Identification of cis-acting sequences and trans-acting factors for targeting the peripherally associated membrane protein, Trm1-II, to the inner nuclear membrane Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Tsung-Po Lai Graduate Program in Molecular Genetics The Ohio State University 2012 Dissertation Committee: Dr. Anita K. Hopper, Advisor Dr. Stephen A. Osmani Dr. Hay-Oak Park Dr. Juan D. Alfonzo Copyright by Tsung-Po Lai 2012 Abstract Appropriate nuclear membrane structure is important in eukaryotic cells. Significantly, many human diseases are caused by failure to correctly localize inner nuclear membrane (INM) proteins to the nuclear rim. To gain an understanding of the targeting mechanism of peripherally associated INM proteins, I employed the budding yeast Saccharomyces cerevisiae as a model system and Trm1-II, a tRNA modification enzyme that is peripherally bound to the INM, as a reporter. In Saccharomyces cerevisiae, the TRM1 gene codes for the tRNA-specific N2, N2- dimethylguanosine methyltransferase. It is located in mitochondria and nuclei by encoding two isomeric forms of the proteins, Trm1-I and Trm1-II, via alternative translation initiation. The longer form, Trm1-I, is exclusively localized to the mitochondria while the shorter form, Trm1-II, is located in the nucleus (90%) and mitochondria (10%). The nuclear pool of Trm1-II is peripherally associated with the inner nuclear membrane (INM). The mechanism of targeting Trm1-II to the INM is unclear. To characterize the amino acids important for targeting Trm1-II to the INM, I employed random and site-directed mutagenesis, and uncovered specific amino acids necessary for Trm1-II to locate at the INM. I defined a sequence of ~20 amino acids that contains information necessary to target Trm1-II to the INM. To address whether the newly defined region necessary for Trm1-II INM location is also ii sufficient to target reporter proteins to the INM, I demonstrated that this short peptide causes the redistribution of reporter proteins from the nucleoplasm to the INM. Thus, I identified the first motif for targeting peripherally associated proteins to the INM in yeast (Lai et al., 2009). A genome-wide study that identified subunits of the N-terminal acetyltransferase C (Nat C): Mak3, Mak10, and Mak31 function in Trm1-II INM targeting and suggested N-terminal acetylation of Trm1-II is necessary for it INM location (Murthi and Hopper, 2005). The data suggest that Trm1-II contains information for INM targeting other than the INM targeting motif. To further address the mechanism of Trm1-II INM location, Trm1-II, non-functional INM motif Trm1-II mutant and Trm1- II from cells without Nat C activity were C-terminally tagged with ZZ domain of Protein A (ZZ) or GFP-ZZ to search for Trm1-II interacting partner(s) at the INM using biochemical approaches. Surprisingly, no specific interacting proteins were uncovered by these studies. Rather, the evidence showed that Trm1-II may interact with lipids via the INM targeting motif and associate with the INM. In addition, I identified that without acetylation at N-terminus Trm1-II has the ability to modify tRNAs and to interact with membrane lipids. The data support a model that the INM targeting motif, rather than N-acetylation, is important for Trm1-II INM targeting by facilitating Trm1-II-lipid interaction at the INM. Since non-functional INM targeting motif Trm1-II mutant contains an amino acid substitution, it raise a possibility that the non-functional INM targeting motif Trm1-II mutant may be a misfolded protein, and thereby affecting Trm1-II-lipid interactions. Further investigation are required iii to confirm that the INM targeting motif is important for Trm1-II-lipid binding and INM targeting. iv Dedication This work is dedicated to my parents, my lovely wife, Huei-Tsu, and my beloved children Brandon and Bryan. You have made me stronger, better and more fulfilled than I could have ever imagined. v Acknowledgments I am deeply indebted to my advisor Dr. Anita K. Hopper for her guidance and support, as well as her dedication to her students and their research. I would like to thank the members of my thesis committee, Dr. Stephen Osmani, Dr. Hay-Oak Park, and Dr. Juan Alfonzo for their valuable inputs and suggestions regarding my projects and my career development. I am very grateful to Dr. Rebecca Hurto for teaching me the techniques and assays necessary for this research and taking time to review this dissertation. I would also like to acknowledge all current and former members of the Hopper lab for their advice and support throughout the length of my project. I would also like to thank Aysha Osmani, Kuo-Fang Shen and I-Ju Lee for their friendship and support. Finally, I must acknowledge my wife, Huei-Tsu, without whose love, encouragement, I would not have finished this thesis. vi Vita 1996 – 2000………………………………………………B. S. Nutrition, Chung Shan Medical University, Taiwan 2000 – 2002………………………………………………M. S. Toxicology, Chung Shan Medical University, Taiwan 2007 – 2011……………………………………............Teaching Asst. The Ohio State University. 