Study of the Role of Moonlighting Proteins: TrappC12 in Membrane Trafficking and Mitosis and TrappC11 in Golgi Integrity and ER‐to‐Golgi Trafficking
Authors: Prematilake Keshika , Sacher Michael 
Published: November 2023

Membrane trafficking is the transport of cargo proteins from one compartment to another through vesicle budding. In eukaryotic cells, vesicle trafficking requires multiple tethering complexes, including TRAPP (TRAnsport Protein Particle) complexes to regulate tethering and fusion of vesicles to membranes. 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 and non‐obligate subunit of human TRAPP complex III, TrappC12, has been identified as a “moonlighting” protein due to its function in mitosis and its association with TRAPP III. Another TRAPP III specific subunit, TrappC11, has also been identified to play a role in the formation of Golgi. In this study, we investigated whether TrappC12 play a direct role in the early secretory pathway and mitosis and whether TrappC11 play a direct role in Golgi fragmentation and the early secretory pathway. To isolate functions of TrappC12 in membrane trafficking and mitosis, we employed a novel and rapid gene silencing method, “knocksideways”, to investigate the immediate effects of inactivating TrappC12 in non‐synchronized cells. We observed TrappC12 was localized away from its functional location in HeLa cells within 20 minutes of knocksideways treatment. The recruitment of TrappC12 away from its site of action arrested HeLa cell line stably expressing TrappC12 at metaphase. Quantification of the effect of knocksideways showed an increase in the number of mitotic cells from (7.7±1.0) % for non‐treated cells to (19.9±3.1) % for knocksideways applied to transfected TrappC12 upon knockdown of endogenous TrappC12. Study of the membrane trafficking function of TrappC12 using knocksideways applied to a membrane trafficking assay, vesicular stomatitis virus glycoprotein (VSV‐G) assay, showed no delay in ER‐to‐Golgi trafficking in western blot. Since TrappC12 does not play a role in the early secretory pathway, further experiments are required to determine TrappC12 function in the late secretory pathway, including intra‐Golgi trafficking, autophagy and Golgi‐to‐plasma membrane trafficking. Golgi compartment changes its morphology during cell cycle to undergo fragmentation during mitosis. The mechanism of how Golgi returns to its normal form could be related to functions of TrappC11. We applied VSV‐G assay to cells upon knockdown of endogenous TrappC11 and cells with mutant variants of TrappC11 and observed a significant delay in ER‐to‐Golgi trafficking and/or fragmented Golgi in western blot analysis, live‐cell imaging, and immunofluorescence. Further experiments are required to determine the distinct role of TrappC11 in ER‐to‐Golgi trafficking and formation of Golgi. These identifications of the possible functions of TrappC12 and TrappC11 can lead to the discovery of potential targets for development of therapeutics for musculoskeletal diseases and intellectual disabilities including LGMB and various myopathies.

Support or Funding Information

Dr. Michael Sacher's laboratory is funded by CIHR (Canadian Institutes of Health Research), NSERC (Natural Sciences and Engineering Research Council of Canada) and CFI (Canada Foundation for Innovation). Dr. Michael Sacher is a member of GRASP (Groupe de Recherche Axé sur la Structure des Protéines).