Enzymology of Golgi Stack Formation

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DESCRIPTION (provided by applicant): We have identified a serine threonine kinase called protein kinase D (PKD) as an essential component in the reactions leading to the fission of transport carriers from the TGN. When the kinase activity of PKD is compromised the transport carriers form from the TGN but fail to undergo fission. The cargo containing carriers grow as a result into large tubes. PKD is recruited to specific regions of the Trans Golgi Network (TGN) via the first cysteine rich domain (C1a). Cia binds to the TGN via Diacyiglycerol (DAG). Reducing the cellular levels of DAG inhibit PKD recruitment to the TGN and protein transport to the cell surface is blocked. Our aim is understand the mechanism by which PKD cycles on and off the TGN. We will identify components involved in the binding and the release of PKD from the TGN. We will reconstitute the generation of PKD containing tubes from the TGN in permeabilized cells. This is aimed towards the purification of components that are involved in the formation of the TGN derived transport carriers. We have generated a cell line, in which we can accumulate the PKD containing tubes and then cause their dissociation by shifting to a permissible temperature. This approach will be used to isolate the PKD containing TGN derived transport carriers. This will help reveal the molecular composition of the TGN derived transport carriers. In addition, we will provide a function for another isoform of PKD3, which we propose, is involved in transport from Golgi to the ER. We will formally test the hypothesis that the organization of the Golgi stacks is regulated through TGN. Vesiculation of the TGN causes vesiculation of the early Golgi cisternae by uncontrolled generation of COPI vesicle. Our studies will reveal novel insights into the process by which transport carriers form from the TON and how a regulated production of these carriers is essential for maintaining the overall Golgi organization.