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INTRODUCTION |
ADP-ribosylation factors
(ARFs)1 comprise a family of
20-kDa GTPases first identified as the protein cofactor required for the ADP-ribosylation of the Gs
protein, catalyzed by the
A1 subunits of the bacterial toxins, cholera toxin or
Escherichia coli heat-labile toxin (LTA1) (1).
The high degree of structural and functional conservation of ARFs
cloned from a wide variety of eukaryotic organisms has allowed a
diversity of experimental approaches addressing the mechanism(s) and
role(s) of ARFs in cell regulation. For example, in the yeast
Saccharomyces cerevisiae, ARFs are required for protein secretion (2), sporulation (3), mitotic growth (4), and respiration
(4)2; in flies, deletion of
the arflike gene (Arl1) causes embryonic lethality (5). The complexity of ARF signaling in any organism or cell
is incompletely understood, and the number of immediate downstream
effectors continues to expand
(6).3 The two best
characterized effectors are the heptameric coat complex, termed
coatomer or COP-I, and phospholipase D1 (PLD1). Indeed, the recruitment
of COP-I by ARF has been proposed as the nucleating step in budding and
subsequent formation of coated vesicles in transport from Golgi stacks
(7, 8) and the activation of PLD1 by ARF has similarly been proposed as
an alternative initiating event in vesicle formation (9).
Vesicular transport is important not only in the cargo that is
selectively shuttled between compartments but also in the maintenance of gradients or differences in the lumenal contents or lipid
composition of the donor and acceptor membranes. The presence of
protein coats on buds, and later vesicles, emanating from a membrane
site is proposed to assist in the formation of the vesicle, in the
selection of cargo destined to enter the nascent vesicle, in the
targeting to the appropriate docking site, and in the regulation of the fusion by regulating the timing and site of uncoating. There are currently five different, multimeric, protein complexes designated as
coats: COP-I, COP-II, AP-1, AP-2, and AP-3. Each of these has been
reported to be recruited to membranes by a member of the ARF family;
COP-I (10), AP-1 (11), AP-2 (12), and AP-3 (13) by an ARF itself and
COP-II by the more divergent SAR1 protein (14). The most extensively
characterized interaction is that between COP-I and ARF proteins as
they were first found to copurify on COP-I-coated vesicles and later
purified ARF was found to be necessary and sufficient to support the
binding of purified coatomer to Golgi membranes upon activation with
the nonhydrolyzable analog GTP
S (7, 10). Thus, the activation of ARF
is proposed as the initiating event, leading directly to recruitment of
coats and later steps that result in the formation of coated vesicles.
With the discovery that ARF proteins are direct activators of PLD1 came
the realization that another model for ARF action in the regulation of
coat protein recruitment is also plausible. PLD catalyzes the
conversion of phosphatidylcholine to phosphatidic acid, and
phosphatidic acid has been shown to increase membrane fluidity; the
lack of the head group is ideal for the changes involved in membrane
reorganization coincident with budding or fission. The model that ARFs
could promote coatomer recruitment and coated vesicle formation through
the activation of PLD was tested and evidence to support it was
provided by Ktistakis et al. (9). The localization of PLD1
to Golgi membranes (15) further strengthens this model. However, not
all ARF-promoted coat assembly is sensitive to inhibition of PLD (12),
and the membrane phosphatidic acid level was not found to increase
during the formation of coatomer-coated vesicles (16).
The functions of ARF proteins have been studied in vivo by
expressing the dominant, activating mutant, Q71L, because this renders
the protein refractory to the hydrolysis of bound GTP which normally
accompanies interaction with ARF GTPase-activating proteins (GAPs).
Induction of the expression of [Q71L]ARF1 (17, 18), [Q71L]ARF3, or
[Q71L]ARF44 in stably
transfected NRK cells caused the vesiculation of Golgi stacks and
expansion of the ER lumen (17). The effects on Golgi morphology are so
dramatic as to be readily seen using light microscopy. These effects on
Golgi and ER structures were assumed to be mediated by the increased
stabilization of COP-I binding at the target membranes but could result
from sustained activation of PLD1. To distinguish between and test each
of the two prevailing models for ARF action in vesicular transport and
to tie the results with in vivo effects of ARF, we sought
second site mutations in [Q71L]ARF3 which would specifically ablate
the activation of PLD1, or the recruitment of COP-I to Golgi membranes,
or the ability to alter the morphology of the Golgi in intact cells. If
such a mutant could be found to lose one but not other activities we
would be able to resolve one or more activities cleanly and test
specific interactions and their consequences in mammalian cells.
