Secretory vesicles bud from the trans-Golgi and move along cytoskeletal elements to sites of active secretion. The exocyst is an ~800 kDa hetero-octameric complex that is thought to function in recognition between the vesicle and its target membrane, as the vesicle is docking to the plasma membrane, prior to membrane fusion. The exocyst interacts with numerous other proteins along the exocytic pathway, so that knowledge of its architecture/structure will be central in understanding the molecular mechanisms underlying exocytosis. Attempts to isolate or express the entire exocyst have been unsuccessful. Instead we will study exocyst subcomplexes, which should be easier to isolate in quantities required for structural studies and which should nevertheless yield information regarding subunit arrangement (Aim 1). Any subcomplexes identified will be monitored for solubility on gel filtration and subject to crystallization trials (Aim 2). The native crystals and/or heavy atom substituted crystals will be used for phasing. Once the structure has been determined, we will study how the proteins interact with other exocyst subunits, to infer overall exocyst architecture (Aim 3). Subunit binding partners to the subcomplex will be identified, and binding partner interaction regions will be narrowed down, through domain analysis. Mutational analysis will be performed to more precisely define surfaces in the subcomplex required for interaction with other exocyst subunits. Additionally, similar experiments will be performed to determine how the activated form of GTPases interacts with exocyst subunits, which will give information regarding exocyst-membrane interactions. These functional studies will allow us to construct a model for exocyst architecture. Public Health Relevance: Malfunctions in the exocytic pathway or the exocyst are relevant to a number of cardiac diseases, such as anemia, hypertension, and diabetes. The exocyst is critical for transferrin receptor recycling (which is impaired in hemoglobin-deficient anemia), secretion of ANP and other hormones (which are increased in hypertension), and for secretion of insulin and GLUT4 trafficking (which are defective in diabetes). A structural understanding of the exocyst will help clarify the function of the exocyst in vesicle tethering, and might give insight into the etiologies of these heart and blood diseases.
| Vasan, Neil; Saglam, Ozlen; Killelea, Brigid K (2012) Metastatic leiomyosarcoma presenting as bilateral, multifocal breast masses. BMJ Case Rep 2012: |
| Vasan, Neil; Hutagalung, Alex; Novick, Peter et al. (2010) Structure of a C-terminal fragment of its Vps53 subunit suggests similarity of Golgi-associated retrograde protein (GARP) complex to a family of tethering complexes. Proc Natl Acad Sci U S A 107:14176-81 |
