The linker domain of the SNARE protein SNAP25 acts as a flexible molecular spacer that ensures efficient S-acylation.

-Acylation of the SNARE protein SNAP25 (synaptosome-associated protein of 25 kDa) is mediated by a subset of Golgi zinc finger DHHC-type palmitoyltransferase (zDHHC) enzymes, particularly zDHHC17. The ankyrin repeat domain of zDHHC17 interacts with a short linear motif known as the zDHHC ankyrin repeat-binding motif (zDABM) in SNAP25 (VVASQP), which is downstream of its -acylated, cysteine-rich domain (CGLCVCPC). Here, we investigated the importance of a flexible linker region (amino acids 93-111, referred to hereafter as the "mini-linker" region) that separates the zDABM and -acylated cysteines in SNAP25.

Detection of Heterogeneous Protein S-Acylation in Cells.

The use of synthetically synthesized azide and alkyne fatty acid analogs coupled with bioorthogonal Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction-based detection methods to study protein S-acylation reactions has replaced the traditional method of using in vivo metabolic radiolabeling with tritiated palmitic acid and has greatly facilitated our understanding of this essential cellular process. Here, we describe the chemical synthesis of myristic (C:14), palmitic (C16:0), and stearic (C18:0) acid-azide probes and detail how they may be utilized as chemical reporters for the analysis of S-acylation of exogenously expressed proteins in cells.

S-acylation regulates the trafficking and stability of the unconventional Q-SNARE STX19.

STX19 is an unusual Q-SNARE as it lacks a C-terminal transmembrane domain. However, it is efficiently targeted to post-Golgi membranes. Here, we set out to determine the intracellular localisation of endogenous STX19 and elucidate the mechanism by which it is targeted to membranes.

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions.

Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module.

LIF-dependent survival of embryonic stem cells is regulated by a novel palmitoylated Gab1 signalling protein.

The cytokine leukaemia inhibitory factor (LIF) promotes self-renewal of mouse embryonic stem cells (ESCs) through activation of the transcription factor Stat3. However, the contribution of other ancillary pathways stimulated by LIF in ESCs, such as the MAPK and PI3K pathways, is less well understood. We show here that naive-type mouse ESCs express high levels of a novel effector of the MAPK and PI3K pathways.

Tracking intracellular uptake and localisation of alkyne tagged fatty acids using Raman spectroscopy.

Intracellular uptake, distribution and metabolism of lipids are tightly regulated characteristics in healthy cells. An analytical technique capable of understanding these characteristics with a high level of species specificity in a minimally invasive manner is highly desirable in order to understand better how these become disrupted during disease. In this study, the uptake and distribution of three different alkyne tagged fatty acids in single cells were monitored and compared, highlighting the ability of Raman spectroscopy combined with alkyne tags for better understanding of the fine details with regard to uptake, distribution and metabolism of very chemically specific lipid species.

The C-terminal domain of zDHHC2 contains distinct sorting signals that regulate intracellular localisation in neurons and neuroendocrine cells.

The S-acyltransferase zDHHC2 mediates dynamic S-acylation of PSD95 and AKAP79/150, which impacts synaptic targeting of AMPA receptors. zDHHC2 is responsive to synaptic activity and catalyses the increased S-acylation of PSD95 that occurs following action potential blockade or application of ionotropic glutamate receptor antagonists. These treatments have been proposed to increase plasma membrane delivery of zDHHC2 via an endosomal cycling pathway, enhancing substrate accessibility.

Substrate selectivity in the zDHHC family of -acyltransferases.

S-acylation is a reversible lipid modification occurring on cysteine residues mediated by a family of membrane-bound 'zDHHC' enzymes. S-acylation predominantly results in anchoring of soluble proteins to membrane compartments or in the trafficking of membrane proteins to different compartments. Recent work has shown that although S-acylation of some proteins may involve very weak interactions with zDHHC enzymes, a pool of zDHHC enzymes exhibit strong and specific interactions with substrates, thereby recruiting them for S-acylation.

Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry.

S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity.

A cluster of palmitoylated cysteines are essential for aggregation of cysteine-string protein mutants that cause neuronal ceroid lipofuscinosis.

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is caused by mutation of the DNAJC5 gene encoding cysteine string protein alpha (CSPα). The disease-causing mutations, which result in substitution of leucine-115 with an arginine (L115R) or deletion of the neighbouring leucine-116 (∆L116) in the cysteine-string domain cause CSPα to form high molecular weight SDS-resistant aggregates, which are also present in post-mortem brain tissue from patients. Formation and stability of these mutant aggregates is linked to palmitoylation of the cysteine-string domain, however the regions of the mutant proteins that drive aggregation have not been determined.

The zDHHC family of S-acyltransferases.

The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthroughs in the S-acylation field. Now, more than a decade since their discovery, major questions centre on profiling the substrates of individual zDHHC enzymes (there are 24 ZDHHC genes and several hundred S-acylated proteins), defining the mechanisms of enzyme-substrate specificity and unravelling the importance of this enzyme family for cellular physiology and pathology.

