Exploring the driving forces influencing S-acylation of peripheral proteins at the Golgi complex

CHUMPEN RAMIREZ, SABRINA; ASTRADA, MICAELA; DANIOTTI, J.L.

Parana

Congreso; 54 Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2018

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

S-acylation of proteins is a post-translational modification that consists in the addition of long chain fatty acids to cysteines, through a reversible thioester bond. For soluble proteins, this modification confers stable attachment to membranes, influencing their trafficking and subcellular distribution. S-acylation is catalyzed by a family of transmembrane Acyl Protein Transferases (PATs) distributed along the secretory pathway, mainly at the Golgi complex. Myristoylation or farnesylation of many soluble proteins constitute the first transient membrane adsorption before being S-acylated. On the other hand, some S-acylated soluble proteins do not possess hydrophobic modifications involved in the initial membrane interaction, as observed in GAP-43, a neuronal protein involved in axonal growth and regeneration. We reported that the signals for GAP-43 S-acylation are contained in the first 13 amino acids (N13GAP-43), including two acylatable cysteines (C3, C4) embedded in a hydrophobic region followed by a basic cluster. By biochemical, fluorescence microscopy and time-lapse imaging experiments, we found that mutation of critical basic amino acids drastically reduced membrane interaction and hence S-acylation of N13GAP-43 both in neuron-like and epithelial cells, without affecting substrate recognition by PATs as revealed in a forced Golgi complex interaction of the mutated protein via binding to PI4P. Moreover, pharmacological depletion of PI4P markedly reduced GAP-43 membrane binding, strongly suggesting that few basic residues around C3 and C4 mediate electrostatic interactions with anionic lipids at the Golgi complex, where GAP-43 is S-acylated.