Jeremy Thorner - US grants

The two haploid cell types (a and Alpha) of the yeast Saccharomyces cerevisiae each secrete specific peptide hormone-like molecules which trigger events leading to fusion ("mating") of the cells, a relatively simple developmental sequence. Because incisive genetic and biochemical studies can be performed in yeast cells, particular those utilizing recombinant DNA, it may be possible to learn the precise molecular details of peptide hormone biosynthesis and mode of action by examining the yeast mating pheromones (a-factor and Alpha-factor). The approaches to be taken include: (1) in vitro alterations of the control regions of the cloned Alpha-factor and a-factor genes, and novel gene fusions to indicator enzymes, to dissect the cell type-specific regulation of transcription of these genes; (2) specific antibodies, inhibitors, and mutations, in conjunction with the cloned genes, to provide probes and substrates for analyzing the synthesis, membrane translocation, post-translational processing, and secretory transport of the pheromone precursor proteins (prepro-Alpha-factor and prepro-a-factor); (3) [125I]-labelled pheromones, covalent cross-linking of reactive pheromone derivatives, affinity chromatography using matrix-bound pheromones, putative genes for Alpha-factor receptor (STE2) and adenylate cyclase (CDC35), and the cloned STE5 gene, to identify pheromone receptors and to examine the mechanism of pheromonal modulation of cyclic AMP synthesis; and, (4) transcript mapping, gene disruption, and DNA sequencing, to characterize the structure and function of cloned genes whose expression is under pheromonal control. In multicellular organisms, peptide hormones are responsible for cell-cell communications that are required during embryonic development and in the coordination of life functions in adults. The under- or over-production of particular peptide hormones, or the inability to sense their presence, have been correlated with numerous human diseases (e.g. dwarfism, Cushing's disease, and diabetes). Understanding of the fundamental aspects of the synthesis and release of peptide hormones, and the regulation of these processes, as well as the biochemical bases of their mechanisms of action, is necessary for effective diagnosis and therapy of such diseases. In addition, knowledge of the biosynthesis of yeast pheromones may allow the production of medically-valuable human hormones by yeast cells on a commercial scale.

The objective of this research is to learn the precise molecular details of both the biosynthesis of peptide hormones and the mechanism of action of peptide hormones. Secreted bioactive peptides (peptide hormones, growth factors, neuroregulatory pep- tides) are indispensable agents of the intercellular interactions that control both embryonic development and the coordination and integration of life functions in the adult. Numerous human diseases are associated with the under- (or over-) production of regulatory peptides, or with the inability to respond properly to such signals. Even the unicellular eukaryotic microorganism Saccharomyces cerevisiae (baker's yeast) secretes and responds to peptide hormones ("mating pheromones")- Because incisive genetic and biochemical studies can be performed in yeast cells, particularly those that utilize recombinant DNA, study of peptide hormone biogenesis and signal transduction in yeast may provide useful insights for understanding the molecular basis of diseases that affect the human neuroendocrine system. Four specific areas of investigation are proposed: (1) The molecular basis of cell type-specific gene expression will be studied by examining the transcriptional regulation of the MF a1 gene (which encodes a precursor, prepro-a-factor, of one of the mating pheromones)- Genetic and biochemical experiments are proposed for further defining both the cis-acting sites and the trans-acting factors responsible for the control of this gene, and for exploring the role of a particular "zinc finger"- containing transcription factor (STE5 gene product). (2) The enzymic basis of pre= cursor maturation at sites comprised of pairs of basic residues will be explored by determination of the three-dimensional structure of a yeast precursor cleaving enzyme (KEX2 gene product) with this specificity and by using probes based on this yeast gene to isolate its mammalian homolog. (3) How a plasma membrane receptor acts as a ligand-triggered molecular switch and how its function is regulated during desensitization will be elucidated using the cloned alpha-factor receptor gene (STE2), its coupled G a subunit (GPAl gene product), and a regulatory factor (SST2 gene product) of as yet unknown function. (4) Hormonal induction of gene transcription will be studied by genetic and biochemical experiments designed to identify and characterize the protein factors that interact with an upstream pheromone-response element (PRE).

