* Department of Developmental and Molecular Biology, Department of Anatomy and Structural Biology, Albert Einstein
College of Medicine, Bronx, New York 10461; and § Department of Pharmacology, and Institute for Cell and Molecular Biology,
State University of New York, Stony Brook, New York 11794
Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.
MAMMALIAN phosphatidylcholine-specific phospholipase D (PLD)1 has been implicated in a
wide range of physiological responses including
metabolic regulation, cell proliferation, mitogenesis, oncogenesis, inflammation, secretion, and diabetes (Exton,
1994 Recently, three laboratories cloned an open-reading
frame encoding PLD activity from Saccharomyces cerevisiae (Rose et al., 1995 In the past few years, genetic and biochemical studies
have shown that ARF-1 plays an essential role in mediating intracellular vesicular transport (Donaldson and
Klausner, 1994 Using a permeabilized cell system derived from rat anterior pituitary growth hormone (GH)- and prolactin
(PRL)-secreting GH3 cells, we demonstrated that recombinant human ARF-1 stimulates the release of nascent
secretory vesicles from the TGN approximately two- to
threefold (Chen and Shields, 1996 Materials
[35S]Pro-mix (>1,000 Ci/mmol) was purchased from Amersham Corp.
(Arlington Heights, IL), [9,10-3H(N)]oleic acid (14 Ci/mmol) was purchased from DuPont-NEN (Boston, MA). Cabbage phospholipase D extract (type V), peanut phospholipase D extract (type III), and neomycin
were purchased from Sigma Chemical Co. (St. Louis, MO). Proteinase K,
guanosine-5 Rabbit antisera to the COOH-terminal peptides of GH and PRL have
been described previously by Austin and Shields (1996a) Cell Culture
GH3 cells were grown in Ham's F10 medium, supplemented with 15%
horse serum, 2.5% bovine fetal serum, 2 mM glutamine, 25 U/ml penicillin, and 25 U/ml streptomycin at 37°C with 5% CO2, as previously described (Stoller and Shields, 1988 Release of Nascent Secretory Vesicles in
Permeabilized Cells
The preparation of permeabilized cells and release of nascent secretory
vesicles from the TGN (vesicle budding assay) was described previously
(Xu and Shields, 1993 Determination of Phospholipase D Activity
Endogenous PLD activity was measured by its transphosphatidylation activity using 1-butanol. The assay was performed according to Wakelam et
al. (1995) Budding of Nascent Secretory Vesicles
The integrity of the nascent secretory vesicles formed in the permeabilized cells was measured one of two ways: (a) by determining the resistance of GH to proteolysis (Xu and Shields, 1993 Human PLD1 Expression in Insect Cells
A cDNA encoding human PLD1a was expressed in insect Sf-9 cells using
a baculovirus expression system from which a cytosolic extract was prepared (Hammond et al., 1995 Immunopurification of Native and Mutant Forms of
Human PLD1
Affinity-purified antibodies generated against two peptides corresponding
to residues 1-15 and 525-541 of PLD1 were covalently coupled to protein
A-Sepharose CL4B, as previously described (Hammond et al., 1997
Human PLD Stimulates Budding of Nascent Secretory
Vesicles from the TGN
Using a permeabilized system derived from rat pituitary
GH3 cells, which secrete GH and PRL, our previous data
showed that ARF-1 stimulated nascent secretory vesicle
budding from the TGN almost threefold (Chen and
Shields, 1996
It was possible that overexpression of human PLD1 in
insect Sf-9 cells modified the cell extract such that vesicle
release was a consequence of the modification rather than
the enzyme activity per se. To exclude this possibility, a
highly purified preparation of native PLD1 was added to
the permeabilized cells (Fig. 2). Human PLD1 was immunopurified to apparent homogeneity from a postribosomal supernatant of Sf-9 cells (Fig. 2 B, inset). As a control, we added an identically purified but inactive mutant form of
PLD1, K898R, possessing Arg at position 898 instead of
Lys (the latter residue is conserved in human, yeast, plant,
and bacterial PLD enzymes) (Fig. 2, A and B). Both preparations of PLD1 migrated on SDS-PAGE as a doublet of
~110,000 Mr. Before use, each purified enzyme preparation was assayed for its ability to hydrolyze PC in a reconstituted liposome assay (Brown et al., 1993 Plant PLD Stimulates Vesicle Release
PLD has been isolated from several plant sources and
shares similarity to the human and yeast enzymes (Hammond et al., 1995
It might be argued that the stimulation of nascent vesicle budding in response to human or plant PLDs resulted
from membrane lysis or leakage of content proteins from
the Golgi apparatus or secretory vesicles in response to
the increased concentration of negatively charged lipids.
To exclude this possibility and to demonstrate the release
of intact, sealed GH- and PRL-containing vesicles in response to PLD activity, we used a high speed centrifugation assay to pellet nascent vesicles (Fig. 4 A) or a protease
protection assay (Fig. 4 B). After treatment with human
PLD1, significantly more GH was recovered in the high
speed pellet (corresponding to nascent secretory vesicles)
than from the control incubations (Fig. 4 A, lanes 2 and 4).
Similarly, in response to plant PLD, approximately twofold more GH was protease resistant than in control permeabilized cells (Fig. 4 B, compare lanes 2 and 4); whereas in the presence of Triton X-100, the GH was degraded
quantitatively (Fig. 4 B, lanes 5 and 6). Together, these results indicated that incubation of permeabilized cells with
either human or plant PLD stimulated the release of intact
membrane-bound secretory vesicles.