Regulatory GTPases have at least two switch regions whose conformation
is sensitive to the bound guanine nucleotide. First described in Ras
proteins (19, 20), these switch I and switch II regions have been
described in G protein subunits (21) and, more recently, in ARFs
(22, 23). Changes in these two switches account in large part for the
change in affinity of GTPases for their effectors and modulatory
proteins, exchange factors, and GAPs. Thus, targeting these switches
for site-directed mutagenesis would likely identify mutants defective
in one or more ARF activities. However, the reverse two-hybrid assay
offers a powerful means of screening thousands of mutants for loss of
specific interactions. Unfortunately, neither PLD1, presumably because
of its hydrophobicity, nor COP-I, because of its heptameric nature, is
amenable to two-hybrid analyses. Instead, we took advantage of recently
described ARF-binding partners from two-hybrid screening to identify
loss-of-interaction mutants with those partners and counterscreened
them for PLD1 activation, COP-I recruitment, and Golgi vesiculation.
Each of the four ARF-binding proteins, LTA1 (6),
POR1/ARFAPTIN2 (24, 25), MKLP1 (6), and GGA13 interacts
preferentially with human [Q71L]ARF3 over wild type ARF3. The mutants
identified in this study have allowed clear separation of PLD1
activation from COP-I recruitment, indicating that activation of PLD1
cannot be the sole means of promoting COP-I recruitment to Golgi
membranes. Surprisingly, neither PLD1 activation nor COP-I recruitment
correlates well with the ability to vesiculate Golgi in live cells,
leading us to propose the presence of another, as yet unidentified,
effector for this action of ARF.
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EXPERIMENTAL PROCEDURES |
Materials--
Unless otherwise specified, chemicals and
reagents were purchased from Sigma. Yeast and bacterial media reagents
were purchased from Difco. Media and reagents for NRK and Chinese
hamster ovary cells were purchased from Life Technologies, Inc. All
lipids were purchased from Avanti Polar Lipids.
Reverse Two-hybrid Assay--
Human ARF3 and the [Q71L]ARF3
mutant were engineered for expression in yeast as fusion proteins with
the addition of the GAL4 binding domain at the COOH terminus, followed
by the epitope from hemagglutinin recognized by the 12CA5 antibody.
Each of four ARF-binding proteins (LTA1, full-length (6,
26); partner of RAC1 (POR1, the NH2-terminal truncation
(70-313)POR1 = POR1
N) (24, 25); mitotic kinesin-like protein 1 (MKLP1, the NH2-terminal truncation mutant (669-960)MKLP1)
(6); and the ARF binder (GGA1, the truncation mutant
(145-531)GGA1))3 was expressed in yeast as a
NH2-terminal fusion protein with the GAL4 activation domain
as described previously. The original expression vectors (pAS1-CYH2 and
pACT2) and yeast strains (Y190 and Y187) were the generous gift of Dr.
Stephen Elledge (26). A plasmid, pBG4D, which allowed expression of the
GAL4 binding domain fusions at the COOH terminus of tester proteins was
the generous gift of Rob Brazas. These cells allow read-out of
two-hybrid interactions as either 
-galactosidase activity or
histidine prototrophy, resulting from expression of the HIS3 gene
product. These two assays were performed as described (26) and give
identical qualitative results, but the -galactosidase assay is
easier to score so that is the one used most often and reported here.
