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Osu1343763753.Pdf (14.8 Targeting of Peripherally Associated Proteins to the Inner Nuclear Membrane in Saccharomyces cerevisiae: The Role of Essential Proteins DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Greetchen M. Díaz Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2012 Dissertation Committee: Anita K. Hopper, Advisor Stephen Osmani Mark Parthun Jian-Qiu Wu Copyright by Greetchen M. Díaz 2012 Abstract The nuclear envelope (NE) is composed of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) which is contiguous with the endoplasmic reticulum (ER). The appropriate location of NE proteins is important in cells. Integral INM proteins are proposed to be synthesized at the ER and then translocated through the nuclear pore complex (NPC). In contrast, peripherally associated INM proteins are proposed to follow a targeting mechanism to the nucleus that is similar to nucleoplasmic proteins. Our research aims to understand the mechanism of targeting of peripherally associated proteins to the INM. We employed yeast as a genetic model and the tRNA modification enzyme, Trm1-II, as a reporter. We screened a collection of temperature sensitive (ts) mutants for defects in galactose-inducible Trm1-II-GFP (Gal-Trm1-II-GFP) INM localization. We found that the majority (46%) of the ts mutations affecting Gal- Trm1-II-GFP localization were in genes that encode proteins involved in ER-Golgi homeostasis. Interestingly, about 35% of the mutated essential genes encode components of the Spindle Pole Body (SPB). In the SPB ts mutants, at the non-permissive temperature, Gal-Trm1-II-GFP accumulates as a spot that localizes to the ER, rather than being evenly distributed around the entire INM as in wild-type cells. Following the dynamics of Gal-Trm1-II-GFP we learned that its inappropriate distribution results from a failure to move from the initial contact with the NE (ONM) throughout the INM. Gal- ii Trm1-GFP accumulates to the ER with time, suggesting that this might be the initial Trm1-II tethering site. Surprisingly, SPB defects also affect targeting of an integral INM protein, but not a soluble nucleoplasmic protein which indicates that there is no defect in import and that appropriate SPBs are required for INM targeting of both integral and peripheral INM proteins, but not nucleoplasmic proteins. Our evidence suggests Gal- Trm1-II-GFP is alternatively transported via soluble mechanism when unable to tether to the ER. We propose a novel mechanism for peripherally associated INM proteins that combines targeting mechanisms for both integral and soluble proteins. We also learned that INM maintenance of Gal-Trm1-II-GFP was altered in SPB defective cells, which suggests that a general defect at the membrane that forms the ER and the NE occurs in SPB defective cells. The possible role of the SPB based in INM targeting and maintenance is discussed. iii This document is dedicated to the memory of my beloved grandmother “Doña” Julia. iv Acknowledgments I would like to thank Dr. Anita K. Hopper for give me the opportunity of being part of a privileged group of people who received her support and guidance. I wish to thank all the members of A.K.H. lab from 2006-12. In particular, my friends in this journey, Rebecca, Hui-Yi, Nripesh, Tsung-Po, Ivy, JingYan and Emily who were always very helpful and supportive of me. I also want to express my appreciation to a former lab member, Athula who not only helped me in my project, but also for being a great friend no matter the distance. My gratitude to the members of my thesis committee: Stephen Osmani, Jian-Qiu Wu and Mark Parthun for their advice, ideas and support. It was an honor for me to have these great people guiding me in this process. In special, I thank Mark for my first rotation opportunity at OSU. Thanks to the MCDB program for the opportunity and the people of the Molecular Genetics Department, particularly to the neighbors at the 8th floor (Riffe) for their friendship and nice environment. Also my thanks to Jim Hopper and his lab members in particular Onur, for all the interesting conversations and exchange of ideas (and reagents!). Thanks to I-Ju Lee for all her help an friendship. I want to thank all the new friends I met here, for everything I shared with them, the smiles, the jokes and happiness, but also thanks to the old friends who gave me their best wishes when I left Puerto Rico and are still there for me. They are so many that I can’t list them here, but they know who they are! In special I would like to thank my great friend Hector for being like a brother, be always there for me and for introduce to my life a special person, Henry. Thanks Henry for his love and support, especially to help me with my crazy ideas. My special thanks to the incredible team “CienciaPR” for their passion to serve and because they motivate me every day to believe that everything is possible. v I give all my unconditional love and gratitude to my very big family in Mississippi, Orlando, Massachusetts and Puerto Rico, because they always wish me the best and they always believe in me. vi Vita June, 2003………………… B.S. Biology, University of Puerto Rico, Mayagüez Campus June, 2006………………….M.S. Biology, University of Puerto Rico, Mayagüez Campus 2006 to present………..Graduate Research Assistant, MCDB, The Ohio State University Fields of Study Major Field: Molecular, Cellular and Developmental Biology vii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii Table of Contents ............................................................................................................. viii List of Tables .................................................................................................................... xv List of Figures .................................................................................................................. xvi Chapter 1: General Introduction ......................................................................................... 1 The Nucleus: Outer Nuclear Membrane ......................................................................... 1 The Nucleus: Inner Nuclear Membrane and the Nucleoplasm ....................................... 2 The Nucleus: Spindle Pole Body .................................................................................... 6 The Nucleus: Nuclear Pore Complex ............................................................................ 10 Nucleocytoplasmic Transport and the Ran Cycle ......................................................... 11 Protein Targeting to the Nucleus ................................................................................... 16 Trm1 as a Reporter for INM Peripheral Targeting ....................................................... 22 Inner Nuclear Membrane Proteins and Disease ............................................................ 26 Yeast as a Genetic Model .............................................................................................. 27 viii Aims of this Study ......................................................................................................... 27 Chapter 2: General Materials and Methods ...................................................................... 29 Yeast strains and media ................................................................................................. 29 Yeast plasmids............................................................................................................... 29 Chemically competent E. coli cells ............................................................................... 31 Plasmid DNA isolation and E. coli transformation ....................................................... 31 Bacterial colony PCR .................................................................................................... 32 Sequencing .................................................................................................................... 32 Yeast plasmid transformations ...................................................................................... 32 Yeast transformation for genomic tagging .................................................................... 33 Isolation of DNA from yeast ......................................................................................... 33 PCR ............................................................................................................................... 34 DNA manipulation ........................................................................................................ 34 Indirect immunofluorescence ........................................................................................ 35 Microscopy and Imaging ............................................................................................... 36 Western Blot .................................................................................................................. 37 Oligonucleotides...........................................................................................................
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