The mating pheromone response pathway of budding yeast, Saccharomyces cerevisiae, is arguably the best understood multi-tiered, mitogen- activated protein kinase (MAPK)-stimulating signaling cascade yet eludicated in any eukaryotic cell. It is now clear, however, that to coordinate the changes in gene expression and cell morphology necessary for mating, response to pheromone requires an elaborate network of interlocking events rather than a simple linear pathway. In addition, feedback mechanisms exist that modulate the efficiency and duration of the events required for signaling at essentially every step. Moreover, this signaling pathway must evoke an appropriate response upon the correct stimulus, yet avoid adventitious activation under inappropriate circumstances. In this regard, it has been shown recently that many of the same components required for pheromone response are also utilized for a different developmental outcome, termed filamentous or invasive growth, that occurs in response to nutrient limitation. How different extracellular signals impinge on the same MAPK cascade, yet are deciphered differently, is not fully understood in any organism. Thus, yeast continues to provide an opportunity to examine fundamental aspects of the organization, specificity, fidelity, and regulation of signal transmission, including how the same signaling components can be coupled to different upstream inputs and downstream responses in the same cell type. Specific aims include: further eludication of the specificity of MEK-MAPK recognition; genetic and biochemical analysis of the regulatory function of the RING-H2 domains of the scaffold proteins, Ste5 and Far1, and their interaction with Gbetagamma (Ste4-Ste18), including development of a new, Gbetagamma-based method for identification of coiled-coil interactions applicable to functional genomics; determination of the crystal structure of the various domains of Ste5 and the mechanism of its regulated nuclear import and export; elucidation of the function and solution structure of Ste50, whose role is as yet ill-defined; biochemical characterization of the novel transcriptional regulators, Dig1 and Dig2, and their function in down- modulation and promoter-specific discrimination in Ste12-dependent gene expression; and, implementation of genetic screens to pinpoint as yet unidentified components unique to the pheromone-response and filamentous-growth signaling pathways. Knowledge gained by studying MAPK signaling in yeast may provide insights ultimately of clinical value for developing therapies against cancers because inappropriate MAPK activation, such as that evoked by well-known oncoproteins (including Ras and Raf), leads to tumor formation.

[unreadable] DESCRIPTION (provided by applicant): The Saccharomyces cerevisiae mating pheromone response pathway is arguably the best understood multi-tiered, MAPK signaling cascade in any eukaryote. However, coordinating the changes in gene expression and cell morphology necessary for mating involves an elaborate network of interlocking events rather than a simple linear pathway. In addition, feedback mechanisms exist to modulate the efficiency and duration of these signaling events at essentially every step. Moreover, this signaling pathway must evoke an appropriate response upon the correct stimulus, yet avoid adventitious activation. Also, it is now appreciated that many components required for pheromone response are also utilized for a different developmental outcome, termed filamentous/invasive growth, in response to nutrient limitation. How different extracellular signals impinge on the same MAPK cascade, yet are deciphered differently, is not understood fully in any organism. Yeast continues, therefore, to provide opportunities to examine basic aspects of the organization, specificity, fidelity, and regulation of MAPK signaling pathways, including how the same components can be coupled to different upstream inputs and downstream responses in the same cell type. Specific aims include: (1) Characterization of Ste5 scaffold protein, including crystal structure determination, elucidating the mechanism of its regulated nucleocytoplasmic transport, and genetic and biochemical analysis of the role of its RING-H2 domain in Ste5 oligomerization, in intra- and intermolecular ubiquitinylation, and in interaction with Gbeta/gamma (Ste4-Ste18). (2) Development of a new method for visualization of signaling protein dynamics in real time in live cells based on exploitation of the fluorescent properties of cyanobacterial phycobiliproteins, also applicable to functional genomics. (3) Genetic and biochemical characterization of the adaptor protein, Ste50, and its interaction with the small GTPase, Cdc42, including solution structure determination. (4) Testing a hypothesis, using yeast RGS protein, Sst2, that DEP domains associate with Gbeta/gamma. (5) Exploring the possibility that the requirement for PKA function in filamentation is to sustain MAPK signaling by blocking MAPK phosphatase action. (6) Investigating the role of phosphoinositides in membrane recruitment of Bem1 adaptor protein and in the mechanisms by which pheromone signaling interdicts both bud formation and cytokinesis. (7) Biochemical characterization of repression and promoter-specific discrimination by transcriptional regulators, Dig1 and Dig2, in Ste12-dependent gene expression. Studying yeast MAPK signaling may provide insights for anti-cancer therapy because inappropriate MAPK activation in humans, evoked by well-known oncoproteins (e.g. Ras and Raf), leads to tumor formation.

