PB1261 - Delayed CCL26 trafficking in the secretory pathway of STX5 depleted Endothelial Cells

Background: The SNARE protein Syntaxin 5 (STX5) is involved in trafficking secretory cargo between the endoplasmic reticulum (ER) and the Golgi. However, the kinetic details behind this remain undefined. The Retention Using Selective Hooks (RUSH) system allows for the coordinated retention and release of a protein of interest. Using this system with a fluorescently-tagged secretory protein probe such as the chemokine CCL26 allows us to investigate the dynamics of the secretory pathway in absence of STX5, its interactors and regulators. Hereby we can characterize their roles in secretion and further unravel the secretory pathway of endothelial cells (ECs).

Aims: To investigate the effect of STX5 depletion on trafficking of secretory proteins in ECs.

Methods: We performed short hairpin (sh)RNA-based STX5 knockdown in human umbilical vein endothelial cells (HUVECs) followed by transient transfection with a streptavidin-KDEL_SBP-GFP-CCL26 construct. The time required for translocation from the ER to the Golgi following addition of biotin was used to quantify and compare progression of CCL26 through the secretory pathway.

Results: A delay of CCL26 trafficking was often observed in STX5 depleted cells (Figure 1). Quantification of reporter translocation time to the Golgi showed a significant difference between shCTRL and shSTX5 transduced HUVECs. In some cells, the absence of STX5 caused (prolonged) ER retention of the probe as Golgi translocation did not occur within 60 minutes (Figure 2).

Conclusion(s): STX5 plays an essential role in the expedited delivery of secretory proteins from ER to the Golgi.