GTP-dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein.
Guanosine triphosphate (GTP) has been implicated in the regulation of Ca(2+)-mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found that, in the presence of GTP, 1.0 microM free Ca(2+) causes maximum secretion-equivalent to that achieved with 100 microM free Ca(2+)-whereas GTPgammaS inhibits Ca(2+)-stimulated secretion. Interestingly, GTP by itself stimulates secretion. These results indicate the existence of a GTP-regulated mechanism of secretion in neutrophils that requires GTP hydrolysis to stimulate secretion in the presence and absence of Ca(2+). The stimulatory effect of GTP is only observed when GTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, gives no stimulatory response, implying that the GTP-dependent secretory apparatus requires at least one cytosolic protein. GTP-dependent secretion can be reconstituted with crude HL-60 and bovine liver cytosol. The reconstituting activity binds to GTP-agarose, suggesting that the cytosolic factor is a GTP-binding protein or forms a complex with a GTP-binding protein. However, it is not a member of the rho or rac families of GTPases. By gel filtration chromatography, the secretion-reconstituting activity eluted at 870 and 200 kDa, but in the presence of GTP, eluted at 120 kDa, indicating that it is part of a high-molecular-weight complex that dissociates in the presence of GTP. Retention of adenosine diphosphate-ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activity to brefeldin A led to our speculation that ARF6 may be the GTPase involved in GTP-dependent secretion, and that activity from a BFA-insensitive ARF6 guanine nucleotide exchange factor reconstitutes secretion.
Rosales
J L
JL
Department of Medicine, Division of Infectious Diseases, San Francisco General Hospital, San Francisco, California 94143, USA. rosales@ucalgary.ca
Ernst
J D
JD
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002463:Cell Membrane Permeability; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism*; D004058:Diffusion; D006160:Guanosine Triphosphate / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D009504:Neutrophils / Q000378:metabolism*; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism*
D011506:Proteins; D006160:Guanosine Triphosphate; D002118:Calcium