roiect Objectives: The S. cerevisiae mating pheromone response pathway is arguably the best understood multi-tiered MARK signaling cascade in any eukaryote. It is now appreciated, however, that coordinating the changes in gene expression and cell morphology necessary for mating involves an elaborate network of nterlocking events, rather than a simple linear pathway. In addition, feedback mechanisms exist to modu- ate the efficiency and duration of these signaling events at essentially every step. Moreover, this signaling oathway must evoke an appropriate response upon the correct stimulus, yet avoid adventitious activation. Furthermore, it is now established that some components required for pheromone response are also utilized both for a different developmental outcome, termed filamentous/invasive growth (triggered by nutrient limita- tion) and for response to hyperosmotic stress. How different extracellular signals impinge on the same MARK elements, yet are deciphered differently, is not fully understood in any organism. For all of these reasons, yeast continues to provide opportunities to examine basic properties of the organization, specificity, Fidelity, regulation and function of MARK signaling pathways, including how certain molecules that participate in signaling can be coupled to different upstream inputs and downstream responses in the same cell type. Specific aims include: (1) Further characterization of two pivotal scaffold proteins, Ste5 and SteSO, including crystal structure determination and, in the case of Ste5, the mechanisms by which its RING-H2 domain contributes to oligomerization, intra- and intermolecular ubiquitinylation, interaction with Gpy (Ste4-Ste18) and other aspects of its function. (2) Further analysis of the function of plasma membrane phosphoinositides in the membrane recruitment of Ste5 (via its PH domain) and the Bem1 adaptor protein (via its PX domain). (3) Further development of a novel FRET-based reporter we devised recently to visualize spatiotemporal features of MARK activation in real time in live cells. (4) Examination of the biochemical basis by which pheromone signaling affects normal septin filament assembly and interdicts cytokinesis. (5) Investigation of how the MARK, Kss1, contributes to control of the apparent IRES elements in the transcripts for gene products required for invasive growth. (6) Determination at the molecular level of the mechanism by which the stress-activated MARK, Hog1, blocks inappropriate activation of the other two pathways that utilize the sameMAPKKK(Ste11). Public Health Relevance: The growth of many human tumor cells can be traced to mutations that lead directly to inappropriate and persistent MARK activation. Thus, studying fundamental aspects of MARK signaling may provide insights for the development of more effective anti-cancer therapies to ameliorate certain prevalent malignancies in people.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Septins are GTP-binding proteins encoded by paralogous genes found in the genomes of nearly every eukaryote examined. These proteins are involved in the remodeling of and demarcation of compartments within the plasma membrane, for example during cytokinesis (Finger, 2005) and in the maturation of dendritic spines in neurons (Caudron and Barral, 2009). Unicellular eukaryote Saccharomyces cerevisiae (baker's yeast) encodes seven septin genes, and Homo sapiens fourteen (Pan et al., 2007). Any given eukaryotic cell type expresses multiple sets of septins, often in a cell type- and developmental stage-specific manner. In all cases known, the protein products of those genes associate in a defined order and assemble into a linear array (McMurray and Thorner, 2008a;Weirich et al., 2008). Moreover, the resulting linear hetero-oligomer ("rod") is a building block that has the capacity to undergo end-on-end polymerization, thereby forming filaments. It is known that these filaments are involved with cell division, but fluorescence microscopy alone has not revealed sufficient details to thoroughly understand the exact mechanism. We propose to use correlated fluorescence and x-ray imaging to determine the location of each of the septins in the yeast, Saccharomyces cerevisiae.

