Vivek Malhotra
e-mail: malhotra@biomail.ucsd.edu
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How and why Golgi membranes fragment in the mammalian cells during mitosis and the process of transport carrier formation from the TGN?
We have reconstituted the fragmentation of the pericentriolar Golgi stacks in permeabilized Normal rat Kidney (NRK) cells by mitotic cytosol prepared from NRK cells that are arrested in mitosis. The Golgi membranes are found in the form of small tubulo-reticular elements dispersed throughout the cytoplasm under these conditions. The fragmentation of the Golgi membranes reconstituted in our permeabilized cells is equivalent to the state of the Golgi membranes in the pre-metaphase/metaphase stage of the mitotic cycle in mammalian cells. The Golgi fragmentation requires Mitogen Activated Kinase Kinase (MEK1) and Polo like Kinase (Plk). We have identified Raf1 as the activator of MEK1 in the Golgi fragmentation process. Our aim is to demonstrate the role of Raf1-MEK1-ERK (MAP Kinase)-GRASP55 (A Golgi associated protein) in the Golgi fragmentation process. We will test our working hypothesis that a novel 400 kD (P400) protein is the downstream target of PlK. We propose that PLK-P400 mediated reaction severs the Golgi membranes from the pericentriolar region . The Raf-MEK-ERK-GRASP55 mediated pathway disconnects Golgi stacks from each other. The net result of these reactions leads to the separation of the Golgi stacks from the pericentriolar region and each other in the form of smaller fragments. In addition we have found that the fragmentation of the pericentriolar Golgi apparatus is necessary for entry of cell into mitosis. We have proposed that the pericentriolar Golgi membranes act as a "sensor" in preparation for entry into mitosis. The mechanism by which the pericentriolar Golgi apparatus regulates this key event is being addressed. Our overall efforts will provide an understanding of the process by which Golgi membranes regulate entry into mitosis and the mechanism by which Golgi membranes are fragmented in preparation for mitosis specific events in the mammalian cells.
We want to understand how transport carriers form from the TGN and the mechanisms that regulate the organization of the Golgi apparatus amidst the tremendous flow of membranes during protein transport. We identified a marine natural product called Ilimaquinone (IQ), which when added to mammalian cells vesiculated Golgi apparatus. The uncontrolled vesiculation by IQ was reconstituted in permeabilized cells and found to involve the trimeric G protein subunits Gßg and a protein kinase called PKD. When the kinase activity of PKD was compromised in cells, protein transport from the TGN to the cell surface was specifically blocked. Under these conditions, the transport carriers initiate the budding process but fail to undergo fission. As a result cargo filled buds grow into large tubules. PKD therefore regulates the process of membrane fission that is specific and necessary for the dissociation of the TGN derived transport carriers. PKD is recruited to specific sites of the TGN via the first cysteine rich domain (C1a) and this interaction requires Diacylglycerol (DAG). There are 3 isoform of PKD. PKD and PKD2 are both involved in the protein transport from the TGN to the cell surface, albeit of different cargo in polarized cells. The exact location and the functions of PKD3 are not known. We want to identify IQ receptor, the mechanism by which cargo stimulates the signal that recruits to the specific region of the TGN the machinery necessary for the deformation of the membrane into a bud, which ultimately undergoes fission by reactions that include PKD and its interaction partners.
Jamora, C., Takizawa, P., Zaarour, R., Denesvre, C., Faulkner, D.J., and Malhotra, V. (1997). Regulation of Golgi structure through heterotrimeric G-proteins. Cell 91: 617-626.
Acharya, U., Mallabiabarrena, A., Acharya, J.K., and Malhotra, V. (1998). Signaling via Mitogen activated protein kinase is required for Golgi fragmentation during mitosis. Cell 92: 183-192.
Glick, B.S, and Malhotra, V (1998). The curious status of the Golgi apparatus. Cell 95: 883-890.
Jamora, C., Yamanouye, N., Trowbridge, I., Lint, J.V., Lauudenslager., J.R., Faulkner, D.J., and Malhotra, V. (1999). Gßg induced Golgi breakdown in through the activation of protein kinase D. Cell 98: 59-68 .
Colanzi, A., Deerinck, T., Ellisman, M.E.H., and Malhotra, V. (2000). A specific activation of the Mitogen Activated Protein Kinase Kinase 1(MEK1) is required for Golgi fragmentation during mitosis. J. Cell Biol. 149: 331-339.
Liljedahl., M., Maeda, Y., Colanzi, A., Ayala, I., VanLint, J., and Malhotra, V. (2001). Protein kinase D regulates the fission of cell surface destined transport carriers from the Trans Golgi network. Cell 104: 409-420.
Maeda, Y., Van Lint, J., and Malhotra, V. (2001). Activation dependent recruitment of Protein kinase D to the trans Golgi network. EMBO J. 20: 5982-5990.
Baron, C.L. and Malhotra, V (2002). Role of Diacylglycerol in PKD recruitment to the TGN and protein transport to the plasma membrane. Science. 295: 325-328.
Sütterlin, C., Hsu, P., Mallabiabarrena, A., and Malhotra.V (2002). Fragmentation and dispersal of the pericentriolar Golgi complex is required for entry into mitosis in mammalian cells. Cell 109: 359-370.
Yeaman, C., Ayala, IWright, J., Ang, A., Maeda, Y., Mellman, I., Nelson, W.J., and Malhotra, V. (2004). Protein kinase D (PKD) regulates basolateral, but not apical plasma membrane protein exit from the Trans Golgi Network. Nature Cell Biology 6: 107-112.
Pecot, M.Y., and Malhotra, V. (2004). Golgi membranes remain segregated from the endoplasmic reticulum during mitosis in mammalian cells. Cell 116: 99-107.
F. Bard, L. Casano, A. Mallabiabarrena, E. Wallace, K. Saito, H. Kitayama, G. Guizzunti, Y. Hu, F. Wendler, R. DasGupta, N. Perrimon and V. Malhotra (2006). Functional genomics reveals genes involved in protein secretion and Golgi organization. Nature 439. 607-609.
Vivek Malhotra received his Ph.D. from Oxford University and was a postdoctoral fellow at Stanford University. He is the recipient of a Pirie-Reid scholarship, a Damon Runyon-Walter Winchell Fellowship, an American Cancer Society Senior Postdoctoral Fellowship and a Basil O'Connor award from the March of Dimes Foundation. Professor Malhotra is an elected fellow to the AAAS. Heis a Senior Investigator for the Sandler's Program for Asthma Research and an established investigator of the American Heart Association. He is the Monitoring Editor for the journal Molecular Biology of the Cell.