For the reverse two-hybrid screen, random mutations were introduced
into the open reading frame of [Q71L]ARF3 performing a PCR under
conditions designed to reduce the fidelity of the polymerase (1/5
normal dATP concentration and addition of 50 µM
MnCl2) (27). A gap was introduced into the plasmid by
restriction enzyme digestion to remove only the ARF coding region while
the PCR product extended an additional 130 base pairs on each end. The
mutagenized PCR product and gapped plasmid were used to transform Y190
cells that already carried plasmids directing the expression of the
ARF-binding partner (yeast strain YAB413 (POR1N), YAB453 (MKLP1),
and YAB466 (GGA1) (6).3 Transformants were replicated on
nitrocellulose membranes and assayed for -galactosidase activity
directly. Colonies giving white or light blue colors were picked and
checked for the expression of full-length ARF3 by detection on
immunoblots of the hemagglutinin tag at the COOH terminus. For each of
the three screens (POR1, MKLP1, and GGA1) about 7,000 transformants
were screened with an average of about 350 white or light blue colonies
picked. 25-50% of these were found to be full-length. Plasmids
expressing a full-length ARF3 were then rescued from yeast and
transformed into Y187 to allow mating with each of the other
ARF-binding partners in Y190 cells. The diploid strains harboring both
plasmids were selected and assayed for protein interactions by the
nitrocellulose filter bound, -galactosidase assay. ARF3 mutants that
retained interaction with at least one of the four binding partners
were sequenced to identify the mutation.
Expression and Purification of Nonmyristoylated and Myristoylated
ARF Proteins--
One set of primers from the ends of the open reading
frame, which incorporate an NdeI site at the initiating
methionine and a stop codon followed by a BamHI site at the
3'-end, were used to amplify the ARF3 mutants from the two-hybrid
vectors to allow subcloning into pET3C. The resulting plasmids were
transformed into BL21(DE3) cells for protein expression at 37 °C, as
described previously (28). For myristoylated ARF proteins, the BL21
cells were cotransformed with a second plasmid, directing expression of
human N-myristoyltransferase (29). In this case, cells were induced at room temperature and after the addition of 200 µM myristic acid (30). The purification of ARF proteins was performed as described
previously (28). The extent of N-myristoylation of each ARF
preparation was determined by reverse phase HPLC analysis on a C4
column loaded in 0.1% trifluoroacetic acid and eluted with a linear
gradient of 0-80% acetonitrile in 0.1% trifluoroacetic acid.
Base-line resolution of the two peaks was readily achieved with
retention times for nonacylated and acylated proteins of typically 46 and 52 min, respectively.
GTPS Binding Assay--
The binding of
[35S]GTPS to ARF proteins was determined using the
nitrocellulose filter trapping method (1, 36) to separate bound and
free ligand, under two different conditions: the "standard" conditions and those used in the assay for PLD1 activity. The main
differences between these conditions are temperature, the concentrations of free magnesium, and the identity and form of lipids/detergents. The standard conditions included incubation at
30 °C of 1 µM ARF, 10 µM
[35S]GTPS, 20 mM Tris-Cl, pH 7.4, 100 mM NaCl, 1 mM dithiothreitol, 1 mM
EDTA, 0.5 mM MgCl2, 3 mM sonicated
L--dimyristoylphosphatidylcholine, and 0.1% (2.5 mM) sodium cholate. Binding under PLD1 assay conditions (31) was performed at 37 °C with 4 µM ARF, 30 µM GTPS, 2.5 mM MgCl2, 1.7 mM CaCl2, 3.5 mM EGTA, 40 µM
EDTA, 80 mM KCl, 1.2 mM NaCl, 20 mM
HEPES, pH 7.5, and 690 µM lipid vesicles (10 mol % dipalmitoylphosphatidylcholine, 86 mol % dioleoylphosphatidylethanolamine, and 4 mol % phosphatidylinositol
4,5-bisphosphate.)
PLD1 Assay--
Hexahistidine-tagged human PLD1 was expressed in
Hi5 cells using a baculovirus construct, the generous gift of Sung Ho
Ryu, Postech University, Pohang, Korea. Membranes from these cells were
prepared according to a procedure described by Brown et al. (37). The PLD1 was then extracted from the membrane by resuspension of
the membrane in 20 mM HEPES, pH 7.5, 500 mM
KCl, 1% n-octyl -D-galactopyranoside, and
protease inhibitors. After 1 h at 4 °C, the resuspension was
cleared by centrifugation at 100,000 × g for 1 h.
The supernatant was diluted with buffer A (20 mM HEPES, pH
7.5, containing 1 mM dithiothreitol, 1 mM EDTA,
and 1% n-octyl -D-galactopyranoside) to a
final concentration of KCl of 100 mM before application to
a 5-ml HiTrap SP column (Amersham Pharmacia Biotech). Proteins were
eluted with a 100-500 mM KCl gradient in buffer A over a
25-min period at 1 ml/min. The fractions containing PLD activity
(fractions 35-43) were pooled to give a final protein concentration of
50 µg/ml and frozen in aliquots in liquid nitrogen before storage at
80 °C.
