Guanosine 5′-[γ-thio]triphosphate induces membrane localization of cytosol-independent phospholipase D activity in a cell-free system from U937 promonocytic leucocytes

Abstract

Activation of phospholipase D (PLD) in phagocytic leucocytes requires protein components present in both the plasma membrane and the cytosol, but the catalytic and regulatory factors are not fully defined. We have characterized the effect of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on the subcellular requirements for reconstitution of PLD activity, using a cell-free system from U937 human promonocytic leucocytes. Incubation of permeabilized cells with 100 microM GTP[S] resulted in a membrane-localized PLD activity which was independent of added cytosol. The PLD activity of membranes from GTP[S]-treated cells was 7-fold greater than the basal activity of control membranes, and could be further augmented by the addition of ATP. This was the first demonstration of a stable agonist-regulated PLD activity in membranes from phagocytic leucocytes which was quantitatively comparable with that seen in a fully reconstituted system. Cytosol from GTP[S]-treated cells had a decreased capacity to support PLD activation, consistent with GTP[S]-induced depletion of a factor essential for reconstitution of PLD activity. Incubation of isolated membrane and cytosol with GTP[S] also resulted in a cytosol-independent PLD activity in the re-isolated membranes. The effect of GTP[S] could be mimicked by guanosine 5'-[beta gamma-imido]triphosphate, but not by aluminium fluoride, consistent with the involvement of a low-molecular-mass GTP-binding protein(s). Incubation of isolated subcellular fractions with GTP[S], followed by removal of unbound nucleotide, suggested that at least one of the GTP-binding proteins involved in the membrane localization of PLD activity was itself present in the membrane fraction. These data were consistent with a model in which activation of GTP-binding protein(s) resulted in the stable assembly of an active PLD signalling complex at the membrane surface.

ADP-ribosylation Factor Translocation Correlates with Potentiation of GTPγS-stimulated Phospholipase D Activity in Membrane Fractions of HL-60 Cells

“…These findings suggest that a least part of the GTP␥S-stimulated PLD activity is dependent on the translocation of one or more cytosolic cofactors, which is in accordance with previously reported results (29,30). One of the required cytosolic cofactors has been shown to be the small GTPbinding protein, ARF.…”

“…In the absence of GTP␥S, ARF was without effect on basal PLD activity. DISCUSSION PLD activation requires both cytosolic and membrane factors (29,30). Activation of PLD by GTP analogues implies a role for GTP-binding proteins.…”

Phospholipases D1 and D2 Coordinately Regulate Macrophage Phagocytosis

“…Unless otherwise stated, materials were from previously published sources (14,16,(31)(32)(33)(34). Polyclonal Abs to PLD1 or PLD2 were raised by immunizing rabbits with specific peptides for each protein (four for PLD1, two for PLD2), as previously described (28).…”

“…A total of 10 6 cells/sample were incubated with 1.0% ethanol for 2 min to permit detection of the PLDspecific transphosphatidylation product, phosphatidylethanol (PEt), followed by complement-opsonized zymosan (COZ, particle:cell ratio of 10: 1), PMA (100 nM), or buffer control, in a total volume of 500 l. Reactions were conducted for 30 min at 37°C, and terminated by addition of 5 vol of chloroform: methanol (2:1, v/v). Lipids were extracted, dried under N 2 , and analyzed by TLC in an ethylacetate:isooctane:acetic acid (9:5:2) solvent system, and PEt was identified by comigration with pure standard (16,33,35). [ 3 H]PEt cpm were quantitated by liquid scintillation spectrometry, and counts normalized for the total amount of 3 H-labeled phospholipid in each experiment.…”

Phospholipase D1 Regulates Phagocyte Adhesion

“…In select experiments, cells were incubated in 0.5-1.0% ethanol for 2 min before stimulation to permit detection of the PLD-specific transphosphatidylation product, phosphatidylethanol (PEt). At the end of the experiment, lipids were extracted in 5 vol of chloroform: methanol (2:1, v/v) and separated by TLC in an ethylacetate:isooctane:acetic acid (9:5:2) solvent system (41,50,59 …”