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Relationship Between Domains of the ER Membrane Protein Atlastin

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Relationship Between Domains of the ER Membrane Protein Atlastin RICE LINIVERSITY Characterization of Structure and Function Relationship between Domains of the ER Membrane Protein Atlastin by Tanvi Desai A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE Doctor of Philosophy James A. McNew, Associate of Biocheryistry & Cell Biology of Phfsics & Astronomy Dr. Michael Stern. Professor &)Cell Biology HOUSTON, TEXAS December 2013 ABSTRACT Characterization of Structure and Function Relationship between Domains of the ER Membrane Protein Atlastin by Tanvi Desai The endoplasmic Reticulum (ER) is an important site for lipid synthesis, protein synthesis and transport. ER fusion is an essential process for its maintenance and biogenesis. Mutations in genes involved in this process cause Hereditary Spastic Paraplegia (HSP). These mutations are shown to affect intracellular trafficking and localization of membrane compartment. One of the important proteins causing early onset of HSP is Atlastin. Previous work in McNew lab at Rice University (Moss et al., 2011b) has shown that atlastin is involved in the homotypic fusion of the ER and the C-terminal cytoplasmic region of atlastin is essential for atlastin mediated fusion. During my studies presented in this thesis, I was able to demonstrate that the C-terminal cytoplasmic region of atlastin destabilizes lipid bilayers to facilitate fusion. The requirement of C-terminal cytoplasmic region is minimal when fusing two fluid (or unstable) lipid bilayers. The C-terminal cytoplasmic region of atlastin forms an amphipathic helix and mutations on the hydrophobic phase of the helix reduce fusion. These mutations are not dominant, as presence of full length atlastin on even one of the fusing lipid bilayers can significantly improve fusion during a heterotypic fusion reaction. Additionally, domain swaps between human atlastin-1 and drosophila atlastin show that the role of C-terminal cytoplasmic region is highly conserved. Also, during my research presented here in, I found that when the transmembrane region and C-terminal cytoplasmic region of human atlastin-1 were swapped with drosophila atlastin, it showed functional similarity. These results show that although atlastins in organisms play an important role in the ER fusion, there are likely species specific differences in how this is achieved. An understanding of atlastin mediated fusion should help in unraveling mechanisms of HSP pathogenesis and other disorders arising from dysfunctional ER. Acknowledgments I would like to thank my professor Dr. James McNew for accepting me in his lab and mentoring me through these five years. He supported me through some of my toughest time during these five years and helped me with my graduate studies. I want to thank my committee members Dr. Janet Braam, Dr. Michael Stern, Dr. Peter Lwigale and Dr. Huey Huang for their perceptive suggestions and constant encouragements. I thank all the members of the McNew Lab Dr. Tyler Moss, Dr. Banafsheh Mirnikjoo, Dr. Joseph Faust and Dr. Avani Verma. I am really grateful to Dr. Tyler Moss, Dr. Joseph Faust and Dr. Avani Verma for teaching me basic techniques in the lab and constant help in troubleshooting. Special thanks to Dr. Avani Verma for collaborating with me on Atlastin project. I would also like to thank Dr. Mary Purugganan for helping me through the writing process. I thank my parents Pragna Desai and Himanshu Desai for their encouragement and faith in me. I would also like to thank my brother, grandparents, mother-in-law and father-in-law for their support during my studies. Most of all I thank my husband Jigar Desai for his love, support and encouragement. He is my friend and critic, who have always stood by me during the toughest of times. Last but not the least, I would like to thank to my three year old son for his love. Contents Acknowledgments..................................................................................................... iv Contents .................................................................................................................... v List of Figures ........................................................................................................... vii List of Equations........................................................................................................ ix Nomenclature ............................................................................................................ x Background and Significance ...................................................................................... 1 1.1. Biological Membrane Fusion.................................................................................... 1 1.2. Membrane Fusion by Dynamin Related Proteins .................................................... 2 1.3. Endoplasmic Reticulum Membrane Fusion ............................................................. 5 1.4. Hereditary Spastic Paraplegia (HSP)......................................................................... 6 1.5. Atlastin and Atlastin paralogs .................................................................................. 7 1.6. Atlastin structure.................................................................................................... 10 1.7. Atlastin Mediated Fusion Requires C-terminal Cytoplasmic Region ..................... 15 1.8. Understanding of Atlastin Structure and Function Relationship ........................... 19 Materials and Methods ............................................................................................ 22 2.1. Cloning.................................................................................................................... 22 2.2. Protein Expression and Purification ....................................................................... 24 2.2.1. Purification of GST-tagged proteins ................................................................ 24 2.2.2. Purification of His-tagged proteins.................................................................. 26 2.3. Production of Proteoliposomes ............................................................................. 27 2.4. Measurement of in vitro fusion ............................................................................. 29 2.5. GTPase Assay.......................................................................................................... 30 Role of the C-terminal Cytoplasmic Region of Atlastin in Fusion ................................ 32 3.1. Mutations in the C-terminal Cytoplasmic Region .................................................. 32 3.2. C-terminal Cytoplasmic Region Destabilizes the Lipid ........................................... 38 3.3. Function of the C-terminal Cytoplasmic Region is conserved Across the Species. 42 3.4. Conclusions............................................................................................................. 44 Role of Human Atlastin-1 Domains in Fusion............................................................. 46 vi 4.1. Mutants Containing Atlastin-1 Domains in Drosophila Atlastin Background ........ 46 4.2. Mutants Containing Atlastin 1 Domains in Drosophila Atlastin Background are Enzymatically Active...................................................................................................... 48 4.3. GTPase Activity of Mutants not enough for in vitro Fusion................................... 49 4.4. Transmembrane Domain and the C-terminal cytoplasmic Region of Human atlastin-1 Shows Functional Similarity to Drosophila Atlastin ...................................... 51 4.5. Conclusions:............................................................................................................ 52 In vitro Interactions of Lunapark Protein with Atlastin .............................................. 54 5.1. Lunapark Protein does not Affect the GTPase Activity of Atlastin ........................ 55 5.2. Lunapark Protein has No Effect on Atlastin Mediated Fusion ............................... 58 5.3. Conclusions:............................................................................................................ 58 APPENDIX “A”: ......................................................................................................... 60 Cellular Uptake and Trafficking of Nanoparticles in vivo............................................ 60 6.1. Background and Significance.................................................................................. 60 6.2. Cloning.................................................................................................................... 64 6.3. Drosophila S2 Cell Lines Expressing Endocytic Markers ........................................ 65 6.4. Intracellular Uptake of Carboxylated Qdots in Presence and Absence of Serum . 67 6.5. Intracellular Uptake of Amine CdSe Qdots in Presence and Absence of Serum ... 72 6.6. Conclusions:............................................................................................................ 76 APPENDIX “B”: ......................................................................................................... 77 Mitochondrial Assembly Regulatory Factor (MARF) .................................................. 77 7.1. Mitochondrial Fusion, Proteins Involved: .............................................................. 77 7.2. Protein Expression and Purification ....................................................................... 80 7.2.1. GTPase Activity ...............................................................................................
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