The Golgi complex is a membrane-bound organelle that serves as a central unit for trafficking, glycosylation, sorting and processing of membrane and secretory proteins in all eukaryotic cells, including hormones, growth factors, antibodies and digestive enzymes. Alterations in Golgi structure and function have been associated with a variety of human diseases, including cancer, autoimmune disease, Huntington's and Alzheimer's diseases, and viral infections. Golgi fragmentation has been observed in many tumor cell lines and tissues, and aberrant glycosylation is a hallmark of cancer. A detailed dynamic model of normal Golgi structure formation and the relationship to its vital cellular function is required before its role in human disease can be understood. The unique Golgi architecture (flattened cisternae arranged into stacks) is believed to rely on the protein network associated with the Golgi, the "Golgi matrix". Very limited information is currently available on the composition and functions of the Golgi matrix. Based on the observation that the Golgi disassembles and reassembles during each cycle of cell division, we hypothesize that the Golgi matrix that generates and maintains the Golgi structure in interphase must be disassembled during mitosis and this process is regulated by extensive mitotic phosphorylation that disrupts protein-protein interactions. This collaboration between a Golgi researcher (Dr. Wang) and a biological mass spectrometry expert (Dr. Andrews) applies a systems approach to investigate the nature of the Golgi matrix by mapping its composition and assembly in the cell cycle and the relationship between phosphorylation and protein-protein interactions. We have developed an in vitro assay that reconstitutes the disassembly of Golgi during mitosis and its reassembly after mitosis. This allows us to prepare interphase and mitotic Golgi membranes of high quantity and high purity for proteomic quantitative analysis and to perform targeted interventional studies. We will use our novel proteomic protocols to quantify protein-protein interaction and protein phosphorylation events. Correlation analysis will allow us to link specific phosphorylation events with protein interactions in the Golgi matrix during the cell cycle, which can be validated and characterized in our in vitro assay. In this study, we will: 1) Use quantitative proteomics to analyze the components of the Golgi matrix in interphase and mitotic Golgi;2) Identify protein-protein interactions in the Golgi matrix and membranes in interphase and mitosis by crosslinking and proteomic analysis;3) Determine the role of phosphorylation on Golgi matrix assembly and disassembly as well as in protein-protein and protein-membrane interactions in vitro and in vivo. In vitro discoveries will be validated in intact cells using our new crosslinker as well as cell biology and biochemical techniques. These studies will provide new insights into the Golgi structure and function in normal cells and its dysfunction in disease states.

Public Health Relevance

The Golgi apparatus is a central cellular organelle for protein processing and secretion and is dysfunctional in a number of diseases including cancer, diabetes, and Alzheimer's disease. This proposal combines new proteomic technologies with novel in vitro reconstitution assays to investigate the components, assembly, and regulation of the Golgi matrix, a protein network that controls Golgi structure formation and function. We anticipate that this study will provide new insights into Golgi function, its dysfunction in diseass, and new protein targets for drug development.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
1R01GM105920-01A1
Application #
8695730
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109