Department of Cell and Developmental Biology (CDB)

Structure Function Relationships of the Golgi Complex and Lipid Droplet formation

Our three projects use the same experimental approaches: (1) discovery of new molecules using mass spectrometry, (2) confirmation of the localization of these molecules using immuno-electron microscopy, and (3) functional studies using basic cell biological assays.

The Golgi is the central organelle of the secretory pathway through which all secretory and plasma membrane proteins transit and are post-translationally modified. For the Golgi project the discovery of new molecules has been completed. A proteomic analysis of the endogenous proteins of the Golgi complex was carried out using the high through-put proteomic strategy (Multi-Dimensional Protein Identification Technology-MudPIT) which rapidly identifies the total protein complement of the fraction including both "known" proteins in the non-redundant protein database as well as the ESTs of "unknown" proteins. This project was carried out with Christine Wu, a former Ph.D. student and currently an Assistant Professor in the Department of Pharmacology. The identification of ESTs allows major unknown Golgi proteins to be cloned and functionally characterized. We have initiated studies to determine the function of some of the “unknown” proteins, and have focused on a new Golgi matrix protein, GMx33.

Additionally we have carried out high resolution (~5 nm) 3-D reconstructions of regions of the Golgi ribbon in various cell types and functional states using intermediate voltage electron microscopy and tomography. This project is carried out in collaboration with colleagues at the Laboratory for 3D Fine Structure on the Boulder campus. Structural features provide hypotheses for detailed biochemical and molecular experiments.

We have an additional collaborative project that focuses on the identification and characterization of ion channels within Golgi membranes with a CDB colleague, John Caldwell and Christine Wu of the Department of Pharmacology. These channels regulate the ionic milieu of the Golgi lumen which is important in providing substrates and appropriate pH to the many enzymes involved in posttranslational modifications of newly synthesized proteins and lipids and providing the proper ionic conditions for sorting of molecules in exit from the Golgi. Knockdown of theses channels in Zebrafish result in embryonic leathality.

These same approaches are being used to study the biogenesis of lipid droplets in lactating mammary epithelial cells. This project is part of Program Project on the normal function of the Mammary Gland led by Steven Anderson of the Department of Pathology.

Together the data from these diverse approaches will help us understand basic functions of the cell and identify the molecules required. Importantly; the data also provides the essential molecular information to interpret numerous disease states.

Marsh BJ, Howell KE. Timeline: The mammalian Golgi - complex debates. Nat Rev Mol Cell Biol. 2002 Oct;3(10):789-95.

Christine C. Wu, Michael J. MacCoss, Gonzalo Mardones, Claire Finnigan, Soren Mogelsvang, John R. Yates, III, and Kathryn E. Howell. Organellar proteomics reveals Golgi arginine dimethylation. Molecular Biology of the Cell 2004, 15: 2907-2919.

John R. Yates, III, Annalyn Gilchrist, Kathryn E. Howell and John J.M. Bergeron. Proteomics of organelles and large cellular structures. Nature Reviews, Molecular Cell Biology 2005, 6: 702-715.

Roger J. Thompson, Hillary C.S.R. Akana, Claire Finnigan, Kathryn E. Howell and John H. Caldwell. Anion channels transport ATP into the Golgi lumen. American Journal of Physiology: Cell Physiology 2006, 290: C499-C514.

Gonzalo A. Mardones, Christopher M. Snyder and Kathryn E. Howell. cis-Golgi matrix proteins move directly to ER exit sites by association with tubules. Molecular Biology of the Cell 2006, 17: 525-538.

Christopher M. Snyder, Gonzalo Mardones, Mark Ladinsky and Kathryn E. Howell. GMx33 associates with the trans-Golgi matrix in a dynamic manner and sorts within tubules exiting the Golgi. Molecular Biology of the Cell 2006, 17: 511-524.

Soren Mogelsvang and Kathryn E. Howell. Global approaches to study Golgi function. Current Opinions in Cell Biology 2006, 18: 438-443.

Kathryn E. Howell. A short history of cell fractionation: The original description of the lysosome. The Biochemist 2006, 28: 31-34.

John H. Caldwell and Kathryn E. Howell. 2008. Pores Galore for the Golgi. Nature Cell Biology 10:, 1125-1126.

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