PROJECT SUMMARY Septins are a family of conserved GTP-binding proteins that assemble into hetero-oligomeric complexes and polymerize into filaments and other supramolecular arrangements. Septin structures associate with the plasma membrane by interacting with a specific phosphoinositide, and are involved in cell compartmentation, in cell division, and other membrane remodeling events. In budding yeast, septins establish a diffusion barrier at the neck between a mother and daughter cell, promote membrane curvature, and act as a scaffold to recruit other proteins to the site of cytokinesis. In humans, septin structures serve very similar functions; they are localized at the cleavage furrow, at the root of the primary cilium, at the base of every dendritic spine, and within the annulus in spermatozoa. Marked alterations of septin gene expression are found in solid tumors of certain tissues and septin gene translocations in mixed lineage leukemias. SEPT9 mutations are one apparent cause of hereditary neuralgic amyotrophy. Substitution mutations preventing GTP binding to SEPT12 disrupt the sperm annulus and cause male infertiity. In-depth structural and functional analysis of septins is necessary to understand the molecular mechanisms governing septin organization and function and obtain new insights to illuminate their pathophysiology. This project aims to apply novel tools and the experimental advantages of yeast to interrogate septin-based structures and eludicate fundamental properties of their organization, regulation and function that should be applicable to the highly homologous septin-based structures in human cells. Our specific aims include experimental tests of the following hypotheses. (1) Post-translational modifications (PTMs) of septins and septin-associated proteins drive the dramatic changes in septin structural organization that occur during passage through the cell division cycle. Therefore, comprehensive analysis by mass spectrometry of PTMs (especially phosphorylation and SUMOylation) that septins undergo during cell cycle progression, and subsequent genetic analysis of the physiological importance of those modifications by site-directed mutagenesis in vivo and a FRET-based method in vitro, will be conducted. (2) Sequential recruitment of septin-binding proteins exert and order the spatio-temporal changes in septin organization and function that impose proper cell morphology. Hence, comprehensive analysis of the in vivo septin interactome using a tripartite split-GFP method will be carried out. In addition, a novel clonable tag for in vivo labeling and protein localization at the ultrastructural level, which will be applied to dynamic analysis of the septin interactome using correlated light (fluorescence) and electron microscopy (CLEM), will be developed; and, (3) Association with PtdIns4,5P2 is obligatory for septin recruitment to the plasma membrane. Thus, to gain unprecedented insight as to how septins associate with a PtdIns4,5P2-containing lipid surface and how specific septin-binding proteins influence that interaction, the newest instrumentation for cryo-EM will be used to visualize these structures at near-atomic resolution.  

