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. 2010 May 11;107(19):8627-32.
doi: 10.1073/pnas.0912306107. Epub 2010 Apr 26.

Tandem fluorescence imaging of dynamic S-acylation and protein turnover

Mingzi M Zhang  1 , Lun K Tsou, Guillaume Charron, Anuradha S Raghavan, Howard C Hang
Affiliations

Tandem fluorescence imaging of dynamic S-acylation and protein turnover

Mingzi M Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

The functional significance and regulation of reversible S-acylation on diverse proteins remain unclear because of limited methods for efficient quantitative analysis of palmitate turnover. Here, we describe a tandem labeling and detection method to simultaneously monitor dynamic S-palmitoylation and protein turnover. By combining S-acylation and cotranslational fatty acid chemical reporters with orthogonal clickable fluorophores, dual pulse-chase analysis of Lck revealed accelerated palmitate cycling upon T-cell activation. Subsequent pharmacological perturbation of Lck palmitate turnover suggests yet uncharacterized serine hydrolases contribute to dynamic S-acylation in cells. In addition to dually fatty-acylated proteins, this tandem fluorescence imaging method can be generalized to other S-acylated proteins using azidohomoalanine as a methonine surrogate. The sensitivity and efficiency of this approach should facilitate the functional characterization of cellular factors and drugs that modulate protein S-acylation. Furthermore, diverse protein modifications could be analyzed with this tandem imaging method using other chemical reporters to investigate dynamic regulation of protein function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Tandem fluorescence approach to imaging S-palmitoylation turnover on proteins. (A) S-Palmitoylation is a reversible and dynamic protein modification. (B) Following metabolic labeling with two chemical reporters, immunopurification and sequential click chemistry reactions with orthogonal detection tags allow simultaneous visualization of the fatty-acylation and protein synthesis. (C) Amino acid chemical reporter for protein synthesis. Fatty acid chemical reporters for N-myristoylation and S-palmitoylation. (D) Clickable fluorescent detection tags.
Fig. 2.
Fig. 2.
Analysis of dynamic S-palmitoylation on Lck by tandem fluorescence imaging. (A) In-gel fluorescence scanning allows tandem detection of az-12 and alk-16 on Lck using alk-Cyfur and az-Rho, respectively. Anti-Lck blot reflects total protein levels. (B) Pulse-chase analysis reveals dynamic S-palmitoylation of Lck. (C) Data from multiple pulse-chase experiments (n = 10). Data points from the same chase times, after normalizing alk-16 to az-12 signals, were compiled and displayed as average values ± SEM. *, nonspecific bands.
Fig. 3.
Fig. 3.
Pervanadate stimulation of T cells accelerates palmitate cycling on Lck. (A) Anti-phosphotyrosine, anti-Lck blots, and (B) alk-16 fluorescence of lysates from unstimulated and PV-treated Jurkat T cells. (C) Anti-phosphotyrosine, anti-Lck blots, and alk-16 fluorescence of immunopurified Lck from unstimulated and PV-treated Jurkat T cells. Mobility shift of immunopurified Lck was observed with PV treatment. (D) Pulse-chase analysis of Lck in the presence of 0.1 mM PV. (E) PV activation data from multiple pulse-chase experiments (n = 7). Data points from the same chase times, after normalizing alk-16 to az-12 signals, were compiled and displayed as average values ± SEM (Inset). (F) Pulse-chase analysis of Lck upon PV treatment with shorter pulse times. (G) PV activation data averaged from two pulse-chase experiments with shorter pulse times.
Fig. 4.
Fig. 4.
Pharmacological perturbation of palmitate turnover on Lck. (A) Pulse-chase analysis of Lck in the presence of chemical inhibitors. (B) Data from multiple pulse-chase experiments (n = 2). Data points from the same chase times, after normalizing alk-16 to az-12 signals, were compiled and displayed as average values ± SEM.
Fig. 5.
Fig. 5.
Use of a more general protein synthesis chemical reporter extends the tandem imaging method beyond N-myristoylated proteins. (A) Pulse-chase analysis of H-RasG12V upon labeling with AHA and alk-16. (B) Data from multiple pulse-chase experiments (n = 5). Data points from the same chase times, after normalizing alk-16 to AHA signals, were compiled and displayed as average values ± SEM.
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