{'fraction_non_alphanumeric': 0.02106084243369735, 'fraction_numerical': 0.0171606864274571, 'mean_word_length': 5.560102301790281, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Substrate recognition by the cell surface palmitoyl transferase DHHC5

Significance Dynamic palmitoylation at the cell surface by the acyl transferase DHHC5 regulates a plethora of physiological processes, from endocytosis to synaptic plasticity. Here we report that DHHC5 is abundantly expressed in cell surface caveolar microdomains in cardiac muscle, and that the Na pump regulator phospholemman is a substrate for DHHC5. Palmitoylation of phospholemman C40 by DHHC5 reduces Na pump activity. DHHC5 interacts with phospholemman independent of its PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, via a region of its intracellular carboxyl tail that has not hitherto been implicated in substrate recognition. This suggests that it may be possible to selectively manipulate DHHC5 activity by blocking the recruitment of specific substrates to the active site by the carboxyl tail. The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme–substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.