PtdIns4P-mediated electrostatic forces influence S-acylation of peripheral proteins at the Golgi complex.

Sabrina Chumpen Ramirez,Micaela R Astrada,Jose L Daniotti

doi:10.1042/bsr20192911

Abstract

Protein S-acylation is a reversible post-translational modification involving the addition of fatty acids to cysteines and is catalyzed by transmembrane protein acyltransferases (PATs) mainly expressed at the Golgi complex. In case of soluble proteins, S-acylation confers stable membrane attachment. Myristoylation or farnesylation of many soluble proteins constitutes the initial transient membrane adsorption step prior to S-acylation. However, some S-acylated soluble proteins, such as the neuronal growth-associated protein Growth-associated protein-43 (GAP-43), lack the hydrophobic modifications required for this initial membrane interaction. The signals for GAP-43 S-acylation are confined to the first 13 amino acids, including the S-acylatable cysteines 3 and 4 embedded in a hydrophobic region, followed by a cluster of basic amino acids. We found that mutation of critical basic amino acids drastically reduced membrane interaction and hence S-acylation of GAP-43. Interestingly, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) at the Golgi complex reduced GAP-43 membrane binding, highlighting a new, pivotal role for this anionic lipid and supporting the idea that basic amino acid residues are involved in the electrostatic interactions between GAP-43 and membranes of the Golgi complex where they are S-acylated.

Highlights

Protein S-acylation, known as palmitoylation, consists of the addition of a lipid molecule to one or more cysteine residues through a thioester bond
Specific version of the Acyl-Biotin Exchange (ABE) assay for identifying S-acylated proteins, we confirmed that N13GAP-43 is S-acylated in Chinese hamster ovary (CHO)-K1 cells, whereas as expected, the mutant N13GAP-43(C3,4S) is not modified (Figure 1B)
We demonstrate that a cluster of basic amino acids surrounding the two S-acylatable cysteines of Growth-associated protein-43 (GAP-43) are involved in the physical contact between the protein and Golgi complex membranes through an electrostatic mechanism

Summary

Introduction

Protein S-acylation, known as palmitoylation, consists of the addition of a lipid molecule to one or more cysteine residues through a thioester bond. This modification, which is widespread in eukaryotes, allows the reversible association of peripheral proteins with membranes or, in the case of integral membrane proteins, modulates their behavior within the plane of the membrane due to their partition to different micro- and nanodomains via interaction with other proteins and lipids [1,2,3]. For instance, H-Ras is first farnesylated in the cytosol, thereby allowing transient association with the cytosolic surface of the endoplasmic reticulum and S-acylation to the Golgi complex [1,15,16]. Many other S-acylated proteins do not contain specific receptors or membrane-targeting motifs, are not modified by the attachment of hydrophobic groups, and

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