DISCRIMINATION BETWEEN TEMPERATURE-SENSITIVE AND BREFELDIN-A-SENSITIVE STEPS IN THE SULFATION, PHOSPHORYLATION, AND CLEAVAGE OF PROGASTRIN AND ITS DERIVATIVES

Maturation of the acid-stimulating hormone gastrin involves precursor cleavage, tyrosine sulfation, serine phosphorylation, and COOH-termina l amidation. We have used brefeldin A and incubation at 22 degrees C t o determine where and when these modifications occur. Immunogold studi es of gastrin cells incubated at 22 degrees C revealed swollen Gels ci sternae, the terminal regions of which were associated with an accumul ation of progastrin immunoreactivity. At 22 degrees C, [H-3]tyrosine a nd [S-35]sulfate were incorporated into progastrin, but Arg(94)-Arg(95 ) cleavage, and Ser(96) phosphorylation, were inhibited. When pulse la beling at 22 degrees C for 120 min was followed by a chase at 37 degre es C, [S-35]progastrin was cleaved at Arg(94)-Arg(95) with a t(1/2) of about 10 min, compared with about 20 min for [H-3]progastrin. Approxi mately 60% of the COOH-terminal cleavage fragment was phosphorylated, but there was little or no incorporation of [P-32]phosphate into proga strin. Addition of brefeldin A during the chase substantially inhibite d cleavage of [H-3]progastrin, but not [S-35]progastrin. However, when pulse labeling was limited to 20 min at 22 degrees C, the presence of brefeldin A in a subsequent chase at 37 degrees C completely inhibite d cleavage of [S-35]progastrin. The data indicate that progastrin sulf ation occurs in the trans-Golgi network, exit from which involves pass age through first a brefeldin A-sensitive and then a temperature-sensi tive step. Cleavage at Arg(94)-Arg(95) and Ser phosphorylation are clo sely linked, occur distal to the temperature-sensitive step, and are f ollowed by amidation in secretory granules. It is known that mature se cretory granules do not phosphorylate progastrin-derived peptides, and so phosphorylation appears to coincides with, and may provide a marke r for, delivery of peptide from trans-Golgi work to immature secretory granules in gastrin cells.