Ubiquitin and Cell Cycle Regulation of Golgi Membrane Dynamics

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DESCRIPTION (provided by applicant): The Golgi complex is a membrane-bound organelle that serves as a central conduit for the processing of secretory proteins in all eukaryotic cells. Alterations in the Golgi structure and function have been associated with a variety of human diseases, including autoimmune disease, Huntington's and Alzheimer's diseases, viral infections and cancer. A better understanding of the relationship between the normal Golgi structure formation and its vital cellular function is required before its role in human disease can be understood. Golgi biogenesis during cell division is mediated by a disassembly and reassembly process. It disassembles into tubularvesicular structures during mitosis, which are partitioned into the daughter cells where they are reassembled into a new Golgi apparatus. Reassembly is mediated by two ATPases (NSF and p97) that fuse the membranes. Our recent discovery that ubiquitin plays a role in p97-mediated Golgi membrane fusion opens a door for a new approach to uncover the underlying mechanism. Ubiquitination occurs during mitotic Golgi disassembly and is required for subsequent reassembly. Reassembly requires the interaction between the p97/p47 and monoubiquitin and the activity of the deubiquitinating enzyme, VCIP135, a cofactor of the p97/p47 complex. We hypothesize that ubiquitination operates as a general mechanism in regulation of Golgi membrane dynamics during the cell cycle. We will use a combination of biochemical and morphological approaches to elucidate how ubiquitination occurs during mitotic Golgi disassembly and how it regulates postmitotic reassembly. The specific aims are: 1) To identify the ubiquitin ligase (E3) and elucidate its function in vitro by inhibition of the enzyme and in vivo by knocking down the protein. 2) To identify the ubiquitinated substrate(s) on the Golgi and confirm it using the available ubiquitin ligase and deubiquitinase. 3) To elucidate the mechanism of ubiquitination in mitotic Golgi membrane dynamics. We will determine the interactions between the enzymes and the substrate(s) in relation to p97-mediated membrane fusion. We will control ubiquitination by manipulation of both the ubiquitin ligase and the deubiquitinating enzyme in cells using shRNA and overexpression techniques, and thus determine the effects on Golgi membrane reassembly at the end of mitosis. These studies will provide new insights into the molecular mechanisms of cell cycle regulation of Golgi membrane dynamics. Public Health Relevance: The Golgi apparatus is a major cellular component that serves as a central conduit for the processing of membrane and secretory proteins, including antibodies, neurotransmitters, hormones, growth factors and digestive enzymes. Dysfunction of the Golgi apparatus has been associated with many diseases including Alzheimer's disease and cancer, in which abnormal protein trafficking and secretion are involved. This proposal studies the biogenesis, structure and function of the Golgi apparatus in order to provide knowledge for understanding the pathogenesis of diseases.