Browsing by Subject "Saccharomyces cerevisiae Proteins"
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Item Characterization of Class D VPS Proteins(2003-03-11) Friedberg, Andrew Seth; Sternweis, Paul C.The vacuole of the yeast Saccharomyces cerevisiae is functionally similar to the mammalian lysosome. The components of the VPS (vacuolar protein sorting) system are responsible for proper delivery of vacuolar biosythetic enzymes. Efforts to dissect the genetics of this system have revealed several classes of mutants, each defective in one transport step in the VPS pathway. The Class D VPS proteins are thought to control anterograde traffic between the late Golgi and late endosome. Although most of these proteins have homologues of known function in other systems, two exceptions are the Vps3p and Vps8p proteins. Analysis of Vps3p reveals that it is associated with a highdensity structure, possibly a coated vesicle or a large protein complex. The Vps8p protein contains a C-terminal H2 RING finger motif, a domain often associated with E3 ubiquitin ligase activity. In vitro analysis reveals that a Vps8p fragment containing this domain has this activity. Deletion of the RING finger reveals that the endocytic marker Ste3p accumulates in an abnormally large late-endosome-derived structure, but that sorting of the soluble vacuolar cargo CPY is relatively unaffected. These results suggest a division of function within the Vps8p molecule.Item The Conserved Oligomeric Golgi (COG) Complex Is Required for Normal Import of Fatty Acids in Saccharomyces Cerevisiae(2004-08-19) Ballard, Johnathan L.; Goodman, Joel M.The goal of my work was to elucidate aspects of the mechanism of trafficking of membrane proteins to peroxisomes. The work described in this document centers around one protein from Saccharomyces cerevisiae, Cog7p. Cog7p is part of the conserved oligomeric Golgi (COG) complex. Results describing a basic function of Cog7p were published well after I began studying this protein. Nevertheless, I use the nomenclature outlined in that work. Cog7p functions in intra-Golgi vesicular transport in concert with seven other proteins. This protein complex is found in both yeast and mammals. We found Cog7p in a different context through a screen to identify proteins that function in the trafficking of membrane proteins to peroxisomes. In the screen a portion of Cog7p was found to interact with the membrane peroxisomal targeting sequence, mPTS, of the Candida boidinii peroxisomal membrane protein, Pmp47. I studied peroxisomal biogenesis in a strain of Saccharomyces cerevisiae in which the COG7 gene had been deleted. I showed that Cog7p was not required for peroxisomal biogenesis, but in so doing, established that Cog7p was required for the proper metabolism of fatty acids in a peroxisome-independent manner. I showed that Cog7p was required for the normal import of fatty acids; without Cog7p, yeast cells imported abnormally high amounts of free fatty acid from the environment. My results are consistent with the hypothesis that one or more protein(s) involved in fatty acid import require the COG complex for proper processing. My work ends before such a protein was identified, but I provide leads that if pursued would contribute to understanding the regulation of fatty acid import into yeast cells.Item Elucidating the Role of Yeast Lipin (PAH1) in Lipid Droplet Biogenesis(2012-07-17) Adeyo, Oludotun; Goodman, Joel M.Lipid droplets are unique organelles important for a host of cellular functions including the storage of neutral lipids, but factors that regulate the biogenesis and maintenance of these organelles remain relatively unknown. The primary focus of this dissertation will be to understand the role of the phosphatidic acid hydrolase (Pah1p) in the biogenesis of lipid droplets in Saccharomyces cerevisiae. Pah1p is an enzyme that converts phosphatidic acid to diacylglycerol, and its absence or elimination of its catalytic activity results in the accumulation of neutral lipids within membranes of the endoplasmic reticulum. Furthermore, lipid droplet formation is facilitated by diacylglycerol through a mechanism that appears to be independent of diacylglycrol’s role as a substrate for triglyceride biosynthesis. Finally, lipid droplets originated from regions of the endoplasmic reticulum where the Pah1p activators were located. The second part of this dissertation will focus on the lipodystrophy related protein Fld1p and its association with lipid droplets. Droplets always associate with Fld1p, and in the absence of lipid droplets, Fld1p is localized as patches distributed throughout the endoplasmic reticulum. In addition, induced lipid droplets originate from these Fld1 patches. I conclude from this work that diacylglycerol facilitates lipid droplet formation and that Fld1p is somehow involved in the biogenesis or maintenance of these organelles.