Supplementary Material for: Gamma-1-Syntrophin Mediates Trafficking of Gamma-Enolase towards the Plasma Membrane and Enhances Its Neurotrophic Activity

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 γ₁-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 γ₁-syntrophin and γ-enolase was observed in neurite growth cones in differentiated SH-SY5Y cells. Silencing of the γ₁-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 γ₁-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 γ₁-syntrophin PDZ domain. Collectively, these data demonstrate that γ₁-syntrophin participates in γ-enolase translocation towards the plasma membrane, a pre-requisite for its neurotrophic activity. By disrupting this γ₁-syntrophin-guided subcellular distribution, cathepsin X reduces γ-enolase-induced neurotrophic signaling.