ARF-1 Stimulates Endogenous Golgi-associated
PLD Activity
The above results, together with the observation that ARF
can activate PLD (Brown et al., 1993
Exogenous PLD Hydrolyzes Endogenous
Golgi Phospholipids
The above experiments, using immunopurified native and
mutant human PLD1, strongly suggest that PLD catalytic
activity mediates vesicle release from the TGN. To determine if exogenously added human PLD hydrolyzed endogenous Golgi membrane phospholipids, GH3 cells were
incubated with [3H]oleate to radiolabel phospholipids,
Golgi membranes were isolated and treated with 1 M KCl
to extract endogenous PLD1 from the membranes (Hammond et al., 1997
Vesicle Budding Requires the Product of PLD Activity
Recent studies from Ktistakis et al. (1996)
Evidence from several laboratories has implicated phospholipid-modifying enzymes and inositol phospholipid
metabolism in mediating various steps of intracellular vesicular transport (Boman and Kahn, 1995 Native but Not Mutant PLD Stimulates
Vesicle Budding
PLD hydrolyzes PC at its terminal phosphodiester bond to
produce PA and choline; PA itself can function as a second messenger. Several cytosolic factors that stimulate
PLD activity have been purified; these include ARF
(Brown et al., 1993 Several controls, in addition to the point mutant, demonstrated that the stimulation of vesicle budding resulted
from PLD enzymatic activity. Firstly, plant PLD (Fig. 3)
and a second form of human phosphatidylcholine-specific
PLD, designated PLD2 (Colley et al., 1997 ARF-1 Stimulates PLD Activity
Both recombinant myristoylated and nonmyristoylated
ARF-1 stimulated the endogenous PLD activity present in
Golgi membranes about two- to threefold (Fig. 5). This
was somewhat unexpected since the myristoylation of
ARF appears neccessary for several of its functions including coat binding (Stamnes and Rothman, 1993 Consistent with our previous results (Chen and Shields,
1996 In the absence of exogenous ARF, PLD was presumably
activated by the low level of endogenous ARF present on
the Golgi membranes (Donaldson and Klausner, 1994 Although PLD activity per se can be stimulated by native ARF and an activated ARF-1 mutant in the presence
of GTP (Fig. 5), this was not sufficient to promote vesicle
budding from the TGN. ATP hydrolysis was also required
(Figs. 1 and 3) and since ARF stimulation of PLD did not
occur in the presence of ATP alone (data not shown),
ATP may therefore be required for other steps in the budding reaction. ATP does however, potentiate GTP At present, we have not determined whether PLD-stimulated vesicle release occurs by a change in the Golgi
membrane lipid composition or by activating a signaling
cascade. In the first case, it is possible that increased PA
levels, generated as a consequence of PLD activity, could
transiently alter the lipid composition and local charge of
the outer bilayer leading to recruitment of coat proteins,
e.g.,
). PLD catalyzes the hydrolysis of phospholipids to
generate phosphatidic acid (PA) and the corresponding
free polar head group. PA can itself be converted to the
second messenger diacylglycerol or lysophosphatidic acid
which activates various downstream signaling events. Consistent with a role in cell signaling, PLD can be activated by small G proteins, intracellular Ca2+, protein kinase C
(PKC), and protein-tyrosine kinases (Liscovitch and
Cantley, 1995
). In mammalian cells, at least two classes of PLD can be differentiated by their susceptibility to regulation by G proteins, or requirements for the phospholipid
phosphatidyl 4,5-bisphosphate (PtdIns-4,5-P2) and fatty
acids (Massenburg et al., 1994
). One of the small G proteins that stimulates PLD activity is ADP ribosylation factor (ARF), which is an ~20-kD GTP binding protein that
is a member of the Ras superfamily (Donaldson and Klausner, 1994
). Interestingly, ARF, Rho, and PKC stimulation of PLD activity requires PtdIns-4,5-P2 as an essential cofactor (Liscovitch et al., 1994
; Pertile et al., 1995
).
PLD is present in Golgi membranes and its activity can be
stimulated by ARF in vitro (Ktistakis et al., 1995
). Furthermore, the ARF-stimulated PLD activity enhanced the
binding of the
-COP subunit of coatomer to isolated
Golgi membranes, suggesting that changes in the membrane lipid composition influences coat recruitment
(Ktistakis et al., 1996
). It has been suggested that ARF
stimulation of PLD plays a role in membrane trafficking
(Brown et al., 1993
; Kahn et al., 1993
; Cockcroft et al.,
1994
; Boman and Kahn, 1995
: Liscovitch and Cantley,
1995
; Bednarek et al., 1996
) although to date, this has not
been demonstrated directly.
; Ella et al., 1996
; Waksman et al.,
1996
); this cDNA encodes a polypeptide of 1,683 amino
acids (predicted mol wt 195,000). Based on the sequence
of the yeast enzyme, one of our laboratories also cloned a
cDNA encoding a human PLD specific for phosphatidylcholine, designated PLD1 (Hammond et al., 1995
). PLD1
cDNA encodes a 1,072-residue polypeptide (Mr = ~120,000) that is predominantly membrane associated, but unlike other phospholipases involved in signal transduction, it lacks SH2, SH3, or pleckstrin domains (Hammond et al., 1995
). Similar to the PLD activity that has
been widely studied, PLD1 requires PtdIns-4,5-P2 as a cofactor and is inactive unless stimulated by members of the
ARF and Rho families of small G proteins or protein kinase C (Hammond et al., 1995
, 1997
). PLD1 localizes to
the ER, Golgi apparatus, and endosomes suggesting that it
may play a role in vesicular trafficking (Colley et al., 1997
).
In contrast, PLD2, the second mammalian PLD cloned, is
not activated by ARF; it localizes to the plasma membrane, and has been proposed to play a role in agonist-
induced actin rearrangement or receptor-mediated recycling (Colley et al., 1997
).
). Numerous studies using the fungal metabolite brefeldin A (BFA), a drug that perturbs ARF
function by inhibiting GTP-GDP exchange, or studies in
which mutant forms of ARF were expressed in cells have
demonstrated that ARF-1 is involved in: (a) maintaining
the structural integrity of the Golgi apparatus; (b) transport from the ER to the Golgi apparatus; and (c) endosome trafficking. In vitro binding studies showed that
ARF in its GTP-bound form recruits the
-COP subunit of coatomer (COP-I) to Golgi membranes, suggesting that
it regulates the formation of coated transport vesicles.
ARF-1 also functions in the late Golgi apparatus; where it
facilitates binding of the
-adaptin subunit of the AP1
clathrin adaptor complex to isolated Golgi membranes in
vitro (Stamnes and Rothman, 1993
; Traub et al., 1993
; Liang and Kornfeld, 1997
). Recently, several ARF-specific guanine nucleotide exchange factors (GEFs) have been
characterized from yeast and mammalian cells (Chardin et
al., 1996
; Morinaga et al., 1996
; Peyroche et al., 1996
; Tsai
et al., 1996
). These appear to fall into two classes, one of
high molecular weight, that is BFA sensitive, and the other
of ~47-55 kD that is BFA insensitive. Interestingly, several of these GEFs posses a domain that is very similar to a
motif present in yeast Sec 7p (Chardin et al., 1996
), a high
molecular weight protein involved in ER to Golgi and intra-Golgi vesicular trafficking (Franzusoff et al., 1991
).