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 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 Molecular Dissection of Bsc2: A Novel Negative Regulator of Triglyceride Lipolysis for a Lipid Droplet Subpopulation(December 2023) Speer, Natalie Ortiz; Goodman, Joel M.; Henne, W. Mike; Friedman, Jonathan R.; Nicastro, DanielaEukaryotic cells store lipids in the form of triglyceride (TG) and sterol-ester (SE) in cytoplasmic organelles called lipid droplets (LDs). Distinct pools of LDs with unique surface proteomes exist in cells, but a pervasive question is how proteins localize to and convey functions to specific LD subsets. Here, we show the yeast protein Bsc2 localizes to a specific subset of TG-containing LDs, and reveal it negatively regulates TG lipolysis. Mechanistically, Bsc2 LD targeting requires TG, and LD targeting is mediated by specific N-terminal hydrophobic regions (HRs) sufficient for Bsc2 function. Molecular dynamics simulations reveal these Bsc2 HRs interact extensively with TG on modeled LDs, and adopt a specific conformation on TG-rich LDs versus SE-rich LDs or a modeled ER bilayer. Bsc2-deficient yeast display no defect in LD biogenesis, but exhibit enhanced TG lipolysis dependent on the major TG lipase Tgl3. Remarkably, over-expression of Bsc2, but not LD protein Pln1, causes TG accumulation without altering SE levels. Finally, we find that Bsc2-deficient cells display altered LD accumulation during stationary phase growth. We propose that Bsc2 is a novel regulator of TG lipolysis that localizes to a subset of TG-enriched LDs and locally regulates TG lipolysis.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 Recognition Mechanisms of Nuclear Localization and Export Signals(2009-06-19) Süel, Katherine Elizabeth; Chook, Yuh MinThe transport of proteins between the nucleus and cytoplasm of cells is mediated by the Karyopherin beta family of proteins. Karyopherin betas recognize their substrates through either a nuclear localization or export signal depending on the direction of transport. Even though there are ten yeast import Karyopherin betas, for the past thirteen years there was only one well characterized nuclear localization signal, the classical nuclear localization signal. However, a second signal, the proline-tyrosine nuclear localization signal recognized by Karyopherin beta2, was recently identified through X-ray crystallography and biochemical studies of Karyopherin beta2 bound to one of its substrates. These studies identified rules for the recognition of the proline-tyrosine nuclear localization signal by Karyopherin beta2. The signal must have overall basic charge, structural disorder and a weak consensus sequence of an amino-terminal basic or hydrophobic-enriched region followed by a carboxyl-terminal arginine residue separated from a proline and tyrosine residue by two to five residues. The proline-tyrosine nuclear localization signal is also recognized by the Saccharomyces cerevisiae homolog of Karyopherin beta2, Karyopherin 104, demonstrating the generality of this import mechanism across eukaryotes. Thermodynamic analyses of the two known substrates of Karyopherin 104, Hrp1p and Nab2p, revealed physical properties governing its binding. The proline-tyrosine nuclear localization signal is an extended signal with significant sequence diversity. The signal is comprised of three binding epitopes, each of which can have varying energetic strengths in different substrates. The multivalent nature of the signal increases the diversity of the signal as well as the difficulty of identifying new substrates. A bioinformatics search identified putative proline-tyrosine nuclear localization signals which were validated through biochemical studies. Additionally, one of the proteins identified, Tfg2p, was verified as a bona fide Karyopherin 104 substrate. Analysis of Tfg2p's cellular localization revealed that its nuclear localization was not solely determined by the presence of a nuclear localization signal, but was also dependent on its retention in the nucleus. Furthermore, crystallographic studies of substrate Snurportin1 bound to the export karyopherin CRM1 revealed that its nuclear export signal has two binding epitopes implying that the multivalent nature of targeting signals may not be limited to the proline-tyrosine nuclear localization signal.Item Seipin Promotes Lipid Droplet Biogenesis(2013-01-17) Hilton, Christopher Lee; Goodman, Joel M.Seipin is an ER membrane protein that is required for adipogenesis in mammalians. Humans lacking functional seipin have virtually no visible adipose tissue. Seipin has been shown to be essential for the later stages of the adipogenic program in mouse pre-adipocytes. In yeast, the absence of seipin (Fld1p) leads to clusters of tiny lipid droplets or “supersized” ones, suggesting a role of the protein in droplet formation. To determine if this is true we created yeast strains that allowed us to “turn on” lipid droplet synthesis by the