This paper explores the capacity of palmitoylation to localize the protein SNAP25 between the cellular membrane and intracellular domains, in specifically recycling endosomes (RE) and the trans Golgi network (TGN). SNAP25 is a peripheral membrane protein responsible for regulating exocytosis and belongs to the SNARE family of proteins. With four cysteines, which comprise the active sites for palmitoylation, these researchers manipulated the availability of these sites to palmitoylation and subsequently measured fluorescent expression of intracellular localization. They tested fluorescence after inhibiting protein synthesis in intracellular regions, mutating cysteine residues, and attached motifs outside of Cys-rich domain. They were able to conclude that palmitoylation can dictate patterning of SNAP25 protein movement across intracellular domains. Additionally, they noted that the cysteine-rich domain is “autonomous and sufficient” for inracellular localization.
Used PC12 cells to modify SNAP25 proteins. Utilized eGFP-tagged SNAP25b proteins after determining equal regional expression between eGFP-SNAP25b proteins and WT SNAP25 proteins. To inhibit protein synthesis, they used Cycloheximide before radiolabelling palmitate and measuring palmitoylation activities of mature proteins. They mutated individual cysteines (85, 88, 90, and 92) with leucine to measure localization with decreased palmitoylation binding sites. To measure effects of motif association to SNAP25, they used a CAAX motif attached after the cysteine-rich domain (1-92). Flueorescent overlap was determined with statistical calculations like quantitative colocalization analysis and triple labelling.
WHY IT’S IMPORTANT:
We are interested in the dynamic action of protein palmitoylation and its specific effects of localizing proteins on the membrane. In our case, we would like to apply this analysis to ion channels in the inner ear hair cells and test how localization may affect the onset of hearing. The next step is applying a similar course of action to the auditory system and more specifically cochlear tissues. This paper also shows good controls as it continues to revisit ideas established early in the experiment, like Golgi localization and increased intracellular localization with cysteine mutation.