GTP-GDP exchange activity is enhanced by inositol phospholipids, particularly PtdIns-4,5-P2, and this is mediated via a pleckstrin homology domain however, the GEF activity resides in the Sec 7 domain (Chardin et al., 1996
). A
Golgi-localized, 49-kD ARF GTPase activating protein
(GAP) has been purified from rat liver cytosol and its
cDNA sequence determined (Cukierman et al., 1995
). Recently, an ARF-1, and -3-binding protein, designated arfaptin, was identified by the yeast two-hybrid system
(Kanoh et al., 1997
); this protein, which is Golgi localized,
only binds to ARF in its GTP-bound form. Together these
studies suggest that ARF and its accessory proteins function in mediating vesicular trafficking by the recruitment
of specific coat proteins to membranes. However, although ARF is clearly central to vesicular transport, its exact role is still unclear.
). In contrast, mutant forms of ARF unable to exchange GDP for GTP or one
lacking the NH2-terminal 17 residues did not stimulate
vesicle budding. In light of observations that ARF can regulate Golgi-localized PLD activity and recruitment of
coatomer to Golgi membranes (Ktistakis et al., 1995
, 1996
)
and the increasing evidence that phospholipid-modifying enzymes play a role in membrane trafficking (Liscovitch
and Cantley, 1995
; De Camilli et al., 1996
), we hypothesized that PLD could be a link between ARF and secretory vesicle release. The availability of human PLD (Hammond et al., 1995
) and a permeabilized cell system that
supports efficient secretory vesicle budding from the TGN (Xu and Shields, 1993
) has enabled us to test this hypothesis directly. Here, we demonstrate that addition of human
PLD to permeabilized endocrine cells stimulated budding
of nascent secretory vesicles from the TGN. Furthermore,
ARF activation of endogenous PLD activity present in the
Golgi apparatus correlated closely with its ability to potentiate nascent secretory vesicle formation. Our data suggest that ARF can regulate vesicle budding from the TGN by
modulating PLD activity.
Materials and Methods
-O-(3-thiotriphosphate) (GTP
S), and adenosine-5
-O-(3-thiotriphosphate) (ATP
S) were obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN).
and Xu and
Shields (1993)
, respectively. The expression, mutagenesis, and purification
of recombinant ARF-1 was performed exactly as described previously
(Chen and Shields, 1996
).
).
; Chen and Shields, 1996
). Approximately 2 × 106
cells were pulse labeled with [35S]Promix for 12 min, and then chased for 2 h
at 19°C to accumulate radiolabeled GH and PRL in the TGN. The cells
were permeabilized at 4°C by incubation in swelling buffer for 5 min, the
buffer was aspirated and replaced with 1 ml breaking buffer (90 mM KCl, 10 mM Hepes, pH 7.2), after which the cells were broken by scraping with
a rubber policeman. The cells were centrifuged at 800 g for 5 min,
washed in 3-5 ml of breaking buffer, and resuspended in 5 vol of breaking
buffer. This procedure resulted in >95% of cell breakage, evaluated by
staining with trypan blue. The permeabilized cells were incubated at 37°C
for 90 min in a 100-µl reaction containing the following reagents: 10 mM
Hepes, pH 7.2, 2.5 mM MgCl2, 0.5 mM CaCl2, 35 mM KOAc, 110 mM
KCl; and an energy regenerating system (ERS; 1 mM ATP, 0.2 mM GTP,
10 mM creatine phosphate, 80 µg/ml creatine phosphate kinase). After incubation, the samples were centrifuged at 13,000 g for 15-30 s. The supernatant containing nascent secretory vesicles (S) and cell lysates (P; residual permeabilized cells) were treated sequentially with anti-GH antisera,
followed by anti-PRL antibodies. The immunoprecipitated polypeptides
were analyzed by SDS-PAGE and the intensity of each band quantitated using a computing densitometer (model 300A; Molecular Dynamics, Inc.,
Sunnyvale, CA). Nascent secretory vesicle budding efficiency was calculated as GH-or PRL-immunoreactive material in the supernatant divided
by the total GH or PRL material in the pellet and supernatant.
with the following modifications. GH3 cells grown to 70% confluency were radiolabeled with 6 µCi/ml [9,10-3H(N)]oleic acid for 24-36 h
after which the cells were harvested, homogenized, and Golgi membranes
prepared using a sucrose equilibrium density gradient (Xu and Shields,
1993
). The Golgi-enriched fractions were incubated in the presence or absence of native or mutant ARF-1, as indicated, under conditions that promote secretory vesicle budding from the TGN. Incubations contained
0.3% 1-butanol to measure the formation of phosphatidylbutanol (PtdBut) in response to PLD activity. After incubation for 1 h at 37°C, samples
were placed on ice and the lipids extracted with methanol and chloroform to give a final ratio of 1:1:0.8 (methanol/chloroform/water). After vortex
mixing, the organic phase containing phospholipids was separated by centrifugation, and dried under vacuum using a Speedvac (Savant Instruments, Farmingdale, UK). The dried samples were resuspended in chloroform/methanol (19:1) and the phospholipids resolved on Whatman
LK5DF TLC plates (Whatman Inc., Clifton, NJ) by developing with an
organic phase consisting of 2,2,4-trimethylpentane/ethylacetate/acetic acid/water (50:110:20:100) (Liscovitch et al., 1994
). The plate was air
dried, treated with EN3HANCE (DuPont-NEN) and exposed to a Kodak
X-Omat AR film (Eastman Kodak Co., Rochester, NY).
); or (b) by a vesicle sedimentation assay (Chen and Shields, 1996
). After the vesicle budding
incubation (for proteinase K protection), samples were treated with 25 µg/ml
proteinase K and 4 mM tetracaine on ice for 30 min in the absence or presence of 1% Triton X-100. PMSF was added to a final concentration of 1.3 mM to inhibit further proteolysis, and the products were incubated with
appropriate antibodies followed by resolution upon SDS-PAGE. After incubation (for sedimentation), permeabilized cells were separated by centrifugation (13,000 g for 30 s) from the vesicle-containing supernatant. The 13,000 g supernatant fraction was centrifuged in a Beckman airfuge (150,000 g for 10 min; Beckman Instruments, Inc., Fullerton, CA) to pellet
nascent vesicles from supernatant material. All samples were treated with
anti-GH antibodies and analyzed by SDS-PAGE.
