Phospholipase A2 Regulation Of Golgi And Endosome Structure And Function
The transport mechanisms of secretory and endocytic pathways are responsible for the coordinated movement of proteins and lipids throughout the cell at the proper time and place. In mammalian cells, there are many proteins dedicated to maintaining organelles and the trafficking of materials between them, though the mechanisms that regulate these processes are far from being fully understood. For over a decade, the role of phospholipase and acyltransferase lipid-modifying enzymes in membrane trafficking has been described, but the identity of the proteins involved had not been determined. Inhibitor studies demonstrated that both PLA2 and LPAT enzymes regulate trafficking events and membrane tubule formation from the Golgi complex and endosomes. In the last year, one LPAT and three PLA enzymes were found to contribute to trafficking and organelle maintenance of the Golgi and endosomes, including the work described here. The function of the PLA2 enzyme complex platelet activating factor acetylhydrolase Ib (PAFAH Ib) was investigated at both the Golgi complex and endosomes. Employing overexpression and knockdown of PAFAH Ib subunits in mammalian cells, I have found that the catalytic [alpha] subunits and the non-catalytic [beta] subunit, LIS1, are important for the maintenance of an intact Golgi ribbon. The catalytic [alpha] subunits are important for the formation of membrane tubules and anterograde trafficking. I have also found that each [alpha] subunit has distinct contributions to regulating Golgi morphology, assembly, and secretory trafficking. Knockdown of one subunit alone is not compensated by the presence of the other. At endosomes, both [alpha] subunits are important for membrane tubule formation and endocytic recycling pathways. Furthermore, the interaction of these subunits with LIS1 appears to regulate the distribution of endocytic compartments within the cell. Additionally, I have used an in vitro Golgi membrane tubule reconstitution assay to investigate the regulation of membrane tubule formation by heterotrimeric G[beta][gamma] subunits. I utilized inhibitors of PLA2 enzymes and the purified G[beta]1[gamma]2 protein complex to demonstrate that G[beta]1[gamma]2 can stimulate Golgi membrane tubules through a PLA2 dependant pathway. This suggests that G[beta]1[gamma]2 may regulate the activity of PLA2 enzymes important for the formation of membrane tubules or positive membrane curvature.
Brown, William J
Quaroni, Andrea; Bretscher, Anthony Paul
Molecular & Cell Biology
Ph.D. of Molecular & Cell Biology
Doctor of Philosophy
dissertation or thesis