DESCRIPTION (provided by applicant): Mechanisms of gene-specific and genome-wide regulation of mRNA turnover Central to the control of eukaryotic gene expression is the precise and rapid regulation of transcript abundance. RNA levels are determined by a balance of both production and degradation, and thus, it is critical to examine not only transcription but also mRNA turnover to dissect the regulatory networks that control gene expression and to understand the kinetics of the cellular response. Whereas much has been learned about the mechanisms controlling transcription, the contribution of mRNA decay to shaping the transcriptome remains poorly understood. The objective of this research proposal is to elucidate the mechanisms by which mRNA turnover regulates gene-specific and genome-wide changes in mRNA levels, and to understand how mRNA decay and transcription are coordinated to induce rapid changes in the transcriptome. The experiments described in this proposal take advantage of a non-invasive metabolic labeling approach that allows us to measure decay rates with great precision for all mRNAs in budding yeast. Yeast provides an excellent model system to characterize the regulation of mRNA turnover, and because the pathways under investigation are highly conserved across species, any mechanistic insights obtained from our studies will be directly relevant to all eukaryotes including humans. We propose a combination of innovative biochemical, genetic and cell biological approaches to address three specific aims: (1) To elucidate mechanisms that induce the turnover of specific groups of mRNAs. (2) To investigate the physiological function of the mRNA decay factor Dhh1 and to understand how Dhh1 controls translational repression and mRNA degradation. (3) To identify mechanisms of genome-wide mRNA turnover regulation. This project will lead to the discovery of novel molecular pathways that regulate mRNA decay and provide fundamental new insight into an important step in the eukaryotic gene expression program critical for all aspects of cellular and organismal physiology. Furthermore, understanding the regulation of mRNA decay will give us critical insight into how this process is misregulated in human disease.

? DESCRIPTION (provided by applicant): Like human cells, budding yeast (Saccharomyces cerevisiae) cells contain protein kinases that control virtually all aspects of their physiology, morphology and development, especially multi-tiered protein kinase cascades, such as mitogen- / messenger-activated protein kinase (MAPK) pathways and the Target of Rapamycin (TOR) complexes, TORC1 and TORC2, and the protein kinases regulated by them. Moreover, all of the classes of protein kinases that evolved in yeast have been conserved in humans. The yeast mating pheromone response pathway (Fus3 MAPK), initiated by a G-protein-coupled receptor (GPCR), is arguably the best understood MAPK pathway in any eukaryote. Likewise, the existence and discrete functions of TORC1 and TORC2 were first delineated in yeast. However, many basic questions remain about how such protein kinase-based signaling pathways are arranged to maintain specificity, how such pathways are integrated, and how they modulate the processes and behaviors under their control, especially coordination of changes in cell growth, plasma membrane expansion and polarity. The overall goals of this project are to develop novel methodological tools for globally interrogating the logic circuitry of protein kinase action and to use them, and the experimental advantages of yeast, to continue to examine fundamental properties of the organization, fidelity, regulation and function of protein kinase signaling pathways, as a means of undercovering additional new principles and processes generally applicable to the highly homologous pathways in human cells. Signaling mediated by a protein kinase often elicits a complex network of interlocking events, rather than a simple linear output; and, it is not well understood how changes in metabolism, gene expression, and biosynthesis (especially membrane lipid synthesis) are properly coordinated in space and time to achieve appropriate, and sometimes dramatic, changes in cell morphology. Moreover, temporal and spatial aspects of protein kinase-evoked signaling are often imposed by negative feedback mechanisms, or must be integrated with the cell cycle machinery, but our understanding of the mechanisms that modulate the efficiency and duration of signaling events, and avoid adventitious activation of the wrong response, is not fully understood in any organism. To address many of these issues experimentally, our specific aims include: (1) mutational and structural analysis of a-arrestin-GPCR recognition and its control by phosphorylation and genome-wide analysis of the targets of the 14 recognized a-arrestins and their phospho-regulation; (2) global screening, and subsequent genetic and biochemical studies of new substrates of the TORC2-regulated protein kinase Ypk1; (3) assembly, organization and control of TORC2 by stress, especially perturbation of plasma membrane lipid composition, and the role of lipid-anchored Ras2 in TORC2 function; and, (4) genetic and biochemical studies of protein kinase control of flippase function in remodeling of plasma membrane lipids during the cell division cycle and in pheromone- and nutrient limitation-induced polarized growth.