2006 – 2012……………………………………...……..Research Asst. The Ohio State University. Publications 1. Mechanism and a peptide motif for targeting peripheral proteins to the yeast inner nuclear membrane. Lai TP, Stauffer KA, Murthi A, Shaheen HH, Peng G, Martin NC, Hopper AK. Traffic. 2009 Sep;10(9):1243-56. 2. Regulation of tRNA bidirectional nuclear-cytoplasmic trafficking in Saccharomyces cerevisiae. Murthi A, Shaheen HH, Huang HY, Preston MA, Lai TP, Phizicky EM, Hopper AK. Mol Biol Cell. 2010 Feb 15;21(4):639-49. vii Fields of study Major Field: Molecular Genetics viii Table of Contents Abstract…………….….……………………….....……………………………………………….………….…ii Dedication………………..……………………………....……………………………..……………………...v Acknowledgments……….…………………………….......…………………………..……………….....vi Vita……………………………………………………………..………………………….………………..…....vii List of Tables……….…………………………………….......…………...…………………..…………...xiv List of Figures………..……………........................................................................………………..xv Chapter 1: General introduction.........................................................................................1 1.1 Overview.................................................................................................................................................1 1.2 Review of the literature ...................................................................................................................3 1.2.1 The nuclear membrane protein………..................….……....................................................3 1.2.2 The NPC and nucleocytoplasmic transport……...............…....……………………….…...6 1.2.3 INM protein targeting ...............................................................................................................8 1.2.4 The INM proteins and nuclear lamina in human diseases……............…...………..11 1.2.5 S. cerevisiae Trm1……………...…………………..............…………….……………………….....11 1.3 Yeast as a model system................................................................................................................13 Chapter 2: General materials and methods..……………..…...............………......……….24 2.1 Yeast strains…………………………………..........……………...……………………………………….24 2.2 Yeast genomic DNA isolation………….................……...………………………………………….25 2.3 General method for plasmid construction….................……………………………………….25 ix 2.4 Oligonucleotides for PCR amplification of DNA fragments….................……………….26 2.5 DNA sequencing………………………………...….................………………………………………….26 2.6 Preparation of E. coli competent cells………….................……………………………………..26 2.7 Chemical transformation of E. coli competent cells……….................…………………….27 2.8 PCR from E. coli colonies…………………………………….................……………………………..27 2.9 Plasmid DNA Purification……………………………….................….……………………...………27 2.10 One-step yeast transformation................................................................................................28 2.11 Preparation of yeast competent cells....................................................................................28 2.12 Yeast transformation...................................................................................................................29 2.13 Isolation of plasmid DNA from yeast using the QIAprep Spin Miniprep Kit........29 2 2.14 m 2G methyltransferase activity assay.................................................................................30 2.15 Harvesting yeast cells for cryogenic disruption...............................................................30 2.16 Cryogenic disruption using planetary ball mill PQ-N04...............................................31 2.17 Conjugation of Magnetic beads with rabbit IgG...............................................................32 2.18 Western Blot analysis..................................................................................................................32 2.19 Fluorescence Microscopy...........................................................................................................33 2.20 SYPRO Ruby staining for protein gel.....................................................................................33 2.21 Coomassie blue staining.............................................................................................................33 2.22 Mass