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The Involvement of NUCLEAR-PORE ANCHOR in SUMO Homeostasis

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The Involvement of NUCLEAR-PORE ANCHOR in SUMO Homeostasis TWO SIDES OF THE PLANT NUCLEAR PORE COMPLEX AND A POTENTIAL LINK BETWEEN RAN GTPASE AND PLANT CELL DIVISION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Xianfeng Xu, B.S. * * * * * The Ohio State University 2007 Dissertation Committee: Approved by Professor Iris Meier, Advisor Advisor Professor Biao Ding Professor Rebecca Lamb Professor David Mackey Graduate Program in Plant Biology ABSTRACT In eukaryotic cells, the nuclear pore complex (NPC) is vital for macromolecular trafficking and thereby the exchange of information between the nucleus and the cytoplasm. While fungal and mammalian NPC has been well studied by proteomics and detailed functional characterization, both the protein components of and various molecular activities associated with the plant NPC are poorly understood. Recent studies have implicated that the plant NPC plays important roles in several processes, including plant-microbe interactions, hormone signaling, and stress tolerance. This highlights a true need to thoroughly understand NPC-associated molecular activities in plants. Here I have identified and characterized two plant NPC-associated proteins, one residing at the inner and the other at the outer side of the NPC. Analyses of these proteins suggest both the conservation and the divergence of the plant NPC from that of other eukaryotes. ii I identified an Arabidopsis thaliana protein called NUCLEAR PORE ANCHOR (NUA); it is a plant homolog of Tpr/Mlp1/Mlp2/Megator. Tpr-like proteins are long coiled-coil proteins associated with the inner basket of the NPC and are involved in mRNA export, unspliced RNA retention, telomere organization, spindle pole assembly, docking of SUMO protease, and spatially regulated gene expression in a number of eukaryotic species but hadn’t been analyzed in plants. Four nua lesions comprise an allelic series with increasing severity for several correlating phenotypes, such as early flowering, increased abundance of SUMO conjugates, and altered expression of flowering regulators. Together with the genetic and physical interaction between NUA and ESD4 (a SUMO protease), these data suggest that NUA is a conserved component of NPC-associated steps of desumoylation in plants. Defects in SUMO homeostasis affect signaling events of flowering time regulation and additional developmental processes. Secondly, I identified a plant-specific family of NPC-associated membrane proteins, WPP-DOMAIN INTERACTING PROTEINS (WIPs). These proteins bind to the WPP domain of the plant Ran GTPase-activating protein RanGAP, and are responsible for the NPC-association of plant RanGAP. RanGAP is essential for the asymmetric distribution of RanGTP and RanGDP, involved in nucleocytoplasmic transport, mitotic spindle assembly, cell cycle control, and nuclear envelope formation. Mammalian RanGAP targeting to the NPC during interphase and to the spindle and kinetochores during mitosis requires interaction of its sumoylated C-terminal domain with nucleoporin Nup358/RanBP2. In contrast, I demonstrated that binding to the coiled-coil domain of WIPs is a plant-unique mechanism for targeting RanGAP to the NPC, which supports a separate evolution of RanGAP targeting in different kingdoms. Moreover, I presented data to suggest that targeting of plant RanGAP appears to involve different mechanisms at different stages of cell cycle and of differentiation. iii Lastly, in light of the increasing evidence indicating that NPC-associated proteins play additional roles during mitosis and cytokinesis, I characterized the localization of Arabidopsis RanGAP1 throughout the cell cycle using immunofluorescence. Arabidopsis RanGAP1 colocalizes with the preprophase band (PPB), concentrates at the kinetochores and spindle midzone, and accumulates at the midline of phragmoplast and/or the nascent cell plate. More strikingly, RanGAP1 was found to be the first marker to demarcate the cortical division site after the PPB disassembles. In addition, the WPP domain of RanGAP1 appears to be necessary and sufficient for the mitotic targeting of RanGAP1, although this localization does not depend on the WIPs. Taken together, by performing detailed studies on two Arabidopsis NPC-associated proteins, I demonstrated two general themes of the plant NPC. First, the plant NPC is an evolutionarily conserved structure with similar protein composition and similar associated molecular activities to those of other eukaryotes, as shown by the conservation of NUA. Second, the plant NPC also diversified to fulfill potential plant-specific cellular and developmental requirements, indicated by the presence of the plant specific NPC-associated WIP family. In