Project I
The Molecular Mechanisms of sorting and exit from the Golgi Complex
We are studying the sorting and exit processes from the Golgi complex using both morphological (Project III) and biochemical approaches (Project I). To put the biochemical project into context, we must summarize the conclusions of the morphological project. Our data suggest molecules moving through the Golgi are sorted and exit directly from three distinct trans-cisternae. This data is in contrast to "common wisdom" that suggest a single trans-most cisterna, the TGN, is the sorting and exit site. Our data also shows a specialized ER adheres to all three trans-cisternae and this ER is predicted to play a significant role in trans-Golgi function.

To test our hypotheses we plan to follow the entire complement of molecules in the vesicles/tubules that exit the Golgi. These vesicles and tubules will be immunoisolated and characterized by proteomics. In order to achieve this goal, the fundamental proteome of the Golgi first must be established and this has been a much bigger project than originally anticipated. The project is carried out in collaboration with John R. Yates, III of the Scripps Institute, who has developed multidimensional protein identification technology (MuDPIT). This technology combines multidimensional liquid chromatography (LC) with tandem mass spectrometry (LC/LC/MS/MS) and identifies soluble, insoluble (matrix-like) and transmembrane proteins in a single analysis. Using this technology Christine Wu has produced substantial data on the Golgi proteome which consists of greater than 500 proteins with approximately 50% of these identified as transmembrane proteins. In addition many ‘unknown/novel’ proteins are identified in the proteome. A number of these novel proteins are being studied to determine their functional role within the Golgi complex. The proteome data generated with MuDPIT also enables identification of posttranslational modifications. We have focused on phosphorylation, methylation and acetylation. In addition, the data includes determination of the orientation of transmembrane proteins within the membrane.

The Golgi proteome contains a massive amount of data that opens whole new areas for understanding the regulation of Golgi function. With the establishment of the Golgi proteome, we are on track to study the protein composition of the vesicle/tubule populations that form from the Golgi using MuDPIT.