Protein palmitoylation is the post-translational modification of proteins via the attachment of palmitate through acyl linkages. The nucleophile sulfhydryl group of cysteines is the common palmitoylation site. Covalent attachment of palmitate occurs on numerous proteins and is usually associated with directing protein localization to the endomembrane system. Detection of protein palmitoylation by in vivo labeling with tritium-labeled palmitic acid typically requires an autoradiographic exposure time of several months, and, thus is not suitable for rapid analyses. Here, we described an easy protocol for quick in vitro detection of protein S-acylation using the Arabidopsis protein kinase, PBS1, as an example. To determine whether PBS1 is modified through thioester linkage to acyl groups, we employed a “biotin switch” assay (Hemsley et al., 2008). This work was first published in Qi et al. (2014), but we expand on the method here. PBS1 functions within the basal immune system of plants, and is a target of the bacterial cysteine protease, AvrPphB (Shao et al., 2002; Zhang et al., 2010). It contains a predicted N-terminal S-acylation motif (MGCFSCFDS), with both Cys-3 and Cys-6 residues predicted to be palmitoylated by CSS-Palm 3.0 (http://csspalm.biocuckoo.org/; Ren et al., 2008). Our method utilizes hydroxylamine-induced cleavage of thioester bonds, which results in free sulfhydryl groups that can then be conjugated to a biotin derivative, 1-biotinamido-4-[4′-(maleimidomethyl) cyclohexanecarboxamido]-butane (Biotin-BMCC). The conjugates are detectable by Western blot with streptavidin-horseradish peroxidase. The whole process of in vitro labelling and detection took less than 3 days, allowing the fast detection of protein modifications via thioester bonds such as palmitoylation.
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