Study of the Role of Moonlighting Proteins: TrappC12 in Membrane Trafficking and Mitosis and TrappC11 in Golgi Integrity and ER‐to‐Golgi Trafficking
Bibliographic record
Abstract
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).
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".