Published: Vol 4, Iss 22, Nov 20, 2014 DOI: 10.21769/BioProtoc.1296 Views: 9599
Reviewed by: Zhaohui LiuAnonymous reviewer(s)
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Abstract
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.
Materials and Reagents
Equipment
Procedure
Representative data
Representative data are shown in Figure 1. This experiment was repeated three times using this protocol with similar results.
Figure 1. S-Acylation mediates PBS1 localization to the PM. A. The predicted N-terminal S-acylation motif in PBS1 is required for plasma membrane localization. N20:sYFP indicates fusion of the first 20 amino acids of PBS1, which contains the predicted palmitoylation motif, to the super yellow fluorescent protein. n20 is the fragment with the G2A/C3AC/6A triple mutations. Both fusion proteins were transiently expressed in Nicotiana benthamiana. Confocal microscopy was performed at 5 h post dexamethasone induction. All images are three-dimensional projections from a Z-stack. The N20:sYFP localized to the plasma membrane and mobile vesicle-like structures were observed, which was reported when acylated proteins were overexpressed (Vilas et al., 2006; Joensuu et al., 2010). However, the n20:sYFP fusion was detected exclusively as cytoplasmic strands. The YFP signal was showed in the false color of green. B. PBS1 is S-acylated. Cell extracts of N. benthamiana tissue expressing N20:sYFP-HA or n20:sYFP-HA were subjected to immunoprecipitation using anti-HA matrix (IP). Immunoprecipitates were subjected to immuno-blot with anti-HA antibodies (IB) as the loading control. At the same time, the immunoprecipitates were treated with 50 mM N-ethylmaleimide to block free sulfhydryl groups, incubated with 1 M hydroxylamine to hydrolyze any Cys-palmitate thioester bonds, and then treated with 1 μm EZ-Link Biotin-BMCC in 50 mM Tris (pH 7.0) to label the newly exposed free sulfhydryl groups resulting from cleaved thioester bonds. The modified immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blot (WB) with streptavidin horseradish peroxidase. Reproduced from Qi et al. (2014) (Copyright American Society of Plant Biologists).
Notes
Recipes
Acknowledgments
This work was supported by the National Institute of General Medical Sciences at the National Institutes of Health (grant no. R01 GM046451 to R.W.I). This protocol was adapted from a previous work by Qi et al. (2014).
References
Article Information
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© 2014 The Authors; exclusive licensee Bio-protocol LLC.
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Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Modification
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