Tandem fluorescence imaging of dynamic S-acylation and protein turnover.
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.
Zhang
Mingzi M
MM
Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, USA.
Tsou
Lun K
LK
Charron
Guillaume
G
Raghavan
Anuradha S
AS
Hang
Howard C
HC
D000215:Acylation / Q000187:drug effects; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D054878:Lipoylation / Q000187:drug effects; D008213:Lymphocyte Activation / Q000187:drug effects; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods*; D010168:Palmitates / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D014638:Vanadates / Q000494:pharmacology
D010168:Palmitates; C076715:pervanadate; D014638:Vanadates; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)