). Briefly, monolayers of Sf-9 cells infected
with recombinant vectors encoding human PLD1 or as a control PLC-
2,
were scraped in 10 mM Tris-HCl, pH 7.5, 1 mM EGTA containing a cocktail of protease inhibitors, and the suspension sonicated to lyse the cells.
The cell lysate was centrifuged at 2,000 g for 10 min at 4°C, the supernatant adjusted to 500 mM KCl, incubated on ice for 1 h, and centrifuged at
100,000 g for 30-60 min. The high speed supernatant was dialyzed briefly against 100 mM KCl, 2.5 mM MgCl2, 20 mM Hepes, pH 7.2, and PLD activity determined by measuring the release of [3H]choline that was hydrolyzed from [3H]phosphatidylcholine-containing liposomes (Brown et al.,
1993
; Hammond et al., 1995
). Aliquots of this postribosomal supernatant were flash frozen in liquid nitrogen and stored at
80°C until used. The
PLD activity for human PLD1, determined by using [3H]phosphatidylcholine as substrate in the presence of GTP
S and ARF (Brown et al., 1993
),
was 5.1 nmoles hydrolyzed/mg per min and was 0.1 nmol hydrolyzed/mg
per min for the control or the PLD1 mutant K898R (see below). It is likely
that endogenous insect cell activators of PLD (e.g., small G proteins or
PKC) are present in this crude extract, because the basal activity of the
enzyme dramatically decreases with immunoaffinity purification (Hammond et al., 1997
).
). This
resin was used to immunopurify a native and a mutant form of human
PLD1. Mutation of Lys 898 to Arg (K898R), which is a lysine conserved
amongst all mammalian, yeast, plant, and bacterial PLD enzymes (Hammond et al., 1995
), abolishes PLD activity in human PLD1, PLD2, and the
yeast enzyme Spo14 (Sung et al., 1997
). Postribosomal supernatants from
Sf-9 cells infected with recombinant baculoviruses encoding native or the
K898R mutant were prepared as outlined above and incubated with the
immunoaffinity resin at 4°C for 1 h. Unbound material was removed by
centrifugation and the resin washed extensively with cell lysis buffer
(Hammond et al., 1995
). PLD was eluted as described (Hammond et al., 1997
) using 100 mM glycine, pH 3.0, containing 1 M KCl, instead of
-D-octylglucoside. The eluate was neutralized immediately, dialyzed against incubation buffer, and assayed for PLD activity (Brown et al., 1993
). The immunopurified PLD migrated as a doublet of ~110,000 Mr (Fig. 2, B, inset)
on our gel system. PLD1 prepared in the absence of detergent was quite
labile and all assays were performed within 24 h of purification. Freshly
prepared native PLD1 had an ARF-stimulated specific activity of 260 nmol phosphatidyl choline (PC) hydrolyzed/mg per min, whereas the activity of the K898R mutant was undetectable. The mutant PLD1 K898R is
completely inactive in vivo but appears to fold correctly and exhibits correct subcellular localization when expressed in fibroblasts (Sung et al.,
1997
).
Fig. 2.
Purified human PLD1 stimulates release of nascent
secretory vesicles. (A) Radiolabeled, permeabilized GH3 cells
were incubated under vesicle budding conditions for 1 h at 37°C
in the absence (control lanes 1 and 2) or presence of the indicated
concentrations of immunopurified human PLD1, (lanes 3-10) or
the mutant (Mt) PLD1 K898R (lanes 11 and 12). After incubation, samples were separated into pellet (P) and supernatant (S)
fractions by centrifugation, the fractions were incubated with
anti-GH antibodies and the immunoprecipitable material analyzed by SDS-PAGE. (B) For the quantitation of vesicle release,
duplicate samples of radiolabeled permeabilized cells were incubated with increasing concentrations of immunopurified native
human PLD1 (Wt) or the inactive PLD mutant, K898R. The control vesicle budding efficiency, in the absence of added human PLD1 was 28%. Inset, silver staining of immunopurified native (Wt), and K898R mutant (Mt) human PLD1 analyzed by SDS-PAGE. , Wild-type PLD1;
, K898R mutant PLD1.
[View Larger Version of this Image (26K GIF file)]
Results
). The stimulation of vesicle release occurred
in the absence of exogenously added cytosol, which suggested that either ARF might function by recruiting prebound coat components or less likely, independently of
coat recruitment. Since PLD was suggested to be a downstream effector of ARF (Brown et al., 1993
; Cockcroft et
al., 1994
), and PLD activity was shown to effect
-COP
binding to Golgi membranes (Ktistakis et al., 1996
), we
reasoned that enhanced vesicle budding might be a consequence of PLD activation. If this were correct, then direct
addition of PLD to the permeabilized cells should also
stimulate vesicle budding. Of the two cloned mammalian
PLDs, only PLD1 localizes to the Golgi and is activated by
ARF (Colley et al., 1997
). Consequently, our initial experiments used a postribosomal supernatant fraction isolated
from insect cells expressing human PLD1. This was added to the permeabilized cells and its effect on nascent vesicle
formation determined (Fig. 1). As previously observed
(Xu and Shields, 1993
; Chen and Shields, 1996
), in control
permeabilized cells vesicle budding was energy dependent
and ~30% efficient (Fig. 1, lanes 1-4). Significantly, PLD1
stimulated vesicle budding approximately twofold (compare Fig. 1, lanes 3 and 4 with 5 and 6) in a reaction that required ATP and GTP (Fig. 1, lanes 7 and 8). Control incubations in which an extract from insect cells expressing a
different phospholipase, phospholipase C-
2, was added
to the system, did not stimulate vesicle budding above
basal levels (Fig. 1, lanes 9 and 10). The stimulated budding of nascent secretory vesicles was dependent on the
concentration of added PLD1-containing extract (Fig. 1 B)
and addition of as little as 3 µg cell extract per ml enhanced vesicle release up to twofold. Increasing the concentration
of the Sf-9 extract containing recombinant human PLD1
stimulated release of GH- or PRL-containing vesicles slightly
to a level of ~60% efficiency (Fig. 1 B). Most significantly,
these data demonstrated that PLD stimulated vesicle budding in a concentration- and energy-dependent reaction.
Fig. 1.
