(Received for publication, November 22, 1994; and in revised form, December 13, 1994)
From the
Proteins in both the cytosol and plasma membrane are needed to reconstitute cell-free phospholipase D activity from phagocytes (Olson, S., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242); membrane factors include a small GTP-binding protein in the Rho family (Bowman, E., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512). ADP-ribosylation factor (ARF) was recently implicated as the cytosolic factor, as it activates phospholipase D in HL-60 membranes. Herein, we show that ion exchange chromatography separates ARF from the major phospholipase D-stimulating cytosolic factor. Both bovine brain ARF and recombinant human ARF-1 stimulated a small amount of phospholipase D activity in the absence of cytosol (about 10% of the response seen with cytosol). With a high concentration of ARF-depleted cytosol, ARF did not further activate. However, at low cytosol, ARF caused marked activation. Thus, ARF synergizes with the cytosolic factor in phospholipase D activation.
Phospholipase D has been implicated as an important signal transducing enzyme in a variety of cells and tissues (1) and is activated by a variety of receptor-linked agonists and by phorbol esters. The enzyme catalyzes the hydrolysis primarily of phosphatidylcholine to generate phosphatidic acid. The latter has been implicated as a biologically active molecule (2, 3, 4, 5) and can also be metabolized via phosphatidic acid phosphohydrolase to form diradylglycerol, a protein kinase C activator. In human neutrophils and HL-60 cells, the major source of diradylglycerol is the phospholipase D/phosphatidic acid phosphohydrolase pathway(6, 7) . In human neutrophils, the diradylglycerol generated through this pathway is essential for cell activation(7) .
Phospholipase
D can be reconstituted in cell-free systems in both membrane and
soluble preparations. Some of these are activated by detergents and
fatty acids(8, 9, 10, 11) , but
their relationship to receptor-linked PLD ()is unclear. A
PLD activity in liver plasma membranes was activated by GTP
S (12) and by phorbol esters by a protein kinase C-dependent,
phosphorylation-independent mechanism(13) . Similar activities
have been noted in homogenates from phagocytic cells(14) , but
activity was absent in isolated plasma membranes. PLD activity in
granulocytes was restored upon recombining cytosol and plasma membrane,
and protein factors in both fractions were needed for both the
GTP
S (15) and phorbol ester responses(16) . The
cytosolic factor had an apparent size of
50 kDa by gel exclusion
chromatography(17) .
Bowman et al.(17) showed that the GTPS-binding protein in the
granulocyte cell-free system is in the plasma membrane, and identified
the protein as a Rho family small GTPase protein, based on inhibition
by Rho GTPase Dissociation Inhibitor. The GTP-binding species in liver
plasma membranes was recently shown to be extracted by Rho GTPase
Dissociation Inhibitor, and activity was restored with recombinant Rho
A(18) . Thus, in both phagocytic cells and in liver, a protein
factor in the plasma membranes is a Rho GTPase.
ADP-ribosylation factor (ARF), another small molecular weight GTP-binding protein in the Ras superfamily, was also implicated as a PLD-activating factor. Using permeabilized, cytosol-depleted HL-60 cells that had lost their PLD activity, brain cytosol reconstituted activity(19) ; the activating factor was purified and identified as ARF (ARF-1 or ARF-3). Brain cytosol also stimulated PLD in isolated HL-60 plasma membranes and membrane extracts, and ARF was likewise isolated as the activating factor(20) .
These studies utilized heterologous systems in which ARF was identified from brain cytosol using HL-60 membranes or permeabilized cells, but it was not shown whether ARF was the primary activating factor in cytosol from granulocytes themselves. We undertook the present studies to investigate whether ARF was present in our human neutrophil fractions, and whether ARF was the same as the cytosolic factor that we identified previously(16) . We find that ARF activity and immunoreactivity, which is present almost exclusively in the cytosol, is distinct from the majority of the cytosolic factor. Unexpectedly, while ARF alone supported PLD activity weakly, it synergized with a low concentration of the cytosolic factor to cause a marked stimulation of PLD activity.
Figure 1:
Expression of
myristoylated human ARF-1 in E. coli. Human ARF-1 was
expressed and purified as described under ``Experimental
Procedures.'' LaneA shows a Coomassie-stained
SDS gel of the purified ARF. Lane B is an autoradiograph of
the purified material obtained from cultures prelabeled with
[H]myristate.
