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Distinct C-Terminal Amino Acid Sequence Motifs Serve As the Targeting Signals for Outer Mitochondrial Membrane and Plastid Outer Envelope TA Proteins

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Distinct C-Terminal Amino Acid Sequence Motifs Serve As the Targeting Signals for Outer Mitochondrial Membrane and Plastid Outer Envelope TA Proteins Distinct C-terminal Amino Acid Sequence Motifs Serve as the Targeting Signals for Outer Mitochondrial Membrane and Plastid Outer Envelope TA Proteins by Howard James Teresinski A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Molecular and cellular Biology Guelph, Ontario, Canada © Howard James Teresinski, January, 2015 ABSTRACT Distinct C-terminal amino acid sequence motifs serve as the targeting signals for outer mitochondrial membrane and plastid outer envelope TA proteins Howard James Teresinski Advisor: University of Guelph, 2014 Dr. Robert T. Mullen Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins that are defined by their single C-terminal transmembrane domain and their ability to insert post-translationally into specific organelles in an Ncytosol-CIMS orientation. While in recent years considerable progress has been made towards understanding the biogenesis of TA proteins in yeast and mammals, relatively little is known about how these proteins are properly partitioned within plant cells, mostly because so few plant TA proteins have been identified. Here we show the results of experiments aimed at cataloguing, in silico, all of the TA outer mitochondrial membrane and plastid outer envelope proteins in Arabidopsis and then identifying and characterising distinct, C-terminal targeting motifs responsible for the proper sorting of at least a subset of these proteins. Collectively, the results of this thesis provide important insight into the molecular mechanisms that underlie the biogenesis of TA proteins in plant cells. Acknowledgments I would first and foremost like to thank my MSc advisor Dr. Robert Mullen for his constant support, encouragement and dedication to ensure I reach my full potential throughout the MSc process. His guidance has not only made me a better scientist but a better person as well. I would also like to thank Dr. Ian Tetlow for serving on my advisory committee and providing valuable expertise throughout my thesis. Also, I would like to thank Dr. Matthew Smith for serving on my MSc advisory committee and for first inspiring my interest in plant biology when he served as my undergraduate thesis supervisor. I would like to express gratitude to all members of the Mullen lab, past and present, for their assistance in the laboratory, but more significantly, for their constant support and friendship along the way. I would also like to thank all of my colleagues in MCB for proving a warm and welcoming environment, and many friends to share the excitements and frustrations of research. To Laura and all of my other friends I have had throughout my thesis process, I truly want to thank you for your terrific company an extraordinary emotional support you have provided. I cannot thank my parents Sandra and Mark Krupiarz enough for their endless support and encouragement throughout my education. I would like to thank my sister Sarah Lato for always reminding me what is truly important in life. I would like to thank my father Howard Teresinski Sr. for instilling in me, a strong work ethic that has allowed me to excel in my studies. I can never express enough gratitude towards these individuals, and everyone who has helped me reach this point. All of the data presented in this thesis was generated by Howard Teresinski and a portion of this thesis is published in Marty, et al. 2014. New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane. (Front. Plant. Sci. 5: 426). Contributions by others include the cloning of all mitochondrial constructs performed by Dr. Satinder Gida, Eric Clendening and Dr. Naomi Marty, and some of the mitochondrial protein bioinformatics analysis and microscopic analysis was performed by Dr. Naomi Marty. Cloning of all constructs used in the analysis of plastid TA proteins was performed by Howard Teresinski with a few exceptions, namely myc-OEP9, myc-At1G19400 and myc-At1G01500 which were cloned by Dr. Priya Dhanoa. The cloning of GFP-OEP6 and the Agrobacterium infiltration experiments were performed by Thuy Nguyen and Dr. Satinder Gidda. iii TABLE OF CONTENTS ABSTRACT ................................................................................................................................................II ACKNOWLEDGMENTS........................................................................................................................ III TABLE OF CONTENTS.......................................................................................................................... IV LIST OF TABLES................................................................................................................................... VII LIST OF FIGURES…........................................................................................................................... VIII ABBREVIATIONS ……………………………………………………….............................................. IX CHAPTER 1: INTRODUCTION………………………………………................................................... 1 1.1 Protein trafficking in eukaryotic cells........................................................................................ 1 1.2 Mitochondria possess a complex morphology and function...................................................... 1 1.3 Post-translational protein trafficking to mitochondria................................................................ 2 1.4 Plastid morphology and function create a unique protein trafficking problem.......................... 6 1.5 Transit peptide-mediated protein targeting to the plastid........................................................... 8 1.6 Tail-Anchored (TA) protein trafficking pathways................................................................... 14 1.6.1 General TA protein trafficking mechanisms......................................................... 16 1.6.2 TA protein trafficking to the ER........................................................................... 17 1.6.3 TA protein trafficking to the peroxisome.............................................................. 20 1.6.4 TA protein trafficking pathways to the OMM....................................................... 21 1.6.5 TA protein trafficking pathways to the plastid...................................................... 22 1.6.6 A possible GET system at the plastid in A. thaliana............................................. 24 1.6.7 Bioinformatic searches have identified putative TA A. thaliana proteins................................................................................................................. 25 1.7 Objectives................................................................................................................................ 26 CHAPTER 2: Materials and methods...................................................................................................... 27 2.1. Bioinformatic analyses of proteomic data and amino acid sequence characteristics...................... 27 2.2. General cloning techniques............................................................................................................. 28 2.2.1. Polymerase Chain Reaction (PCR) for DNA amplification.................................................. 28 iv 2.2.2. Reverse Transcriptase - PCR (RT-PCR) for DNA amplification from Arabidopsis suspension cell cDNA........................................................................................................ 28 2.2.3. PCR for QuikChange® (QC) Site-Directed mutagenesis...................................................... 29 2.2.4. Overlap Extension PCR for the generation of DNA for recombinant cloning...................... 29 2.2.5. Annealing synthetic nucleotides for recombinant cloning..................................................... 29 2.2.6. Restriction enzyme digestion of DNA for recombinant cloning............................................ 30 2.2.7. Ligation of vector and insert DNA fragments........................................................................ 31 2.3. Plasmid construction...................................................................................................................... 31 2.3.1. Vectors used for mitochondrial TA protein trafficking experiments..................................... 31 2.3.2. Vectors used for plastid protein trafficking experiments....................................................... 32 2.4. Preparation of chemically-competent DH5α E. coli cells.............................................................. 35 2.5. Tobacco BY2 suspension cells and biolistic bombardment........................................................... 36 2.6. Staining and microscopy of BY2 suspension cells......................................................................... 36 2.7. Agrobacterium tumefaciens transformation.................................................................................... 38 2.8. Nicotiana benthamiana growth conditions..................................................................................... 38 2.9. Transient N. benthamiana transformation via A. tumefaciens........................................................ 39 CHAPTER 3: Results and Discussion...................................................................................................... 40 3.1. Bioinformatic analysis of TA proteins in the A. thaliana deduced proteome................................. 40 3.2. A subset of TA OMM proteins
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