Human PLD1 stimulates release of nascent secretory
vesicles. (A) GH3 cells were pulse labeled with [35S]Promix for 12 min at 37°C, chased for 2 h at 19°C, and permeabilized. The permeabilized cells were incubated for 1 h at 37°C in the absence of
added PLD (lanes 1-4, 9, and 10), or in the presence of 15 µg/ml
of an Sf-9 insect cell lysate expressing either PLD1 (lanes 5-8) or
a control (Ctrl) PLC-2 specific for inositol phospholipids (lanes
9, and 10). After incubation, samples were separated into pellet
(P) and supernatant (S; nascent vesicle) fractions by centrifugation, immunoprecipitated with rabbit antibodies to GH or PRL
(not shown) and the immunoprecipitable material analyzed by
SDS-PAGE. ERS, energy regenerating system. (B) Quantitation
of vesicle budding. Permeabilized cells were incubated with the
indicated concentrations of Sf-9 cell lysate expressing PLD1 and
the release of nascent secretory vesicles determined. Data are the
average of two experiments.
, PLD1 GH vesicle budding;
,
PLD1 PRL vesicle budding. Vesicle budding efficiency was calculated as GH or PRL immunoreactive material in the supernatant
(S) divided by the total GH or PRL material (pellet + supernatant, P + S).
[View Larger Version of this Image (23K GIF file)]
); whereas native PLD was highly active (PC hydrolysis was 260 nmol/
mg per min; see Materials and Methods), the K898R mutant was inactive (Sung et al., 1997
; and data not shown).
Addition of increasing concentrations of native PLD1
stimulated release of nascent secretory vesicles about twofold (Fig. 2 B) and maximal vesicle budding occurred with
0.6-1 µg PLD1/ml, equivalent to ~5 nM enzyme. Most significantly, addition of the enzymatically inactive mutant
form of PLD1 to the permeabilized cells did not stimulate
vesicle budding above control levels even at the highest
concentration used (Fig. 2 B).
). We argued that if PLD1 stimulated vesicle budding as a result of its activity then PLD enzymes
from other species (including plant) should have a similar
effect (Fig. 3). PLD isolated from cabbage (type V) or
peanut (type III) was added to the permeabilized cell system and their effect on GH- and PRL-containing vesicle budding determined. Both species of plant PLD stimulated vesicle release from the TGN approximately twofold
(Fig. 3 B). Similar to the mammalian enzyme, stimulation
of vesicle budding by cabbage PLD was also energy dependent (Fig. 3 A, lanes 7 and 8) and required ATP hydrolysis since there was minimal budding with GTP alone or in the presence of the nonhydrolyzable analogue ATP
S
and GTP (Fig. 3 A, lanes 9 and 10, and 11 and 12, respectively). Heat inactivation or pretreating either cabbage or
peanut PLDs with proteinase K inhibited their ability to
stimulate vesicle formation above background levels (data
not shown) demonstrating that increased vesicle release
was dependent on an active enzyme.
Fig. 3.
Plant PLDs stimulate nascent vesicle budding from
mammalian TGN. (A) Permeabilized cells were incubated in the
absence (lanes 1-4) or presence (lanes 5-12) of 40 U/ml cabbage
PLD type V or peanut PLD type III (lanes 13 and 14) without
() or with (+) ATP and GTP as indicated. Samples in lanes 11 and 12 were incubated in the presence of 1 mM ATP
S plus
GTP. After incubation, samples were separated into pellet (P)
and nascent secretory vesicles (S) by brief centrifugation and the
fractions treated with anti-GH or anti-PRL (not shown) antibodies.
(B) For the quantitation of vesicle budding in response to plant
PLDs, permeabilized cells were incubated with the indicated concentrations of plant PLD and the release of nascent GH (
,
)
and PRL-containing secretory vesicles (
,
) determined. Data
are the average of three experiments.
, Cabbage PLDV-GH
vesicle budding;
, cabbage PLDV-PRL vesicle budding;
,
peanut PLDIII-GH vesicle budding;
, Peanut PLDIII-PRL vesicle budding. Vesicle budding efficiency was calculated as GH
or PRL immunoreactive material in the supernatant (S) divided by the total GH or PRL material (pellet + supernatant, P + S).
[View Larger Version of this Image (24K GIF file)]
Fig. 4.
Human and plant
PLDs stimulate budding of
intact nascent secretory vesicles. (A) Sedimentation of
nascent secretory vesicles containing GH. Permeabilized cells incubated in the
absence () or presence (+)
of 15 µg/ml of Sf-9 cell extract expressing human PLD1. After incubation, the
permeabilized cells (lanes 1 and 3; CP) were separated by
centrifugation (13,000 g for
20 s), from the vesicle-containing supernatant. This was
further centrifuged in a
Beckman airfuge (150,000 g,
10 min) to pellet nascent vesicles (lanes 2 and 4; SP). All
the samples were treated
with anti-GH antibodies and
analyzed by SDS-PAGE. (B)
Resistance of GH-containing vesicles to proteolysis. After the
budding assay performed without (lanes 1, 2, 5, and 6) or with
cabbage PLD (lanes 3 and 4), samples were incubated with 25 µg/ml proteinase K at 4°C for 30 min in the absence (lanes 1-4)
or presence of 1% Triton X-100 (lanes 5 and 6). The pellet (P)
and supernatant (S) fractions were separated by brief centrifugation and treated with anti-GH antibodies; identical results were
obtained for PRL containing vesicles (data not shown).
