Supplementary Material for: Gamma-1-Syntrophin Mediates Trafficking of Gamma-Enolase towards the Plasma Membrane and Enhances Its Neurotrophic Activity
2011-03-01T00:00:00Z (GMT) by
Syntrophins are scaffold proteins that can bind several signaling molecules and localize them to the plasma membrane. We demonstrate here that in neuroblastoma SH-SY5Y cells, brain-specific γ<sub>1</sub>-syntrophin binds the neurotrophic factor γ-enolase through its PDZ domain, and translocates it to the plasma membrane, as shown by immunoprecipitation, surface plasmon resonance, fluorescence colocalization and flow cytometry. Extensive colocalization of γ<sub>1</sub>-syntrophin and γ-enolase was observed in neurite growth cones in differentiated SH-SY5Y cells. Silencing of the γ<sub>1</sub>-syntrophin gene by RNA interference significantly reduced the re-distribution of γ-enolase to the plasma membrane and impaired its neurotrophic effects. We demonstrated that an intact C-terminal end of γ-enolase is essential for its γ<sub>1</sub>-syntrophin-assisted trafficking. The cleavage of two amino acids at the C-terminal end of γ-enolase by the carboxypeptidase cathepsin X prevents binding with the γ<sub>1</sub>-syntrophin PDZ domain. Collectively, these data demonstrate that γ<sub>1</sub>-syntrophin participates in γ-enolase translocation towards the plasma membrane, a pre-requisite for its neurotrophic activity. By disrupting this γ<sub>1</sub>-syntrophin-guided subcellular distribution, cathepsin X reduces γ-enolase-induced neurotrophic signaling.