We have reconstituted the formation of Golgi stack formation from small vesicles of 60-90 nm average diameter. We have identified a cytosolic fraction containing two proteins of 120 and 66 kD mol. wt (p120/66), which is required for very early stages of the assembly process. Our results suggest that these proteins may be involved in untethering of VGMs from a cytosolic anchor. The untethered or mobilized VGMs then fuse by an NSF and SNAP dependent process to form larger vesicles of about 200-300 nm diameter. We will formally test the role of p120/66 in mobilizing VGMs for their assembly into stacks. In addition, we propose to identify p120/66 interacting partners. In the presence of an NSF like ATPase called p97, the large vesicles fuse and the resulting membranes are flattened into disc shaped cisternae. We suggest the p97 may be involved in post fusion flattening of the larger vesicles. In addition, we have started fractionating peripheral Golgi membrane proteins and find a fraction, after three column chromatographic steps to contain the stack formation activity. We will continue with this fractionation scheme to identify the essential components. A cytosolic protein fraction containing polypeptides of 55 and 33 kD (p55/33) in the presence of GTP inhibits Golgi vesiculation by IQ. We propose that these proteins may be exchanging GTP onto a Golgi membranes associated protein. The characterization of these proteins, therefore, promises to reveal the Golgi associated recipient of GTP, which as a result inhibits the effects of IQ.
Our aims therefore promise to reveal novel insights into the mechanism by which Golgi stacks form from VGMs and the process by which the Golgi may prevent its complete vesiculation by utilizing a GTP dependent reaction.
| Nakagomi, Saya; Barsoum, Mark J; Bossy-Wetzel, Ella et al. (2008) A Golgi fragmentation pathway in neurodegeneration. Neurobiol Dis 29:221-31 |
| Guizzunti, Gianni; Brady, Thomas P; Malhotra, Vivek et al. (2007) Trifunctional norrisolide probes for the study of Golgi vesiculation. Bioorg Med Chem Lett 17:320-5 |
| Takizawa, P A; Yucel, J K; Veit, B et al. (1993) Complete vesiculation of Golgi membranes and inhibition of protein transport by a novel sea sponge metabolite, ilimaquinone. Cell 73:1079-90 |
| Veit, B; Yucel, J K; Malhotra, V (1993) Microtubule independent vesiculation of Golgi membranes and the reassembly of vesicles into Golgi stacks. J Cell Biol 122:1197-206 |