[View Larger Version of this Image (14K GIF file)]
; Cockcroft et al.,
1994
; Ktistakis et al., 1995
, 1996
), suggested that ARF
stimulation of nascent vesicle budding might occur via
PLD activation. If this hypothesis was correct, then endogenous Golgi PLD activity should be enhanced under conditions that promote vesicle budding from the TGN as a
result of active ARF. To test this idea, GH3 cells were incubated with [3H]oleic acid to radiolabel phospholipids
and a Golgi membrane fraction was isolated; this was incubated in the absence or presence of native and mutant
ARF-1 under vesicle budding conditions (Fig. 5). Since
transphosphatidylation activity is a diagnostic property of
PLD activity (Wakelam et al., 1995
), we included low concentrations of 1-butanol (0.3%) in the incubations and determined the Golgi-associated PLD response to native and
mutant ARF polypeptides by measuring the formation of
PtdBut using TLC (Wakelam et al., 1995
). Under basal
vesicle budding conditions, i.e., in the presence of energy (Fig. 5 A, lane 2), PtdBut was generated and its formation
was stimulated nearly twofold upon incubation with myristoylated ARF (Fig. 5, A and B, lanes 2 and 4). Most significantly, those ARF polypeptides that have previously been
shown (Chen and Shields, 1996
) to stimulate vesicle budding, i.e., the "activated" ARF mutant Q71L (Fig. 5 A,
lanes 5 and 10), which hydrolyzes GTP inefficiently, or native ARF (Fig. 5 A, lane 7) stimulated PLD activity approximately threefold (Fig. 5 B). By contrast, PLD activity
was enhanced only slightly above basal levels when samples were incubated with an ARF-GDP mutant T31N or
one lacking the NH2-terminal 17 amino acids
NT (compare Fig. 5 A, lane 3 with 8 and 9). Control incubations demonstrated that the PtdBut generated was a consequence of PLD activity since this material was greatly diminished when: (a) an energy system was excluded from
the incubation (lane 1); (b) neomycin was present in the
incubation (lane 11) consistent with previous work demonstrating that low concentrations of neomycin inhibit
PLD activity by binding PtdIns-4,5-P2, an essential cofactor for PLD activity (Liscovitch et al., 1994
; Whatmore et
al., 1994
; Pertile et al., 1995
); and (c) no PtdBut was
formed in the absence of butanol (lane 12). Taken together, these data demonstrated that under conditions
where native ARF enhances vesicle budding approximately
twofold (Chen and Shields, 1996
; Fig. 5 A, lanes 4 and 5)
there is also a concomitant stimulation of endogenous Golgi
localized PLD activity.
Fig. 5.
Human ARF-1 stimulates endogenous PLD activity in
isolated Golgi membranes. GH3 cells were radiolabeled with
[3H]oleic acid, homogenized, and a Golgi-enriched membrane
fraction isolated by floatation on a sucrose gradient (See Materials and Methods). The isolated Golgi membranes were incubated
at 37°C for 1 h with an energy generating system (lanes 2-12) in
the absence () or presence (+) of the indicated components including 0.3% butanol to measure PLD activity by formation of
phosphatidylbutanol (lanes 1-11). When present native and mutant recombinant ARF-1 polypeptides were present at a final
concentration of 1.5 µM (lanes 4-10). After incubation, total
phospholipids were extracted and resolved by TLC (See Materials and Methods). Arrowhead, phosphatidylbutanol standard.
Myr, myristoylated ARF-1; ARF, non-myristoylated ARF-1; Q71L, myristoylated ARF mutant (defective in GTP hydrolysis,
active in budding);
NT, a mutant lacking residues 1-17 (inactive
in budding); T31N, myristoylated ARF-1 (defective in GDP-
GTP exchange, inactive in budding). (B) Quantitation by densitometry of fluorograms similar to that in A. The intensities of
each spot corresponding to PtdBut was determined using a computing densitometer (model 300A; Molecular Dynamics). Data
are the average of three experiments and are normalized to the
+ERS sample (lane 2).
[View Larger Version of this Image (28K GIF file)]
; Fig. 6). The salt-treated Golgi membranes were incubated with Sf-9 cell extracts under vesicle
budding conditions in the presence of 0.3% 1-butanol. The
ability of human PLD1 to hydrolyze endogenous phospholipids was then determined by measuring the formation of
PtdBut (Fig. 6). Incubation of the Golgi membranes with
recombinant myristoylated ARF had no effect on lipid hydrolysis since the endogenous PLD had been effectively
removed from the membranes by high salt treatment (Fig.
6, lane 1). Addition of the PLD1 containing Sf-9 extract
(Fig. 6, lane 2) but not the control extract (Fig. 6, lane 5)
hydrolyzed the phospholipids to generate PtdBut. As previously observed (Brown et al., 1993
; Cockcroft et al.,
1994
; Hammond et al., 1995
), PLD activity was enhanced
in the presence of GTP
S and myrARF (Fig. 6, lanes 2 and 3). Most importantly, these results demonstrated that
human PLD1 was active in hydrolyzing endogenous Golgi
membrane phospholipids, and that under these conditions
vesicle budding was enhanced.
Fig. 6.
Human PLD1 hydrolyzes Golgi membrane
phospholipids. GH3 cells
were incubated for 24 h with
[3H]oleic acid and Golgi
membranes isolated by
floatation on a sucrose gradient (Austin and Shields,
1996). The isolated membranes were incubated with
1 M KCl at 4°C for 10 min to
remove endogenous PLD.
The salt-treated membranes were pelleted by centrifugation at
150,000 g for 50 min and resuspended in incubation buffer. Equal
aliquots of the salt-washed membrane suspension (~100,000 cpm) were incubated with 0.3% butanol to generate phosphatidylbutanol in the presence of ~1 µg/ml of the Sf-9 cell extract
expressing human PLD1 (lanes 2-4) or the control extract (lane
5) in a reaction containing (lanes 1, 2, 4, 5) or lacking (lane 3) recombinant myristoylated ARF-1 (m-ARF*). When present,
ARF was used at a final concentration of ~1 µM and GTPS at
30 µM.
[View Larger Version of this Image (29K GIF file)]
demonstrated
that PLD activity mediated
-COP recruitment to isolated
Golgi membranes and that coatomer binding was abrogated in the presence of low concentrations of primary alcohols (i.e., when formation of PA was prevented). Our
preceding experiments demonstrated that the endogenous
phospholipids in Golgi membranes were substrates for endogenous (Fig. 5) as well as exogenously added PLD (Fig. 6), and that this lipid hydrolysis correlated with vesicle budding from the TGN. We argued therefore, that if PLD
hydrolysis of PC, to yield PA, was neccessary for vesicle
budding, then inhibition of PA production should also inhibit nascent vesicle release. To test this hypothesis, we exploited the observation that only primary alcohols but not
secondary or tertiary alcohols participate in the PLD
transphosphatidylation reaction. Permeabilized cells were
incubated with 1% butanol, 2-propanol, and tertiary butanol and the effect on vesicle budding determined (Fig. 7,
A and B). 1-Butanol inhibited vesicle release by ~50%
(higher concentrations
1.5% inhibited vesicle budding
quantitatively; however this was a nonspecific effect resulting from partial protein precipitation). Significantly,
inhibition of vesicle budding with 1% 1-butanol led to production of PtdBut (Fig. 7, C) as the major product of PLD1 activity. In contrast, incubation in the presence of
2-propanol or t-butanol, which do not participate in the
transphosphatidylation reaction, had no effect on vesicle
budding (Fig. 7, B and C). Taken together, these data suggest that enzymatically active PLD1 and the PA product
of PC hydrolysis are required for nascent secretory vesicle
release from the TGN.
