the transcriptional activity of the master transcriptional activator of sulfur metabolism genes, Met4, via oligo-ubiquitination when sulfur metabolite levels are high. When yeast are starved of sulfur, Met30 ceases to ubiquitinate Met4, releasing it to be deubiquitinated and transcriptionally active to boost levels of a network of sulfur metabolic genes known as the MET regulon to restore sulfur metabolite levels. While the molecular activities of both Met30 and Met4 have been extensively studied over the last two decades, the biochemical basis for sulfur-sensing by the Met30 E3 ligase has remained unknown. Herein, I reveal the biochemical details by which Met30, the master regulator of sulfur metabolism, senses the availability of sulfur metabolites to modulate its E3 ligase activity to regulate sulfur metabolism in yeast. Utilizing a combination of yeast genetics and biochemical assays, I show that Met30 uses redox-active cysteine residues in its C-terminal WD-40 repeat region to modulate binding between itself and its substrate Met4 in accordance with the availability of sulfur metabolites. These insights represent significant advances in the understanding of sulfur metabolic regulation in yeast.Item Quantitative Studies of Composition and Formation of Yeast P Bodies(2021-05-01T05:00:00.000Z) Xing, Wenmin; Nam, Yunsun; Rosen, Michael K.; Frederick, Kendra; Thomas, Philip J.Eukaryote cells organize their internal spaces into distinct compartments to achieve precise spatiotemporal regulation of biochemical reactions. One level of organization is achieved through membrane borders that form classical organelles such as nuclei and mitochondria. However, another widespread type of structures concentrates distinct molecular components without being enclosed by membranes--these are termed biomolecular condensates. Quantitative studies are lacking to mechanistically understand condensates within the complicated cellular environment. Toward this aim, I developed live cell imaging methods to quantitatively measure protein partitioning into condensates. Using P bodies, an archetypal biomolecular condensate that concentrate proteins and RNA, I first generated a quantitative inventory of the major proteins in yeast P bodies. I found that only 7 proteins are highly concentrated in P bodies while the 24 others examined are appreciably lower. P body concentration correlates inversely with cytoplasmic exchange rate. Based on the results, I proposed that the compositions of natural condensates can be classified into scaffold-like and client-like components based on their distinct partitioning and interaction network. To understand compositional specificity, I showed that sequence elements driving Dcp2 enrichment into P bodies are distributed across the protein, and that these elements act cooperatively. Multiple distributed enrichment elements provide a thermodynamic framework for regulating compositional specificity of P bodies. I further illustrated that changing the molecular interactions could shift phase boundaries, suggesting that behaviors of biomolecular condensates are dictated by molecular interactions. Taken together, my work provides a quantitative view of compositions and formation of natural biomolecular condensates.Item Switching the Fate of mRNAs for Mitochondrial Biogenesis(2017-03-02) Lee, Chien-Der; Liu, Yi; Tu, Benjamin; McKnight, Steven L.; Conrad, NicholasmRNAs encoding mitochondrial biogenesis proteins are co-regulated in a manner closely linked to metabolism. In yeast growing in glucose, mitochondrial biogenesis is repressed, but must be induced upon glucose depletion to enable energy production using alternative carbon sources such as ethanol or acetate through mitochondrial respiration. Yeast cells growing in glucose constitutively transcribe nuclear-encoded mitochondrial ribosomal mRNAs at a basal level. However, instead of sharing a common upstream activating sequence for transcription, those mRNAs all harbor a common sequence motif within their 3'UTRs. Puf3p, an RNA-binding protein, can directly bind to this class of mRNA transcripts to promote degradation in glucose medium. However, the function of Puf3p upon glucose depletion is not clear. In the first part of this study, I show how Puf3p responds to glucose availability to switch the fate of its bound transcripts that encode proteins required for mitochondrial biogenesis. This regulation allows cell to quickly respond to glucose depletion by switching the degradation fate of those mRNAs to translation. Thus, yeast can activate pre-existing mRNA without relying on de novo transcription for mitochondrial biogenesis. I then show Puf3p is subjected to phosphorylation downstream of a glucose sensing pathway. Puf3p is hypophosphorylated in glucose medium; however, upon glucose depletion, Puf3p becomes heavily phosphorylated within its N-terminal region of low complexity, associates with polysomes, and promotes translation of its target mRNAs. In the second part of this study, I show that phosphorylation of Puf3p is required for translational activation of its bound mRNAs. Strikingly, a Puf3p mutant that prevents its phosphorylation no longer promotes mRNA translation but also becomes trapped in intracellular foci in an mRNA-dependent manner. These findings suggest how the inability to properly resolve Puf3p-containing RNA-protein granules via a phosphorylation-based mechanism might be toxic to a cell. The toxicity might be due to sequestration of translational factors in the Puf3p RNA protein granule in a manner reminiscent of neurodegenerative disease-related protein aggregation.Item Yeast Ataxin-2 (Pbp1) Condensates Regulate TORC1 Activity and Autophagy in Response to Cellular Redox State(2018-11-26) Yang, Yu-San; O'Donnell, Kathryn A.; Tu, Benjamin; DeBerardinis, Ralph J.; Potts, Patrick RyanYeast ataxin-2, also known as Pbp1 (Poly(A) binding protein-binding protein 1), is an intrinsically disordered protein that has earlier been implicated in stress granule formation, RNA biology, and neurodegenerative disease. However, the normal endogenous function of Pbp1 and ataxin-2 remains poorly understood. In this dissertation, I identified Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Unlike the autophagy-deficient atg mutants that harbor severe growth defects, pbp1 null mutants exhibited significantly increased cell growth despite lack of autophagy. I discovered that Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region of Pbp1 forms reversible cross-β fibrils that facilitate phase transition of the protein into either liquid-like or gel-like states in vitro and enables self-association of full-length Pbp1 into pelletable assemblies in vivo. Sequence analysis revealed that Pbp1 LC region contains an unusually high frequency of methionine residues (24 methionines in 150 a.a.) compared to the rest of the yeast proteome. I showed that the phase separation of Pbp1 is mediated by these methionine residues, which are sensitive to H2O2-mediated oxidation and mitochondrial toxins in living cells. I also observed that the phase separation of Pbp1 mediated by its C-terminal LC region is responsive to the activity state of mitochondria and required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy in vivo. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling. These observations offer a mechanistic explanation describing how reversible formation of condensates formed from the LC region of Pbp1 has evolved as a sensor of cellular redox state.