Abstract
S-Palmitoylation is the only reversible post-translational lipid modification. Knowledge about the DHHC palmitoyltransferase family is still limited. Here we show that human ZDHHC6, which modifies key proteins of the endoplasmic reticulum, is controlled by an upstream palmitoyltransferase, ZDHHC16, revealing the first palmitoylation cascade. The combination of site specific mutagenesis of the three ZDHHC6 palmitoylation sites, experimental determination of kinetic parameters and data-driven mathematical modelling allowed us to obtain detailed information on the eight differentially palmitoylated ZDHHC6 species. We found that species rapidly interconvert through the action of ZDHHC16 and the Acyl Protein Thioesterase APT2, that each species varies in terms of turnover rate and activity, altogether allowing the cell to robustly tune its ZDHHC6 activity.
Highlights
Cells constantly interact with and respond to their environment
Using the Acyl-RAC method to isolate palmitoylated proteins (Werno and Chamberlain, 2015), we verified that the endoplasmic reticulum (ER) chaperone calnexin (Lakkaraju et al, 2012), the E3 ligase gp78 (Fairbank et al, 2012), the IP3 receptor (Fredericks et al, 2014) and the transferrin receptor (Trf-R) (Senyilmaz et al, 2015) are ZDHHC6 targets (Figure 1B)
Calnexin and Trf-R were no longer captured by Acyl-RAC in the HAP1 ZDHHC6 KO cells, confirming that they are exclusively modified by ZDHHC6, while capture of the IP3 receptor and gp78 was reduced but not abolished indicating that they can be modified by other palmitoyltransferases (Figure 1B) consistent with previous findings (Fairbank et al, 2012; Fredericks et al, 2014)
Summary
Cells constantly interact with and respond to their environment This requires tight control of protein function in time and in space, which largely occurs through reversible post-translational modifications of proteins, such as phosphorylation, ubiquitination and S-palmitoylation. The latter consist in the addition on an acyl chain, generally C16 in mammals, to cysteine residues, thereby altering the hydrophobicity of the protein and tuning its function (Blaskovic et al, 2014; Chamberlain and Shipston, 2015). The list of proteins undergoing palmitoylation is ever increasing ([Blanc et al, 2015], http://swisspalm.epfl.ch/) and the modification is found to be important in numerous key cellular processes including neuronal development and activity (Fukata and Fukata, 2010), cardiac function (Pei et al, 2016), systemic inflammation (Beard et al, 2016), innate immunity to viruses (Mukai et al, 2016), cell polarity (Chen et al, 2016), EGF-signalling (Runkle et al, 2016), protease activity (Skotte et al, 2017) and cancer (Coleman et al, 2016; Thuma et al, 2016)
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