GTP-dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein

Guanosine triphosphate (CTP) has been implicated in the regulation of Ca2+- mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found th at, in the presence of GTP, 1.0 muM free Ca2+ causes maximum secretion-equi valent to that achieved with 100 muM free Ca2+-whereas GTP gammaS inhibits Ca2+-siimulated secretion. interestingly, GTP by itself stimulates secretio n. These results indicate the existence of a CTP-regulated mechanism of sec retion in neutrophils that requires GTP hydrolysis to stimulate secretion i n the presence and absence of Ca2+. The stimulatory effect of GTP is only o bserved when CTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, g ives no stimulatory response, implying that the GTP-dependent secretory app aratus requires at least one cytosolic protein. CTP-dependent secretion can be reconstituted with crude HL-60 and bovine liver cytosol. The reconstitu ting activity binds to GTP-agarose, suggesting that the cytosolic factor is a GTP-binding protein or forms a complex with a GTP-binding protein. Howev er, it is not a member of the rho or rac families of GTPases. By gel filtra tion chromatography, the secretion-reconstituting activity eluted at 870 an d 200 kDa, but in the presence of CTP, eluted at 120 kDa, indicating that i t is part of a high-molecular-weight complex that dissociates in the presen ce of CTP. Retention of adenosine diphosphate-ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activ ity to brefeldin A led to our speculation that ARF6 may be the GTPase invol ved in GTP-dependent secretion, and that activity from a BFA-insensitive AR F6 guanine nucleotide exchange factor reconstitutes secretion, (C) 2000 Wil ey-Liss, Inc.