The PLD assay was performed as described by Lopez et al.
(31) with slight modification. Briefly, the substrate was prepared in
the form of lipid vesicles composed of 10 mol % [3H-methylcholine]dipalmitoylphosphatidylcholine,
86 mol % dioleoylphosphatidylethanolamine, and 4 mol % phosphotidylinositol 4,5-bis-phosphate at a final concentration of 690 µM. Myristoylated ARF and GTPS were preincubated in
the presence of lipid vesicles at 37 °C for 40 min prior to the
addition of PLD1. The reaction was stopped by the addition of 1 ml of
chloroform:methanol:HCl (50:50:0.3), followed by adding 350 µl of 1 M HCl, and 5 mM EGTA. A portion of the aqueous
phase (500 µl) was analyzed for the release of
[3H]choline. PLD activity was expressed as the amount of
choline released/min/mg of solubilized membrane protein.
COP Recruitment Assay--
Bovine brain cytosol was prepared
according to the procedure of Serafini and Rothman (32). From 350 g of frozen bovine brain (Pel-Freez Biologicals), we obtained 140 ml of
bovine brain cytosol at 16.0 mg/ml protein. Cytosol (15 ml) was
resolved on a Sephacryl S-300, HiPrep 26/60 column (Amersham Pharmacia
Biotech) which had been equilibrated previously with 50 mM
HEPES-KOH, pH 7.4, 200 mM KCl, and 10% glycerol. Fractions
(2 ml) were collected and the presence of COP-I or ARF proteins
analyzed by immunoblotting, using the EAGE anti--COP (33) or R-1023,
anti-ARF1/3 polyclonal rabbit antisera (34). Fractions found to be free
of ARF and enriched in coatomer were pooled and snap frozen in aliquots
for use in the coatomer recruitment assay.
Golgi-enriched membranes were prepared from Chinese hamster ovary cells
as described in Beckers and Rothman (35). Membranes were washed with
0.5 M KCl on ice before use in the recruitment assay.
ARFs and the enriched coatomer preparation were cleared by
centrifugation at 100,000 rpm for 30 min in a TLA 100.1 rotor (Beckman) before use in the assay. All reactions were performed in siliconized microcentrifuge tubes. Golgi membranes (10-12 µg of
protein/reaction) were incubated with 4 µM myristoylated
ARF proteins and 0.6 mg/ml coatomer-enriched cytosol at 37 °C for 20 min in a final volume of 100 µl in the presence of 25 µM of GTPS or GDPS, 2.5 mM
MgCl2, 1 mM dithiothreitol, 0.2 M
sucrose, 15 µg/ml bovine serum albumin, 1 mM ATP, 2 mM creatine phosphate, and 8 IU/ml creatine phosphokinase. The reaction was terminated by centrifugation of the membranes through
a 300-µl cushion of 20 mM HEPES, pH 7.4, 0.5 M sucrose, and 20 mM KCl. The pellet was washed
with 20 mM HEPES, pH 7.4, and dissolved in 15 µl of SDS
sample buffer. The proteins were resolved in gradient (8-16%)
polyacrylamide gels and transferred to nitrocellulose membranes to
allow immunoblotting using the rabbit polyclonal antibody to -COP
(R-23200; 33). Immunoreactivity was visualized with horseradish
peroxidase-conjugated anti-rabbit IgG antibody (Amersham Pharmacia
Biotech) and the enhanced chemiluminescence substrate from Amersham.
[Q71L]Arf3 was included as a positive control on each gel to help
control for variability seen in the electrophoretic transfer to
nitrocellulose. Nevertheless, some variation in background (that seen
with no nucleotide or GDPS added) recruitment of -COP was
observed. For this reason the amount of -COP recruited to membranes
with GTPS was compared with that seen with GDPS. These
experiments were repeated at least three times, and the ability of each
mutant to recruit -COP in a GTPS-dependent manner was
qualitatively the same in each case.
