Figure 7. ERAT acylates GAM-proghrelin, requires certain amino acids at the N-terminus of the acylated protein, and is specific for Ser5.

(A) ERAT acylates GAM-proghrelin, but not His-tagged proghrelin, nor other precursors. Two µg of each peptide precursor were incubated with HEL cell P2 protein in the presence or absence of 0.1 mM Zn⁺⁺. Lanes 1 and 2, His-tagged proghrelin; lanes 3 and 4, GAM-proghrelin; lanes 5 and 6, mouse POMC; lanes 7 and 8, rat proenkephalin; lanes 9 and 10, ACTH. Left panel, Coomassie-stained gel of reaction mixtures showing the presence of equal quantities of each substrate; right panel, autoradiogram of the same gel, to identify [¹⁴C]octanoylated bands. (B) ERAT requires certain amino acids at the N-terminus of the acylated protein. Two µg of the peptides des-acyl ghrelin (lane 1) or GAM-des-acyl ghrelin (lane 2) were tested as potential substrates for ERAT. Left panel, autoradiogram of reaction mixtures to identify [¹⁴C]octanoylated bands; right panel, Coomassie-staining of the same gel to demonstrate the quantity of peptide. (C) ERAT is specific for Ser5: lack of acylation of the GAM-proghrelin mutants and preproghrelin. Acyltransferase activity was tested using GAM-proghrelin (lane 1), each mutant (S5A, lane2; S6A, lane 3; and S5,6A, lane4) and preproghrelin (lane 5) under the standard reaction conditions. Lower panel, autoradiogram to identify [¹⁴C]octanoylated bands; upper panel, Coomassie staining of the same gel in order to demonstrate the presence of equal quantities of each substrate.