S-Acylation of transmembrane proteins in the early secretory pathway

Eukaryotic cells are composed of many different compartments and pathways that are specialised to perform specific functions. The secretory pathway modifies and sorts the thousands of different membrane proteins that are produced in a cell. Following their synthesis, membrane proteins enter the secretory pathway and move through the endoplasmic reticulum and Golgi compartments en route to the plasma membrane. During these transport steps, proteins undergo a number of chemical modifications such as the attachment of sugar or lipid groups - and these modifications often play an important role in directing proteins to a specific cellular location or enhancing their folding and stability. Whereas glycosylation pathways in the secretory pathway that mediate the attachment of sugar groups to proteins have been extensively studied, the S-acylation of proteins (attachment of lipid groups) is poorly understood. In particular, how are the thousands of membrane proteins passing through the secretory pathway selected for S-acylation and how do the endoplasmic reticulum and Golgi coordinate this process?

S-Acylation is mediated by "zDHHC" enzymes and there are twenty-three of these enzymes in humans. The vast majority of zDHHC enzymes are present at the endoplasmic reticulum and Golgi and our previous work suggested that these enzymes are either specialised to mediate the S-acylation of a specific and restricted pool of proteins or instead have a broad specificity that allows them to modify a diverse set of proteins- however the pools of proteins modified by individual zDHHC enzymes are poorly defined. This project will take a compartment-centric view of S-acylation and use methods that permit the reversible trapping of membrane proteins at the endoplasmic reticulum, allowing us to exert fine control over their movement between this compartment and the Golgi. This will be combined with high-sensitivity assays of S-acylation to map out the compartment-specific S-acylation patterns of a diverse array of membrane proteins (i.e. whether they are S-acylated at the endoplasmic reticulum or after release from this compartment). To understand how membrane proteins are selected for S-acylation by different compartments, we will undertake a detailed analysis of the amino acid sequence requirements that underpin compartment-specific S-acylation patterns. On top of this, we will study eight different zDHHC enzymes present at the endoplasmic reticulum to decipher how they contribute to the S-acylation capacity and specificity of this organelle. A specific focus here will be on the enzyme zDHHC6, which our recent work suggests may be a broad specificity enzyme that mediates S-acylation of a range of membrane proteins at the endoplasmic reticulum.

These complementary analyses will provide an important breakthrough in our understanding of how the S-acylation of a large and diverse array of membrane proteins is coordinated by different cellular compartments and will generate new fundamental insight into protein modification during transport through the secretory pathway. In addition to this fundamental new knowledge, this work is likely to have longer-term impact as there is growing interest in targeting protein S-acylation pathways as a treatment for different human diseases including cancer, neurological disorders and infectious diseases. Understanding the roles of different compartments and enzyme isoforms in S-acylation of different classes of membrane protein will contribute important information that ensures that this process can be fully and appropriately exploited as a therapeutic target.

S-Acylation is a widespread regulatory post-translational modification involving the reversible attachment of fatty acids onto cysteine residues. Thousands of different proteins are modified by S-acylation including ion channels, receptors, transporters, scaffolds, enzymes, membrane fusion proteins and signalling molecules. S-acylation is mediated by the zDHHC enzyme family (encoded by 23 different ZDHHC genes in the human genome) and these polytopic membrane proteins are enriched at the endoplasmic reticulum and Golgi.

Despite the role that S-acylation plays in regulating the trafficking, stability and function of a diverse array of essential proteins, there is very little known about how membrane proteins are selected for S-acylation during their transport through the secretory pathway. Indeed, analyses of individual zDHHC enzymes has so far failed to provide an understanding of how S-acylation reactions are coordinated more widely in the cell. This proposal will take a different compartment-centric view of S-acylation to provide new insight into how S-acylation reactions are coordinated in the secretory pathway. Compartment trapping techniques will be combined with high-sensitivity S-acylation assays to (i) identify membrane proteins that are modified at the endoplasmic reticulum or following release from this organelle, (ii) identify sequence-specific determinants that underlie compartment-specific S-acylation patterns of membrane proteins, and (iii) examine the contribution of ER-localised zDHHC enzymes to compartment-specific S-acylation patterns and identify determinants of substrate S-acylation by zDHHC6 as an exemplar of a broad specificity ER enzyme active against multiple membrane proteins. Collectively, these analyses will provide important new knowledge on the post-translational modification of membrane proteins in the secretory pathway.