Keshika Prematilake and Michael Sacher
Concordia University, Canada
McGill University, Canada
Posters & Accepted Abstracts: J Proteomics Bioinform
Eukaryotic vesicle trafficking requires multiple tethering complexes including TRAPP (transport protein particle) complexes which in mammalian cells, regulate tethering and fusion of vesicles to membranes in early secretory pathway. TRAPP complexes consist of core subunits and non-obligate subunits which are associated with heterogeneous diseases including myopathies such as Limb-Girdle Muscular Dystrophy (LGMB). A novel non-obligate subunit of human TRAPP complex, TrappC12, has been identified as a ├ó┬?┬?moonlighting├ó┬?┬? protein with functions in membrane trafficking and mitosis. We employed a novel and rapid gene silencing method, knocksideways, to investigate the immediate effect of inactivating TrappC12 in a non-synchronized cell population. We observed TrappC12 was localized away from its functional location in HeLa cells within 20 minutes of applying knocksideways. The recruitment of TrappC12 away from its site of action arrested HeLa cell line stably expressing TrappC12 at metaphase. We observed a two fold increase in mitotic cells in treated cells. Study of the membrane trafficking function of TrappC12 using knocksideways method applied to a membrane trafficking assay, vesicular stomatitis virus glycoprotein (VSV-G) assay, showed no delay in ER-to- Golgi trafficking in biochemical assays. VSV-G assay applied to another non-obligate subunit of TRAPP complex associated with LGMB, TrappC11, showed delay in ER-to-Golgi trafficking in biochemical assays, live-cell imaging and immunofluorescence. Further experiments are required to determine TrappC12 function in late secretory pathway including intra-Golgi trafficking, autophagy and Golgi-to-plasma membrane trafficking. Identification of possible functions of TRAPP subunits can lead to discovery of potential targets for development of therapeutics for musculoskeletal diseases including myopathies.
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