Our long term goal is to understand the mechanism of protein transport to and within the Golgi complex. To understand the physical and molecular basis for transport, an in vitro reconstitution approach is essential. A cell-free system that reconstitutes transport between Golgi cisternae has been used to identify a large number of proteins required for transport, but has failed to unambiguously define the physical mechanism of protein movement between cisternae. To overcome the limitations of this original assay, new cell-free assays for the analysis of transport from other compartments within the Golgi complex have been developed. The characteristics of protein transport between Golgi cisternae in these new assays are distinct from those of the original assay. Preliminary analyses suggest that about half of the transport appears to require Golgi coated bud formation, whereas half is independent of bud formation. Two mechanisms to account for this can be proposed. Transport might normally proceed by transient fusion between tubules. Coat proteins are needed to make buds for new tubule formation and to facilitate fission of cisternae, but not for pre-existing tubules to fuse. Alternatively, transport may be mediated by coated vesicle intermediates, and Golgi tubules produced during isolation of these membranes may be competent to fuse with other cisternae in vitro. These new transport assays will be used to directly test these models for transport with both isolated Golgi complexes, and intact Golgi complexes within cells. Using this multifaceted approach, it should be possible to delineate the physical and biochemical mechanisms of transport within the Golgi complex. Our understanding of the genetic basis of human diseases makes it clear that some mutations affect the trafficking of mutant proteins through the Golgi complex to the cell surface. A clearer understanding of the mechanisms and regulation of protein trafficking within the Golgi will contribute to the rational design of pharmacological therapeutics for these types of genetic diseases.
| Pullikuth, Ashok K; Weidman, Peggy J (2002) In vitro transport on cis and trans sides of the Golgi involves two distinct types of coatomer and ADP-ribosylation factor-independent transport intermediates. J Biol Chem 277:50355-64 |
| Happe, S; Weidman, P (1998) Cell-free transport to distinct Golgi cisternae is compartment specific and ARF independent. J Cell Biol 140:511-23 |
| Mironov, A A; Weidman, P; Luini, A (1997) Variations on the intracellular transport theme: maturing cisternae and trafficking tubules. J Cell Biol 138:481-4 |
