TRANSLOCATION OF ARF1 TO THE SECRETORY GRANULES IN RAT PAROTID ACINAR-CELLS

We investigated the interaction of ADP-ribosylation factor (Arf) with the secretory granules in rat parotid acinar cells. The 20.5-kDa small -molecular-mass GTP-binding protein in the cytosolic fraction of rat p arotid acinar cells was identified as ADP-ribosylation factor1 by usin g a pan-Arf monoclonal antibody and isotype-specific polyclonal antibo dies for Arf proteins 1, 3, 5, and 6, Incubation of the cytosolic frac tion with isolated secretory granule membranes in the presence of GTP gamma S resulted in the translocation of Arf1 from the cytosolic fract ion to the secretory granule membranes. The translocation was not obse rved in the presence of GDP beta S in place of GTP gamma S, indicating that the process is GTP-dependent, The immunoelectron microscopy expe riment confirmed Arf1 is translocated to the secretory granules. A pri or treatment of the granule membranes with trypsin inhibited the trans location of Arf1 at 2 mM Mg2+, but had no effect in the absence of Mg2 + (condition of spontaneous conversion of Arf-GDP to Arf-GTP), Thus, t he trypsin-sensitive nucleotide exchange activity for Arf1 is probably associated with the secretory granule membranes, These results demons trate Arf1 translocates to the secretory granules in rat parotid acina r cells. (C) 1998 Academic Press.