regulated expression of enzymes that create either triacylglycerol (TAG) or sterol ester (SE), the main neutral lipid components of droplets, in a droplet-null background with seipin (4KO) or without it (4KOfld1Δ). Using fluorescence microscopy, I showed that the number of newly formed TAG fluorescent bodies (individual droplets or clusters of unresolvable small droplets) decreased but their size increased in the absence of seipin. The large fluorescent bodies in 4KOfld1Δ were fluorescently dimmer and had an irregular perimeter compared to those in the 4KO strain, while their intracellular membranes stained with BODIPY had brighter fluorescence, suggesting that seipin is involved in the packaging of TAG. Electron microscopy showed that the TAG fluorescent bodies were clusters of small droplets. Levels of whole-cell TAG were generally similar during droplet formation, although somewhat lower at early time points. Seipin deletion had a milder effect on formation of SE fluorescent bodies. We conclude that seipin plays a direct role in normal lipid droplet assembly. Finally, in several side projects, I leaned about a possible role of seipin in droplet protein composition, the effects of different detergents on the seipin homo-oligomer, and the lack of a role of seipin in ER stress.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 A TOG:αβ-Tubulin Complex Structure Reveals Conformation-Based Mechanisms for a Microtubule Polymerase(2012-12-04) Ayaz, Pelin 1983-; Yu, Hongtao; Albanesi, Joseph P.; Rosen, Michael K.; Rice, Luke M.Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use alpha/beta-tubulin-interacting TOG (tumor overexpressed gene) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of alpha/beta-tubulin, but how they do so has remained unclear. In this study, we first introduce the polymerization blocked mutants of alpha/beta-tubulins that we developed as unique tools for biochemical studies of alpha/beta-tubulins to avoid the difficulties that has arisen from the self-assembly tendency of tubulins, then we report the structure of the TOG1 domain from Stu2p bound to the plus end polymerization blocked yeast alpha/beta-tubulin we created to facilitate crystallization. Our structure and further biochemical characterizations of the TOG1:alpha/beta-tubulin complex showed that TOG1 binds alpha/beta-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a “curved” conformation of alpha/beta-tubulin that cannot be incorporated into microtubules, contacting α- and β-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with alpha/beta-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of alpha/beta-tubulin, and how they selectively recognize the growing end of the microtubule.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.Item The Yeast Transcription Factor GAL4: A Model for Understanding Eukaryotic Transcription(2009-06-18) O'Neal, Melissa Ann; Kodadek, Thomas J.The 26S proteasome regulates numerous cellular pathways, including transcription, through proteolytic and non-proteolytic methods. The Kodadek and Johnston laboratories recently established a novel function for the proteasomal ATPases: the destabilization of activator-DNA complexes. This is independent of proteolysis but requires direct activator-ATPase interactions as well as ATP hydrolysis. The Gal4 mutant Gap71, which is hyper-sensitive to destabilization from a GAL promoter, was instrumental to this discovery. Gal4, but not Gap71, was mono-ubiquitylated in a HeLa nuclear extract and in vivo, suggesting that mono-ubiquitylation of an activator is critical to resisting destabilization by the proteasomal ATPases. To gain a better understanding of these events, the three amino acid substitutions in the Gap71 DNA-binding domain were individually cloned and analyzed for their contributions to the function of Gal4. The data showed that Serine 22 and Lysine 23 but not Lysine 25 were important for the efficiency of the activator. The charge at Lysine 23 was found to be important for Gal4-based transcription and subsequent in vitro work revealed that Gal4 was not only phosphorylated at Serine 22 but that this phosphorylation event was essential for the function of the activator. Many times a phosphorylation event precedes a mono-ubiquitylation event on an activator. Knowing the kinase and ligase machinery that modifies Gal4 would permit us to further test our model. As a result, I designed selection screens in an attempt to isolate the kinase and/or ligase machinery components that modify Gal4. While these particular enzymes were not identified, other novel genes were found to negatively affect the galactose utilization pathway, MSU1 and SPS1. Altogether, the data demonstrated that two post-translation events, phosphorylation and mono-ubiquitylation, prevent an activator-DNA complex from being disrupted, leading to an elegant model in which the proteasomal ATPases act as an important check point in transcription.