Medicine and Health Sciences Commons^™

Ksp1 Is An Autophagic Receptor Protein For The Snx4-Assisted Autophagy Of Ssn2/Med13, Sara E Hanley, Stephen D Willis, Steven J Doyle, Randy Strich, Katrina F Cooper

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Ksp1 is a casein II-like kinase whose activity prevents aberrant macroautophagy/autophagy induction in nutrient-rich conditions in yeast. Here, we describe a kinase-independent role of Ksp1 as a novel autophagic receptor protein for Ssn2/Med13, a known cargo of Snx4-assisted autophagy of transcription factors. In this pathway, a subset of conserved transcriptional regulators, Ssn2/Med13, Rim15, and Msn2, are selectively targeted for vacuolar proteolysis following nitrogen starvation, assisted by the sorting nexin heterodimer Snx4-Atg20. Here we show that phagophores also engulf Ksp1 alongside its cargo for vacuolar proteolysis. Ksp1 directly associates with Atg8 following nitrogen starvation at the interface of an Atg8-family interacting …

Dpc29 Promotes Mitochondrial Translation Post-Initation In Saccharomyces Cerevisiae, Kyle Andrew Hubble

Graduate School of Biomedical Sciences Theses and Dissertations

Although the cytosolic and bacterial translation systems are well studied, much less is known about translation in mitochondria. In the yeast Saccharomyces cerevisiae, mitochondrial gene expression is predominately regulated by translational activators. These regulators are thought to promote translation by binding the elongated 5’-UTRs on their target mRNAs. Since mammalian mitochondrial mRNAs generally lack 5’-UTRs, they must regulate translation by other mechanisms. As expected, most yeast translational activators lack orthologues in mammals. Recently, a mitochondrial gene-specific translational activator, TACO1, was reported in mice and humans. To better define its role in mitochondrial translation I examined the yeast TACO1 orthologue, DPC29. …

Cdk8 Kinase Module Modifies Expression Of Specific Translation-Related Proteins Before And After Stress, Brittany Friedson, Katrina Cooper

Rowan-Virtua Research Day

Translation is tightly coupled to growth status. Efficient protein synthesis is necessary for cell growth in nutrient rich environments, while global translation inhibition combined with selective translation of stress-responsive mRNAs helps limit growth in times of stress. Environmental stress cues which inhibit the nutrient-sensing complex TORC1 are known to reduce general translation, but how does the cell alter protein synthesis machinery to adapt to these conditions? A few mechanisms to promote cell survival in nitrogen starvation include post-translational modification and selective degradation of specific mRNA-binding translation factors, as well as inhibition of activators of genes whose products are required for …

Effects Of Trans-Acting Factors On The Translational Machinery In Yeast, Brandon M. Trainor

Graduate School of Biomedical Sciences Theses and Dissertations

Synthesis of proteins, or translation, is a complex biological process requiring the coordinated effort of numerous protein and RNA factors. Central to translation is the ribosome, a complex macromolecular complex consisting of both ribosomal RNA (rRNA) and ribosomal protein (r-protein). Ribosomes are essential and are one of the oldest and most abundant biomolecules across all forms of life. In addition to the ribosome, translation requires messenger RNA (mRNA), transfer-RNA conjugated to an amino acid (aa-tRNA), translation factors, and energy in the form of ATP and GTP. Translation universally occurs in four major stages, initiation, elongation, termination, and recycling, with initiation …

Iron-Dependent Cleavage Of Ribosomal Rna During Oxidative Stress In The Yeast Saccharomyces Cerevisiae, Jessica A Zinskie, Arnab Ghosh, Brandon M Trainor, Daniel Shedlovskiy, Dimitri G Pestov, Natalia Shcherbik

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Stress-induced strand breaks in rRNA have been observed in many organisms, but the mechanisms by which they originate are not well-understood. Here we show that a chemical rather than an enzymatic mechanism initiates rRNA cleavages during oxidative stress in yeast (Saccharomyces cerevisiae). We used cells lacking the mitochondrial glutaredoxin Grx5 to demonstrate that oxidant-induced cleavage formation in 25S rRNA correlates with intracellular iron levels. Sequestering free iron by chemical or genetic means decreased the extent of rRNA degradation and relieved the hypersensitivity of grx5Δ cells to the oxidants. Importantly, subjecting purified ribosomes to an in vitro iron/ascorbate …

