LOW-MOLECULAR-WEIGHT GTP-BINDING PROTEINS IN HL-60 GRANULOCYTES - ASSESSMENT OF THE ROLE OF ARF AND OF A 50-KDA CYTOSOLIC PROTEIN IN PHOSPHOLIPASE-D ACTIVATION

Phospholipase D (PLD) activation by guanine nucleotides requires prote in cofactors in both the plasma membrane and the cytosol, HL-60 cytoso l was fractionated by ammonium sulfate and gel-permeation chromotograp hy, Two cytosolic protein fractions were found to reconstitute the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) stimulated PLD in a rec onstitution assay consisting of H-3-labeled HL-60 membranes and eluted column fractions. The major peak of reconstituting activity was in th e region of 50 kDa, and a second discrete peak of PLD reconstitution a ctivity was observed in the region of 18 kDa, Rho GDP/GTP exchange inh ibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rad. RhoA and Rac2 were entirely complexed with Rho GDI and eluted with an apparent molecular mass of 43 kDa by gel filtration chromatography, The partial overlap between cytosolic Rac2 and RhoA with the 50-kDa peak of recon stituting activity was not consistent with the participation of cytoso lic Rho-related GTPases in the activation of PLD by guanine nucleotide s. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family of small GTP binding proteins, reduced GTP gamma S-sti mulated PLD activity in HL-60 homogenates. The stimulatory exchange fa ctor, Smg GDS, which is active on Rho and Rac, could be partially sepa rated from the PLD-stimulating factor(s) by gel-permeation chromatogra phy. Moreover, recombinant Smg GDS failed to stimulate GTP dependent P LD activity, Cytosolic ADP-ribosylation factor (ARF) was exclusively l ocated in the 18-kDa peak of reconstitution activity, Faint amounts of membrane-bound ARF were also detected using the monoclonal antibody 1 D9, The effects of the 50-kDa and 18-kDa PLD inducing factors on the s alt-extracted PLD activity were synergistic. The weak stimulatory effe ct of ARF alone suggested that the GTP gamma S stimulated PLD activity is dependent on the presence of another protein(s), presumably ARF-re gulatory proteins. We propose that a membrane-bound GTP-binding protei n, possibly ARF, may be involved in the activation of PLD when combine d with the component(s) of the 50 kDa fraction.