New links between S-acylation and cancer.

S-acylation (also known as palmitoylation) is a major post-translational protein modification in all eukaryotic cells, involving the attachment of fatty acids onto cysteine residues. A variety of structural and signalling proteins are modified in this way, affecting their stability, membrane association and intracellular targeting. The enzymes that mediate S-acylation are encoded by genes belonging to the large (> 20 genes) ZDHHC family.

Palmitoylation and the trafficking of peripheral membrane proteins.

Palmitoylation, the attachment of palmitate and other fatty acids on to cysteine residues, is a common post-translational modification of both integral and peripheral membrane proteins. Dynamic palmitoylation controls the intracellular distribution of peripheral membrane proteins by regulating membrane-cytosol exchange and/or by modifying the flux of the proteins through vesicular transport systems.

Palmitoylation-induced aggregation of cysteine-string protein mutants that cause neuronal ceroid lipofuscinosis.

Recently, mutations in the DNAJC5 gene encoding cysteine-string protein α (CSPα) were identified to cause the neurodegenerative disorder adult-onset neuronal ceroid lipofuscinosis. The disease-causing mutations (L115R or ΔL116) occur within the cysteine-string domain, a region of the protein that is post-translationally modified by extensive palmitoylation. Here we demonstrate that L115R and ΔL116 mutant proteins are mistargeted in neuroendocrine cells and form SDS-resistant aggregates, concordant with the properties of other mutant proteins linked to neurodegenerative disorders.

Endoplasmic reticulum localization of DHHC palmitoyltransferases mediated by lysine-based sorting signals.

Intracellular palmitoylation dynamics are regulated by a family of 24 DHHC (aspartate-histidine-histidine-cysteine) palmitoyltransferases, which are localized in a compartment-specific manner. The majority of DHHC proteins localize to endoplasmic reticulum (ER) and Golgi membranes, and a small number target to post-Golgi membranes. To date, there are no reports of the fine mapping of sorting signals in mammalian DHHC proteins; thus, it is unclear how spatial distribution of the DHHC family is achieved.

The palmitoyl transferase DHHC2 targets a dynamic membrane cycling pathway: regulation by a C-terminal domain.

Intracellular palmitoylation dynamics are regulated by a large family of DHHC (Asp-His-His-Cys) palmitoyl transferases. The majority of DHHC proteins associate with endoplasmic reticulum (ER) or Golgi membranes, but an interesting exception is DHHC2, which localizes to dendritic vesicles of unknown origin in neurons, where it regulates dynamic palmitoylation of PSD95. Dendritic targeting of newly synthesized PSD95 is likely preceded by palmitoylation on Golgi membranes by DHHC3 and/or DHHC15.

Differential palmitoylation regulates intracellular patterning of SNAP25.

SNAP25 regulates membrane fusion events at the plasma membrane and in the endosomal system, and a functional pool of the protein is delivered to recycling endosomes (REs) and the trans Golgi network (TGN) through an ARF6-dependent cycling pathway. SNAP25 is a peripheral membrane protein, and palmitoylation of a cluster of four cysteine residues mediates its stable association with the membrane. Here, we report that palmitoylation also determines the precise intracellular distribution of SNAP25, and that mutating single palmitoylation sites enhances the amount of SNAP25 at the RE and TGN.

DHHC palmitoyl transferases: substrate interactions and (patho)physiology.

S-palmitoylation is a reversible post-translational modification that occurs on diverse cellular proteins. Palmitoylation can affect proteins in many different ways, including regulating membrane attachment, intracellular trafficking, and membrane micro-localisation. Intracellular palmitoylation reactions are mediated by a family of recently identified aspartate-histidine-histidine-cysteine (DHHC) palmitoyl transferases.

The intracellular dynamic of protein palmitoylation.

S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation.

Palmitoylation of the SNAP25 protein family: specificity and regulation by DHHC palmitoyl transferases.

SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b.

S-acylation by the DHHC protein family.

A family of 23 DHHC (Asp-His-His-Cys) proteins that function as mammalian S-acyltransferases has been identified, reinvigorating the study of protein S-acylation. Recent studies have continued to reveal how S-acylation affects target proteins, and have provided glimpses of how DHHC-substrate specificity might be achieved.

Regulation of SNAP-25 trafficking and function by palmitoylation.

The SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) protein SNAP-25 (25 kDa synaptosome-associated protein) is essential for regulated exocytosis in neuronal and neuroendocrine cells. Whereas the majority of SNARE proteins contain transmembrane domains, SNAP-25 is instead anchored to membranes by the palmitoylation of a central cysteine-rich region. In this review, we discuss the mechanisms of SNAP-25 palmitoylation and how this modification regulates the intracellular trafficking and exocytotic function of this essential protein.