An amphipathic α-helix directs palmitoylation of the large intracellular loop of the sodium/calcium exchanger

Figure 1.. Palmitoylation of NCX1 is largely independent of primary sequence close to the palmitoylation site. Palmitoylated proteins were purified by resin-assisted capture and immunoblotted alongside their respective unfractionated cells lysates. In all experiments palmitoylation is expressed relative to expression. A, single amino acid substitutions with alanine do not influence NCX1 palmitoylation with the exception of D741A, which modestly reduces palmitoylation. B, charge substitutions and insertions before or after Cys-739 are without impact on NCX1 palmitoylation. UF, unfractionated cell lysate; Palm, palmitoylated proteins. *, p < 0.05 versus WT; **, p < 0.01 versus WT, n = 5–6.

Figure 2.. Palmitoylation and trafficking of human polymorphisms of NCX1. A, substitution of Ser-738 with a bulky phenylalanine residue increases the fraction of NCX1 palmitoylated in HEK cells, but P737L is without effect. B, polymorphisms P737L and S738F are without impact on the delivery of NCX1 to the cell surface. EV, empty vector transfected cells; UF, unfractionated cell lysate; palm, palmitoylated proteins **, p < 0.01 versus WT, n = 7.

Figure 3.. Palmitoylation of YFP-NCX1 requires an amphipathic α-helix on the C-terminal side of the palmitoylation site. A, schematic of the NCX1 intracellular loop, indicating the positions of the calcium-binding domains (CBD), catenin-like domain (CLD), and amphipathic α-helix (AαH). The positions of truncations are indicated below the schematic. B, impact of truncations on the palmitoylation of YFP-NCX1. Only removal of amino acids 744–765 causes palmitoylation of Cys-739 to be abrogated. C, sequence of NCX1 residues 739–765, highlighting the abundant aromatic amino acids (φ). The underlined region, 744–765, is required for palmitoylation of NCX1 in B. The boxed region is predicted to form an α-helix by JPred4. A projection of this α-helix is shown, with the polar face highlighted in black. UF, unfractionated cell lysate; Palm, palmitoylated proteins. **, p < 0.01 versus 266–765 (WT), n = 5.

Figure 4.. Palmitoylation of YFP-NCX1 is abolished by mutations in the C-terminal α-helix. Introduction of a single negative charge at position 746 (F746E) reduces but does not eliminate palmitoylation of YFP-NCX1, whereas palmitoylation is completely abolished either by introducing a negative charge in position 750 (F750E), two negative charges (F746E/F750E), or the helix-breaking insertion of three prolines (M744P/H745P/F746P). Deletion of the entire α-helix (Δ740–756), the first two turns of the helix (Δ740–746), or the second two turns of the helix (Δ747–753) also prevents or reduces YFP-NCX1 palmitoylation. UF, unfractionated cell lysate; Palm, palmitoylated proteins. **, all p < 0.01 versus WT, n = 4.

Figure 5.. Point mutations and palmitoylation of full-length NCX1. A, point mutations F746E/F750E, Δ740–756, Δ740–746, and M744P/H745P/F746P prevent palmitoylation of full-length NCX1, whereas F750E reduces full-length NCX1 palmitoylation, and Δ747–753 and F746E are without effect. B, of the mutations assessed in A, only F746E/F750E impedes progression of full-length NCX1 through the secretory pathway. C, NCX1 is palmitoylated at Cys-731 when the region usually on the C-terminal side of Cys-739 is instead positioned adjacent to Cys-731. D, C-terminal fusion of NCX1 residues 738–756 is sufficient to direct palmitoylation of YFP. E, C-terminal fusion of NCX1 residues 738–756 anchors YFP to intracellular membranes in a manner indistinguishable from YFP-NCX1. Scale bar, 20 μm. *, p < 0.05 versus WT; **, p < 0.01 versus WT, n = 5.

Figure 6.. Alanine mutations on the hydrophilic face of the amphipathic α-helix impair NCX1 palmitoylation. Substitution of Asp-741, His-745, and Thr-748 but not Lys-752 reduce palmitoylation of full-length NCX1. Substitution of all four residues on the hydrophilic face with alanine (All4A: D741A/H745A/T748A/K752A) reduces NCX1 palmitoylation by ∼85%, UF, unfractionated cell lysate; Palm, palmitoylated proteins. **, p < 0.01 versus WT, n = 4.