Fig. 7.
Primary alcohols
suppress release of nascent
vesicles from the TGN. (A)
Radiolabeled, permeabilized
GH3 cells were either untreated (lanes 1 and 2) or incubated with 1% butanol
(lanes 3 and 4), 2-propanol
(lanes 5 and 6), or tertiary
butanol, (t-Butanol; lanes 7 and 8) under vesicle budding
conditions (See Materials and Methods). After 1 h incubation at 37°C, samples
were centrifuged briefly at
13,000 g and the pellet (P)
and supernatant (S) fractions incubated with anti-GH antibodies. The GH immunoreactive products were analyzed by SDS-PAGE. (B) For the quantitation of vesicle budding, duplicate samples identical to those shown in A were analyzed by SDS-PAGE and the GH band intensity quantitated
by densitometry (Materials and Methods). Percent budding was
calculated as GH immunoreactive material in the supernatant
(S) divided by the total GH material (pellet + supernatant, P + S). (C) TLC analysis of phosphoatidylbutanol formation by endogenous Golgi PLD. Cells were incubated overnight with
[3H]oleic acid and Golgi membranes isolated by sucrose density
centrifugation. The radiolabeled membranes were incubated under vesicle budding conditions for 1 h at 37°C with the indicated
alcohols (final concentration, 1%), after which the total lipids
were extracted with chloroform-methanol and the products analyzed by TLC (Materials and Methods). PtdBut, migration of
phosphatidyl butanol; the asterisk indicates the probable mobility of phosphatidic acid. (Note this spot is almost absent from
samples incubated with 1-butanol).
[View Larger Version of this Image (20K GIF file)]
Discussion
; Liscovitch and
Cantley, 1995
; De Camilli et al., 1996
). Earlier work (Herman et al., 1992
; Schu et al., 1993
; Stack and Emr, 1994
)
demonstrated that the yeast VPS34 gene encodes a PtdIns-3-kinase that is essential for protein transport from the
Golgi apparatus to the vacuole. In mammalian cells, phosphoinositol (PI)-3 kinases are involved in regulating endocytic trafficking of plasma membrane receptors and in
transport of lysosomal proteases from the Golgi apparatus
to lysosomes (for review see Shepherd et al., 1996
). A second class of proteins that affect phospholipid metabolism, phosphoinositol transfer proteins (PI-TP) also mediate vesicular transport. The SEC14 gene encodes a PI-TP (Bankaitis et al., 1989
, 1990
) and yeast cells expressing a temperature-sensitive SEC14 allele are blocked in intra-Golgi
transport at the nonpermissive temperature. Significantly,
mammalian homologues of Sec14p have been implicated
in two stages of the late secretory pathway. Firstly, a
Sec14p-related protein was purified from rat PC12 cells
and its addition to permeabilized PC12 cells stimulated
Ca2+-mediated exocytosis in vitro (Hay and Martin, 1993
).
Secondly, a 34-kD protein purified from bovine adrenal
medulla which possesses PI-TP activity is required for nascent secretory vesicle budding from the TGN in vitro
(Ohashi et al., 1995
); the yeast Sec14p could substitute for
the mammalian PI-TP in both reactions.
; Cockcroft et al., 1994
; Houle et al.,
1995
; Siddiqi et al., 1995
), Rho A (Malcolm et al., 1994
;
Bourgoin et al., 1995
; Kwak et al., 1995
) and Cdc42 (for review see Frohman and Morris, 1996
). The essential role ARF1 plays in vesicle trafficking (Donaldson and Klausner, 1994
; Rothman, 1994
), its regulation of PLD (Brown
et al., 1993
; Kahn et al., 1993
; Cockcroft et al., 1994
; Liscovitch and Cantley, 1995
), the presence of an ARF-regulated form of PLD in Golgi membranes (Ktistakis et al.,
1995
) and the recent demonstration that ARF-activated PLD activity enhances coatomer binding to Golgi membranes (Ktistakis et al., 1996
) suggested ARF may stimulate vesicle budding from the TGN via PLD activation.
The data of Figs. 1-3, and 5 argue that this hypothesis is
correct. A cytosolic extract obtained from insect Sf-9 cells
expressing human PLD1 stimulated vesicle budding (Fig.
1). It was possible, although unlikely, that other components of the extract rather than PLD activity per se caused
vesicle release. To exclude this possibility, we used an immunopurified preparation of PLD1 and this stimulated
vesicle budding potently with maximal activity at ~5 nM
(Fig. 2). Given that the purified enzyme is very labile in
the absence of detergent (most of its activity is lost by 24 h;
Hammond, S.M., and A.J. Morris, unpublished observations), it is likely that the enzyme is considerably more potent in promoting vesicle release than our data suggest.
The specificity of this reaction was futher demonstrated by
the use of a purified but enzymatically inactive point mutant of PLD1. This mutant posseses only a minor change in
that a highly conserved lysine 898 (present in mammals,
yeast, and plant PLDs) was changed to arginine. Addition
of this mutant to the permeabilized cell system had no effect on vesicle budding (Fig. 2). Furthermore, a control
Sf-9 cell extract expressing an unrelated phospholipase, PLC-
2 specific for the hydrolysis of inositol phospholipids, also had no effect on vesicle budding (Fig. 1). Together, these results demonstrated that recombinant human PLD potently stimulated vesicle budding in a reaction
that was dependent on enzyme concentration and energy.
), stimulated
vesicle release (data not shown). Secondly, the human
PLD1 enzyme was active in hydrolyzing endogenous
Golgi phospholipids (Fig. 6); and thirdly, the transphosphatidylation reaction inhibited vesicle budding (Fig. 7).
Thus, when 1-butanol, but not tertiary butanol or any secondary alcohols was present in the budding reaction, vesicle release was inhibited significantly, suggesting that the
product of PC hydrolysis (PA) is required for vesicle release. In this context, addition of choline to the permeabilized cells had no effect on vesicle release (data not shown). Furthermore, since both human and plant PLDs
stimulated vesicle budding (the latter are not ARF regulated) and the similarity between these enzymes is confined largely to their putative active sites (Hammond et
al., 1995
), this suggests that enzyme activity per se promotes vesicle release. Consistent with this idea, incubation of Golgi membranes with Streptomyces chromofuscus
PLD, which is also not ARF regulated, increased coatomer binding about fourfold (Ktistakis et al., 1996
). Our
data provide direct evidence that in addition to PI-TPs and
PI-3 kinases, a third class of phospholipid modifying proteins, namely PLDs, can mediate vesicle trafficking (in this
case budding from the TGN).
