Figure 2.

Identification of the NCX1 palmitoylation site. A) Membrane topology of mature NCX1.1 following cleavage of the signal peptide. The start and end of the large intracellular loop, and positions of the mutated cysteines are numbered. B) Individual cysteines and pairs of cysteines were mutated to alanine and transiently expressed in HEK cells. All mutants were palmitoylated with the exception of C739A. All acyl-RAC reactions were blotted for flotillin 2 (Flot2) to confirm efficient purification of palmitoylated proteins. C) Individual cysteines were reintroduced to NCX1 with a cys-less intracellular loop (cysteines 383, 387, 485, 557, 731, and 739 all mutated to alanine). Palmitoylation of NCX1 requires the presence of C739 only. M, mock transfected; Palm, proteins purified using acyl-RAC; RVM, rat ventricular myocytes; UF, unfractionated cell lysate.