Tandem fluorescence imaging of dynamic S -acylation and protein turnover

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.bioorthogonal ligation | chemical reporters | click chemistry | S-palmitoylation | fatty acylation P rotein S-palmitoylation (S-acylation) targets proteins to discrete intracellular membrane compartments, controls protein stability, and mediates protein-protein interactions (1, 2). Furthermore, the reversibility of S-acylation offers spatial and temporal control of protein function akin to protein phosphorylation (Fig. 1A). Notably, the differential S-palmitoylation and membrane targeting of H-and N-Ras isoforms have been shown to activate discrete signaling pathways for cellular growth and differentiation (3-6). Accelerated deacylation of G-protein subunits and G-protein coupled receptors upon receptor stimulation (7,8) suggest active mechanisms regulating S-acylation/deacylation cycles. In addition, receptor activity-regulated palmitate turnover of PSD-95 is proposed to modulate synaptic strength and plasticity in postsynaptic neurons by mediating receptor clustering (9). Development of the acyl-biotinyl exchange protocol (10-12) and fatty acid chemical reporters (13-16) enabled improved detection and identification of many S-palmitoylated proteins from diverse biological pathways in eukaryotes (17). Nonetheless, it remains to be determined if S-acylation is dynamic and regulated for identified fatty-acylated proteins.Quantitative analysis of S-acylation/deacylation cycle on proteins would provide further insight into the biological significance and function of dynamic S-palmitoylation, but tools to efficiently visualize palmitate turnover on proteins are limited. While photoactivation/bleaching of S-palmitoylated proteins fused to fluorescent reporters allow visualization of protein dynamics in cells (4,5,18), these methods yield indirect readouts of protein S-acylation. Acyl-biotinyl exchange enables nonradioactive det...

Substrate recognition by the cell surface palmitoyl transferase DHHC5

“…An appreciation is now growing that protein palmitoylation turns over rapidly (in minutes) for certain proteins (4)(5)(6)(7)(8). For example, dynamic surface membrane protein palmitoylation by DHHC5 underlies a novel form of endocytosis, massive endocytosis (MEND), in which up to 70% of the cell surface membrane is internalized (7,8).…”

Mutations in the X-linked Intellectual Disability Gene, zDHHC9, Alter Autopalmitoylation Activity by Distinct Mechanisms

“…The major form of protein palmitoylation, S-palmitoylation, occurs via a thioester linkage on cysteine residues. Myristoylation and prenylation are irreversible modifications, whereas palmitoylation is reversible through proposed protein palmitoylthioesterases (1). The reversible nature of the thioester linkage provides for a dynamic acylation/deacylation cycle that is required for subcellular trafficking and function of palmitoylated proteins (2,3).…”

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

“…Noncanonical amino acids have been used to measure the kinetics of protein and nucleosome turnover (38,39), visualize local protein synthesis in neuronal subcompartments (40,41), and identify the products of stimulus-induced protein synthesis in axons and dendrites (42,43). Here we introduce an additional level of control in the metabolic labeling of proteins made in mammalian cells, by using a noncanonical amino acid that requires a mutant aminoacyltRNA synthetase for activation.…”