Figure 2: Immunochemical visualization of ARF in the cytosol and plasma membrane from human neutrophils. The indicated quantities of plasma membrane (PM), cytosol, recombinant human ARF-1 (rARF), or recombinant human Rac1 (rRac1) were electrophoresed on polyacrylamide gels, transferred to nitrocellulose, and visualized by immunostaining as described under ``Experimental Procedures'' using antibody to the human recombinant ARF-1.
Figure 3:
Chromatographic separation of ARF from the
phospholipase D cytosolic factor from human neutrophils. Cytosol from
human neutrophils was chromatographed on DEAE-cellulose, as under
``Experimental Procedures.'' Concentrated fractions were
assayed for ADP-ribosylation activity (open squares) or
stained for ARF immunoreactivity (inset). Fractions were also
assayed for GTPS-dependent PLD-supporting activity by assaying in
the presence of plasma membrane (filledsquares). The
experiment shown is representative of four.
Figure 4:
Synergistic activation of phospholipase
D by ARF and cytosolic factor. The concentrations of purified bovine
brain ARF (bARF, upperpanel) or purified
recombinant human ARF-1 (rARF) were varied as indicated, and
phospholipase D-catalyzed transphosphatidylation was monitored by
measuring phosphatidylethanol formation (PEth), expressed as a
percentage of the total radiolabeled lipids (see ``Experimental
Procedures''). Activity was measured in the presence of plasma
membranes and GTPS (10 µM), either in the absence of
cytosol (filled circles), or the presence of 15 µg (filledsquares) or 50 µg (filledtriangles) of ARF-depleted cytosol. Errorbars represent the range or standard error of two to
three determinations for each point.
ARF in the
absence of ARF-depleted cytosol caused a small stimulation of PLD,
which was 10% or less of that seen with high cytosol. ()At a
high concentration of ARF-depleted cytosol, PLD activity was maximal
and was not further stimulated by either bovine ARF or recombinant
ARF-1. However, at a low concentration of cytosol, both bovine and
recombinant ARF produced a marked stimulation. The two forms of ARF
were approximately equipotent, stimulating maximally at less than 1
µM ARF. The bovine ARF in combination with cytosol
stimulated to the same degree as a high concentration of cytosol, while
the recombinant ARF-1 was somewhat less effective, perhaps due to
incomplete myristoylation or to the presence of more effective isoforms
that might be present in the bovine preparation. Non-myristoylated ARF
had no effect in this concentration range. The stimulation by ARF was
synergistic rather than additive with cytosol, indicating that ARF
cooperates in the function of the cytosolic factor.
The present study demonstrates that the major cytosolic factor in human peripheral blood neutrophils is distinct from ARF. The PLD activity is seen in the absence of added ARF, indicating that cytosolic ARF is not essential for the activity. While it is possible that the very small amount of ARF which can sometimes be detected in plasma membrane preparations may account for some activity, this seems unlikely; we estimate that less than 1 nM ARF would be contributed by the presence of plasma membrane, whereas a minimum of 100 nM ARF is required to observe any stimulation with cytosol. Thus, an essential role for ARF in phospholipase D activity is not supported by the present studies.
However, ARF is shown herein to act cooperatively with the cytosolic
factor, so that in the presence of ARF, a low concentration of cytosol
suffices for near-maximal activation. The mechanism by which ARF
synergizes with the cytosolic factor remains unclear. ARF functions not
only in stimulation of cholera toxin-dependent ADP-ribosylation of G
protein subunits(27) , but also in Golgi membrane
trafficking (28, 29, 30, 31) . ARF
translocates from cytosol to a putative receptor on Golgi vesicles and
triggers coatamer binding. No general themes have yet emerged regarding
ARF mechanisms, except that in all its functions, membranes, and/or
phospholipids are involved. Given the well known ability of GTP-binding
proteins to complex with their effector enzymes, it is reasonable to
suggest that ARF functions by forming a complex either with the
cytosolic factor or with a PLD-related protein in the plasma membrane.
ARF is also known to interact directly with phospholipids, and it is
possible that it is functioning by binding to either the substrate (e.g. to make phosphatidylcholine more accessible to
phospholipase D) or a regulatory lipid such as phosphatidylinositol
4,5-bisphosphate. However, until the identity and function of the
cytosolic factor as well as any other unknown membrane proteins is
revealed, any proposed mechanisms remains speculative. Thus, in
summary, three or more proteins: a membrane-associated Rho-family
GTPase, cytosolic ARF, and a 50-kDa cytosolic factor, participate in
GTP
S-dependent PLD activity in human neutrophils.