; Traub et
al., 1993
) and guanine nucleotide exchange activity (Morinaga et al., 1996
; Liang and Kornfeld 1997
). However,
myristoylation is not an absolute prerequisite for ARF
function; for example, although myristate facilitates binding of ARF-GDP to membrane phospholipids, and this
enhances GDP-GTP exchange, the interaction between
ARF and its GEF was not myristate dependent (Franco et al., 1996
). In addition, GAP-stimulated hydrolysis of GTP
bound to ARF is independent of myristoylation (Ding et
al., 1996
) and most recently, the binding of arfaptin to
ARF-3 was also shown to be independedent of myristoylation (Kanoh et al., 1997
). Furthermore, although PLD activation by ARF-5 and -6 required myristoylation, there
was much less difference between the ability of myristoylated and nonmyristoylated ARF-1 to enhance PLD activity (Massenburg et al., 1994
). Similarly, our results suggest
that ARF stimulation of PLD can occur in the absence of
myristate; however, our preliminary data suggest that native myristoylated ARF stimulates PLD at about a fivefold
lower concentration than non-myristoylated ARF (Siddhanta, A., A. Elgort, and D. Shields, manuscript in preparation). These results suggest that myristoylation may potentiate ARF action rather than being absolutely required
for its function.
), an "activated" ARF mutant (Q71L) that potently
enhances vesicle budding, stimulated PLD maximally. In
contrast, the
NT and T31N ARF mutants that do not
stimulate vesicle budding had no significant effect on PLD
activity. These data demonstrated a direct correlation between the ability of ARF to stimulate endogenous PLD
activity and to enhance nascent secretory vesicle release
from the TGN. It might be argued that endogenous ARF
present in the insect cell lysate, rather than the human
PLD1 activity, stimulated vesicle budding. However, this
was not the case since the control Sf-9 extract had no effect on vesicle budding (Fig. 1).
) or
by other PLD-activating proteins such as Rho family
members, Rac, cdc42, or PKC (Frohman and Morris, 1996
). At present however, we do not know if ARF activation of PLD occurs by a direct interaction between these
two polypeptides. Evidence from several laboratories has
shown that ARF and PLD co-chromatograph through several purification steps suggesting a direct interaction
(Brown et al., 1993
; Siddiqi et al., 1995
) and ARF and purified PLD do interact in phospholipid micelles (Hammond et al., 1997
). Interestingly, during v-SRC activation of PLD, PLD appears to interact directly with the Ral GTPase (Jiang et al., 1995a
,b). Given these results, we speculate that ARF and PLD might interact directly on the TGN
membrane to effect nascent secretory vesicle release.
S activation of PLD (Geny and Cockcroft, 1992
; Dubyak et al.,
1993
) and recent evidence suggests that ATP hydrolysis is
required by phosphoinositol-4-phosphate-5-kinase (Pertile et al., 1995
) in the final step of PtdIns-4,5-P2 synthesis. Thus, in part ATP may be neccesary to generate the PtdIns-4,5-P2 cofactor that activates PLD. In adddition, ATP
hydrolysis might be necessary to provide the energy for
vesicle scission which releases the nascent vesicle from the
TGN membrane.
-adaptin and clathrin to result in vesicle budding
(Fig. 8). In the second scenario, PLD would function as a
signal transducer by producing PA (Song et al., 1994
;
Briscoe et al., 1995
; Jiang et al., 1995a
). The PA produced
in this reaction could then function as a second messenger
by being hydrolyzed to a number of possible intermediates
such as diacylglycerol which could further trigger intracellular signaling events (e.g., activation of PKC). Consistent
with this idea, PKC itself has been shown to stimulate vesicle budding (Xu et al., 1995
; Singer et al., 1996
), and recently PKC inhibitors were shown to prevent vesicle budding from isolated Golgi membranes (Simon et al., 1996
).
Significantly, ARF-1 was also implicated in phosphotyrosine-mediated vesicle budding (Austin and Shields,
1996); whether this occurs by activation of phospholipase
D or by another mechanism is currently under investigation. Most significantly, our results have identified a novel
mechanism for vesicle budding in which a Golgi phospholipid modifying enzyme, PLD can be activated by input
from multiple signals, and enhances release of nascent
secretory vesicles from the TGN.
Fig. 8.
Possible role of
PLD in nascent secretory
vesicle budding. (A) Hydrolysis of phosphatidylcholine
(PC) in the TGN mediated by ARF-activated PLD leads
to a high local concentration
of phosphatidic acid (PA, ~);
this could alter the composition of the lipid bilayer transiently, perhaps changing its
physical properties. These
putative lipid microdomains,
enriched in PA, facilitate enhanced coat recruitment, possibly the Golgi-specific AP-1 adaptor complex, resulting in budding of nascent secretory vesicles
from the TGN in a reaction requiring ATP and GTP. (B) PA is
rapidly hydrolyzed to a number of possible intermediates which
may be converted to other metabolites that could initiate a signaling cascade, the end product of which (X) mediates coat recruitment.
[View Larger Version of this Image (35K GIF file)]
Received for publication 14 October 1996 and in revised form 12 June 1997.
Please address all correspondence to Dennis Shields, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Tel.: (718) 430-3306. Fax: (718) 430-8567.This work was supported by National Institutes of Health (NIH) grant DK21860, and in part by a grant from the Juvenile Diabetes Foundation to D. Shields, and by NIH grants GM 50388 to A.J. Morris and HD 29758 to M.A. Frohman. Core support to D. Shields was provided by an NIH Cancer Center grant P30CA13330.
ARF, ADP ribosylation factor; BFA, brefeldin A; ERS, energy regenerating system; GAP, GDPase activating protein; GEF, guanosine nucleotide exchange factors; GH, growth hormone; PA, phosphatidic acid; PI-TP, phosphoinositol transfer protein; PKC, protein kinase C; PLD, phospholipase D; PRL, prolactin; PtdBut, phosphatidylbutanol; PtdIns-4,5-P2, phosphatidyl 4,5-bisphosphate.
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