addition, a potential link between the Ran cycle and the plant-unique mode of cytokinesis is proposed based on the mitotic targeting of Arabidopsis RanGAP1. Together, my research contributes to a better understanding of the plant NPC and a better appreciation of plant cytokinesis. iv Dedicated to my family, my love! v ACKNOWLEDGMENTS I was extremely fortunate to get the opportunity to work with my advisor, Prof. Iris Meier. I thank her for the continuous guidance, support and motivation, which will benefit me throughout my future career. I am grateful to all my committee members, Prof. Rebecca Lamb, Prof. Biao Ding and Prof. David Mackey for their time, advice and encouragement. I also want to thank Prof. Biao Ding for his generosity in sharing the confocal microscope, without which this dissertation would not have been possible. I would also like to thank all my lab-mates through the years: Dr. Tomasz Calikowski, Dr. Sun Yong Jeong, Dr. Shalaka Patel, Dr. Annkatrin Rose, Heather Wang, Qiao Zhao, Dr. Jelena Brkljacic, Sowmya Venkatakrishnan, Sivaramakrishnan Muthuswamy, Thushani Rodrigo-Peiris, Chao Sylvia He, Grace Fry, Kelly Lake, Nicholas Holomuzki, and other undergraduate assistants. I am grateful to their warmth, encouragement, and helpful discussion. Also I want to thank Dr. Annkatrin Rose, Sivaramakrishnan Muthuswamy, Dr. Sun Yong Jeong, Sowmya Venkatakrishnan, and Qiao Zhao for showing some of their data on the collaborative project described in chapter 1. I am thankful to Thushani Rodrigo-Peiris and Heather Wang for citing some of her unpublished data. I am sincerely thankful to Dr. Tea Meulia for the delightful collaboration on the electron microscopic studies. I also appreciate all the expertise that she showed me about general microscopy. vi I am grateful to all the people in the Plant Biotech Center, for critical feed-back on my research. I thank all our former and current staff members, Denise Blackburn-Smith, Jill Hartman, Rene Madsen, and Laurel Shannon from the Department of Plant Cellular and Molecular Biology; Melinda Parker, Diane Furteny, Dave Long and Scott Hines from the Plant Biotech Center; Joe Takayama from our greenhouse facility; and James Mann, Debbie Crist, Emma Knee, and Luz Rivero from The Arabidopsis Biological Resource Center (ABRC). Last but not least, I thank my family and all the people who care about me, for their love and support. Especially, I am deeply in debt to Chao Sylvia He for all her love, understanding and tremendous help in my research. vii VITA Dec. 28, 1983 ……………………………... Born in Anhui, P.R.China 2002 ………………………………………. B.S in Biotechnology College of Life Science Peking University, Beijing, P.R.China 2002 – present …………………………….. Graduate Teaching/Research Associate Plant Cellular and Molecular Biology The Ohio State University, Columbus, Ohio PUBLICATIONS Meier I., Xu X.M., Brkljacic J., Zhao Q., and Wang H-J. Going Green: Plants’ Alternative Way to Position the Ran Gradient. Journal of Microscopy, in press. Xu X.M., Rose A., and Meier I. NUA Activities at the Plant Nuclear Pore. Plant Signaling & Behavior, in press. Xu X.M., Meulia T., and Meier I. Anchorage of Plant RanGAP to the Nuclear Envelope Involves a Novel Family of Plant Nuclear Pore-Associated Transmembrane Proteins. Current Biology, 2007, 17:1157–63. Xu X.M., Rose A., Muthuswamy S., Jeong S.Y., Venkatakrishnan S., and Meier I. NUCLEAR-PORE ANCHOR, the Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator, Is Involved in mRNA Export, SUMO Homeostasis and Affects Diverse Aspects of Plant Development. Plant Cell, 2007, 19(5):1537-48. FIELD OF STUDY Plant Biology viii TABLE OF CONTENTS Page Abstract …………………………………………………………………………………….………….ii Dedication………………………………………………………………………………………..…….v Acknowledgments……………………………………………………………………………….……..vi Vita…………………………………………………………………………………………….……...viii List of Figures………………………………………………………………………………………...xiv List of Tables…………………………………………………………………………………….……xvi Chapters: 1. NUCLEAR PORE ANCHOR, the Arabidopsis homolog of Tpr/Mlp1/Mlp2/Megator, is involved in SUMO homeostasis and affects diverse aspects of plant development……………………………...1 1.1 Abstract……………………………………………………………………………………....2 1.2 Introduction…………………………………………………………………………………..3 1.3 Materials and Methods……………………………………………………………………….6 1.3.1 Plant material and growth conditions……………………………………………..6 1.3.2 Flowering time measurements…………………………………………………….6 1.3.3 PCR-based genotyping of T-DNA insertion lines…………………………………7 1.3.4 Arabidopsis transformation………………………………………………………..7 1.3.5 RT-PCR……………………………………………………………………………8 1.3.6 Sumoylation assays………………………………………………………………..8 1.3.7 Immunolocalization………………………………………………………………..8 ix 1.4 Results………………………………………………………………………………………..9 1.4.1 Identification of NUCLEAR-PORE ANCHOR (NUA), an Arabidopsis protein similar
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