Comparative Analysis Of Mutant Huntingtin Binding Partners In Yeast Species., Yanding Zhao, Ashley A Zurawel, Nicole P Jenkins, Martin L Duennwald, Chao Cheng, Arminja N Kettenbach, Surachai Supattapone

Anatomy and Cell Biology Publications

Huntington's disease is caused by the pathological expansion of a polyglutamine (polyQ) stretch in Huntingtin (Htt), but the molecular mechanisms by which polyQ expansion in Htt causes toxicity in selective neuronal populations remain poorly understood. Interestingly, heterologous expression of expanded polyQ Htt is toxic in Saccharomyces cerevisiae cells, but has no effect in Schizosaccharomyces pombe, a related yeast species possessing very few endogenous polyQ or Q/N-rich proteins. Here, we used a comprehensive and unbiased mass spectrometric approach to identify proteins that bind Htt in a length-dependent manner in both species. Analysis of the expanded polyQ-associated proteins reveals marked enrichment of …

Acetic Acid Induces Sch9p-Dependent Translocation Of Isc1p From The Endoplasmic Reticulum Into Mitochondria, António Rego, Katrina F Cooper, Justin Snider, Yusuf A Hannun, Vítor Costa, Manuela Côrte-Real, Susana R Chaves

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Changes in sphingolipid metabolism have been linked to modulation of cell fate in both yeast and mammalian cells. We previously assessed the role of sphingolipids in cell death regulation using a well characterized yeast model of acetic acid-induced regulated cell death, finding that Isc1p, inositol phosphosphingolipid phospholipase C, plays a pro-death role in this process. Indeed, isc1∆ mutants exhibited a higher resistance to acetic acid associated with reduced mitochondrial alterations. Here, we show that Isc1p is regulated by Sch9p under acetic acid stress, since both single and double mutants lacking Isc1p or/and Sch9p have the same resistant phenotype, and SCH9 …

Guidelines And Recommendations On Yeast Cell Death Nomenclature, Didac Carmona-Gutierrez, Maria Anna Bauer, Andreas Zimmermann, Andrés Aguilera, Nicanor Austriaco, Kathryn Ayscough, Rena Balzan, Shoshana Bar-Nun, Antonio Barrientos, Peter Belenky, Marc Blondel, Ralf J Braun, Michael Breitenbach, William C Burhans, Sabrina Büttner, Duccio Cavalieri, Michael Chang, Katrina F Cooper, Manuela Côrte-Real, Vítor Costa, Christophe Cullin, Ian Dawes, Jörn Dengjel, Martin B Dickman, Tobias Eisenberg, Birthe Fahrenkrog, Nicolas Fasel, Kai-Uwe Fröhlich, Ali Gargouri, Sergio Giannattasio, Paola Goffrini, Campbell W Gourlay, Chris M Grant, Michael T Greenwood, Nicoletta Guaragnella, Thomas Heger, Jürgen Heinisch, Eva Herker, Johannes M Herrmann, Sebastian Hofer, Antonio Jiménez-Ruiz, Helmut Jungwirth, Katharina Kainz, Dimitrios P Kontoyiannis, Paula Ludovico, Stéphen Manon, Enzo Martegani, Cristina Mazzoni, Lynn A Megeney, Chris Meisinger, Jens Nielsen, Thomas Nyström, Heinz D Osiewacz, Tiago F Outeiro, Hay-Oak Park, Tobias Pendl, Dina Petranovic, Stephane Picot, Peter Polčic, Ted Powers, Mark Ramsdale, Mark Rinnerthaler, Patrick Rockenfeller, Christoph Ruckenstuhl, Raffael Schaffrath, Maria Segovia, Fedor F Severin, Amir Sharon, Stephan J Sigrist, Cornelia Sommer-Ruck, Maria João Sousa, Johan M Thevelein, Karin Thevissen, Vladimir Titorenko, Michel B Toledano, Mick Tuite, F-Nora Vögtle, Benedikt Westermann, Joris Winderickx, Silke Wissing, Stefan Wölfl, Zhaojie J Zhang, Richard Y Zhao, Bing Zhou, Lorenzo Galluzzi, Guido Kroemer, Frank Madeo

