Grant: Discovery Grants Program - Individual

The movement of proteins and lipids between cellular compartments is crucial for the establishing and maintaining the distinct nature of the compartments. Many diseases are linked to improper protein targeting, thus understanding how this works will provide insight into certain disease states. In the endomembrane system, the movement is mediated by small vesicles that emerge from a donor compartment and must fuse and deliver their contents to the appropriate acceptor compartment. One of the most upstream events in this process is vesicle recognition that involves large protein complexes referred to as tethering factors. One such tethering factor is a family of related complexes called TRAPP. In yeast, there are three such complexes referred to as TRAPP I, II and III. Each complex has been implicated in different transport events, with TRAPP I functioning in transport between the endoplasmic reticulm (ER) and Golgi, TRAPP II functioning in transport at the late Golgi and TRAPP III functioning in autophagy. To date, only two TRAPP complexes have been identified in humans, representing the equivalents of the yeast TRAPP II and III complexes. Using random mutagenesis, we identified a series of yeast mutations in the gene encoding the Trs23 subunit, found in all three yeast TRAPP complexes. We focused on a single mutation that deleted a yeast-specific domain not found in higher eukaryotes that we called the SMS domain. This domain was not essential for the function of the protein in yeast but did affect the assembly of TRAPP I in a recombinant system and in vivo. Thus we hypothesize that yeast TRAPP I does not exist in vivo and is only seen upon cell lysis. If this is true it will have important implications for the role of TRAPP in membrane trafficking events, suggesting that TRAPP complexes can be involved in multiple events. This would further imply that there is post-translational regulation of the activity of TRAPP complexes. The present proposal seeks to address these questions using a multifaceted approach. We will first establish new, simple and quantitative assays to measure transport between the ER and Golgi and between the Golgi and plasma membrane in yeast. This will allow us to test the function of all three TRAPP complexes for their ability to support traffic between these compartments. We will assess whether cytosol taken from starved cells (undergoing autophagy) alters the activity of the complexes. We will also focus on a subunit called Tca17 for which evidence from higher eukaryotes suggests it may play a regulatory role in TRAPP function. Indeed, we have found a genetic interaction between the TCA17 gene and a protein phosphatase, suggesting a role for phosphorylation in TRAPP function. The results of these studies will result in a paradigm shift as to how tethering factors function and whether they are indeed specific for one particular transport event.