Palmitoylation of Exocytic Proteins: Role in Membrane Compartmentalization, Intracellular Trafficking, and Function

Certain cells contain small sacs or ‘vesicles‘ filled with important cargo, such as hormones or neurotransmitters. Appropriate stimuli trigger the fusion of these vesicles with the cell membrane, resulting in cargo secretion from the cell. A large number of proteins are required for membrane fusion, and it is essential to determine how these various proteins are regulated. Interestingly, we have shown that the attachment of palmitate (a fatty acid) to certain proteins modifies their localisation in the cell membrane and regulates their ability to support membrane fusion. This proposal will precisely determine how palmitate addition regulates the activity of proteins that mediate membrane fusion, and identify whether dynamic addition/removal of palmitate is important for fusion activity. Furthermore, we will determine how palmitate regulates the sorting of proteins to specific locations in the cell. These analyses will provide valuable and novel information on the regulation of membrane fusion by palmitate, and will also serve as an important paradigm to understand how palmitate regulates the sorting and membrane distribution of proteins in general. The results generated from this study will hopefully contribute to the design of treatments for conditions such as epilepsy and other brain disorders, and metabolic disorders such as diabetes.

The fusion of intracellular secretory vesicles with the plasma membrane (exocytosis) mediates cellular secretion of essential molecules such as neurotransmitters and hormones. Exocytosis in neuronal cells is dependent upon the interaction of the plasma membrane SNAREs, SNAP25 and syntaxin 1A, with the vesicle SNARE, VAMP2. Indeed, the formation of this ‘SNARE complex‘ may directly catalyze membrane fusion. A large number of other proteins function in exocytosis, including the chaperone cysteine-string protein (CSP). Despite a good understanding of the proteins and protein-protein interactions that mediate exocytosis, far less is known about how lipid modifications, such as protein palmitoylation, regulate this process.
We identified a relationship between the number of potential palmitoylation sites in SNAP25, or its ubiquitous homologue SNAP23, and the extent of exocytosis. These effects of palmitoylation on exocytosis may be mediated by compartmentalisation of SNAP25/23 into specific micro-domains of the plasma membrane. To extend upon these novel observations, we will utilise mass spectrometry to analyze the extent of SNAP25/23 palmitoylation, and whether differentially palmitoylated pools of these proteins are present in cells. Immunogold labelling will then be used to precisely map the micro-localisation of SNAP25/23 at the plasma membrane, and determine how this is modulated by palmitoylation. Furthermore, we will examine the activity-dependent regulation of SNAP25/23 palmitoylation, and of other cellular proteins, and determine the importance of this for exocytosis. Finally, total internal reflection microscopy and amperometry will be used to pinpoint the aspect(s) of exocytosis that is modulated by specific SNAP25/23 cysteine mutants or palmitoylating enzymes.
In addition to regulating micro-localization and function of SNAP25/23, we propose that palmitoylation also regulates the intracellular sorting of SNAP25 and CSP. Indeed, recent work has highlighted an important role for palmitoylation in the intracellular trafficking of certain proteins. However, little is known about how palmitoylated proteins that lack identifiable primary membrane targeting determinants, such as SNAP25 and CSP, are trafficked within the cell. To address this, we will use a combination of confocal imaging, fractionation and inhibitor studies to identify the intracellular trafficking pathways of SNAP25 and CSP, and determine how trafficking and correct sorting is regulated by palmitoylation. In addition, we will determine how palmitoylation code regulates correct intracellular sorting to specific pre/post-synaptic sites in neurons. Finally, the role of palmitoylation in regulating the exocytic function of CSP will be tested.
Overall, these analyses will provide novel data on the role of palmitoylation in regulating protein trafficking, micro-localization and function.