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic …

One-Step Hot Formamide Extraction Of Rna From Saccharomyces Cerevisiae, Daniel Shedlovskiy, Natalia Shcherbik, Dimitri G Pestov

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Current methods for isolating RNA from budding yeast require lengthy and laborious steps such as freezing and heating with phenol, homogenization with glass beads, or enzymatic digestion of the cell wall. Here, extraction with a solution of formamide and EDTA was adapted to isolate RNA from whole yeast cells through a rapid and easily scalable procedure that does not require mechanical cell lysis, phenol, or enzymes. RNA extracted with formamide-EDTA can be directly loaded on gels for electrophoretic analysis without alcohol precipitation. A simplified protocol for downstream DNase treatment and reverse transcription reaction is also included. The formamide-EDTA extraction of …

Endonucleolytic Cleavage In The Expansion Segment 7 Of 25s Rrna Is An Early Marker Of Low-Level Oxidative Stress In Yeast, Daniel Shedlovskiy, Jessica A Zinskie, Ethan Gardner, Dimitri G Pestov, Natalia Shcherbik

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The ability to detect and respond to oxidative stress is crucial to the survival of living organisms. In cells, sensing of increased levels of reactive oxygen species (ROS) activates many defensive mechanisms that limit or repair damage to cell components. The ROS-signaling responses necessary for cell survival under oxidative stress conditions remain incompletely understood, especially for the translational machinery. Here, we found that drug treatments or a genetic deficiency in the thioredoxin system that increase levels of endogenous hydrogen peroxide in the yeast Saccharomyces cerevisiae promote site-specific endonucleolytic cleavage in 25S ribosomal RNA (rRNA) adjacent to the c loop of …

Med13p Prevents Stress-Independent Mitochondrial Hyperfragmentation And Aberrant Apoptosis Activation In Saccharomyces Cerevisiae By Controlling Cyclin C Nuclear Localization, Svetlana Khakhina

Graduate School of Biomedical Sciences Theses and Dissertations

During aging, and as a result of environmental changes, cells are exposed to elevated levels of reactive oxygen species (ROS). High ROS levels induce lipid oxidation, protein aggregation, mitochondrial hyperfragmentation, DNA damage and programmed cell death (PCD), also called apoptosis. PCD is a highly regulated process and its misregulation has been linked to neurodegenerative diseases and cancer development.

Our hypothesis is that cyclin C plays a role in the initiation of apoptosis. During normal conditions, cyclin C represses the transcription of stress response genes (SRG). In response to stress, cyclin C translocates to the cytoplasm where it facilitates mitochondrial hyperfragmentation …

Cell Cycle-Dependent Binding Of Yeast Heat Shock Factor To Nucleosomes, Christina Bourgeois Venturi, Alexander M. Erkine, David S. Gross

Scholarship and Professional Work – COPHS

In the nucleus, transcription factors must contend with the presence of chromatin in order to gain access to their cognate regulatory sequences. As most nuclear DNA is assembled into nucleosomes, activators must either invade a stable, preassembled nucleosome or preempt the formation of nucleosomes on newly replicated DNA, which is transiently free of histones. We have investigated the mechanism by which heat shock factor (HSF) binds to target nucleosomal heat shock elements (HSEs), using as our model a dinucleosomal heat shock promoter (hsp82-ΔHSE1). We find that activated HSF cannot bind a stable, sequence-positioned nucleosome in G₁-arrested …

The Skn7 Response Regulator Of Saccharomyces Cerevisiae Interacts With Hsf1 In Vivo And Is Required For The Induction Of Heat Shock Genes By Oxidative Stress, Desmond C. Raitt, Anthony L. Johnson, Alexander M. Erkine, Kozo Makino, Brian Morgan, David S. Gross, Leland H. Johnston

Scholarship and Professional Work – COPHS

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and …