Transient Expression of ARF3 Mutants in NRK Cells--
The
coding regions of each of the ARF3 mutants were subcloned into the
mammalian expression vector pcDNA3 (Invitrogen) at the
NdeI and XbaI sites to allow constitutive
expression under control of the cytomegalovirus promoter. Purified
plasmids (120 ng/ml) and fluorescein isothiocyanate-dextran (1.2 mg/ml;
Molecular Probes) were injected into NRK cells, grown on coverslips,
using an Eppendorf automatic microinjector system. Injections were
targeted to the nucleus with the aid of a 400 × light microscope.
After injection the cells were incubated at 37 °C for 7 h
before indirect immunofluorescence staining was performed using the
polyclonal antibody to mannosidase II as primary and Texas
red-conjugated anti-rabbit IgG antibody as secondary antibody.
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RESULTS |
To test the relationships between PLD1 activation and COP-I
recruitment, as well as each of these activities of ARFs to other cellular responses, we sought point mutations in an ARF which would
lose one or more of these functions to allow the molecular dissection
of ARF signaling both in vitro and in intact cells. The size
and hydrophobicity of PLD1 and heptameric nature of COP-I make them
unusable as targets in yeast two-hybrid assays as a means of monitoring
interactions with ARFs. Thus, we screened for loss-of-interaction
mutations with a recently identified set of ARF effectors and then
looked among them for specific defects in PLD1 activation and COP-I recruitment.
Generation of Human ARF3 Mutants--
This approach was made
possible by the recent identification of at least four different
binding partners that bind ARF3 preferentially in the activated
GTP-bound state and demonstrate activity in the yeast two-hybrid assay.
Reverse two-hybrid screens were performed to identify mutants of human
ARF3 which specifically lost the interaction with one effector but
retained the interaction with others. Three separate reverse two-hybrid
screens were performed with POR1, MKLP1, and GGA1, as described under
"Experimental Procedures." In each of the reverse two-hybrid
screens the effector was expressed as a fusion protein with the GAL4
activation domain. The ARF3 was also expressed as a fusion protein,
with the GAL4 binding domain at the COOH terminus, followed by a
hemagglutinin epitope at the COOH terminus. In addition, the ARF3
fusion protein in each case contained the GTPase-deficient, activating,
Q71L mutation because this leads to higher levels of GTP-bound ARF in
cells and promotes interactions with effectors in the two-hybrid assay. Random mutagenesis of [Q71L]ARF3 was achieved by PCR under conditions of reduced polymerase fidelity. Loss-of-interaction mutants were identified as white or pale blue colonies in the -galactosidase assay, performed on nitrocellulose filters soaked in
5-bromo-4-chloro-3-indolyl--D-galactopyranoside. The
presence of a full-length ARF3 fusion protein in each of these colonies
was confirmed by immunoblotting, using the 12CA5 antibody to detect the
hemagglutinin epitope at the COOH terminus. Plasmids expressing
full-length mutants were rescued from yeast and used to transform
strain Y187 prior to mating with strains carrying each of the four
binding partners of [Q71L]ARF3. Diploid strains were then assayed for
-galactosidase activity. Only mutants retaining interaction with at
least one effector were considered further. This eliminated those
mutations that cause the protein to lose the ability to bind GTP or to
become unfolded or unstable. Plasmids from surviving positives were
then sequenced to identify mutations. In each of the three screens
about 7,000 transformants were screened, and an average of 300 white or
pale blue colonies were isolated. Of these, between 25 and 50% encoded
full-length ARF3. Combining results from the three screens, a total of
150 plasmids expressing full-length ARF3 were rescued, and the open
reading frame from a total of 72 plasmids was sequenced; 46, 10, and 16 from the POR1, GGA1, and MKLP1 screens, respectively. Of these, 39 had a single new mutation in [Q71L]ARF3. These single mutations resulted in missense coding in 23 residues.
We focused our attention originally on those mutations found in regions
homologous to switch I and switch II of Ras proteins because these are
known to be critical in protein-protein interactions among members of
the RAS superfamily and confirmed in ARF proteins by the solution of
crystal structures of ARF bound to its exchange factor or GAP (22, 23).
Eight of the 23 mutations were found within these switch domains, as
shown in Table I. Four switch II mutants,
R79G, Y81H, Q83R, and T85A, were each identified originally by the loss
of interaction with POR1. Two switch II, Y81C, I74T, and one switch I
mutant, I49T, were from the MKLP1 screen, with two of these three
mutants independently isolated twice. The switch I mutant F51Y was
found among those mutants that lost interaction with GGA1. The S62G
mutation was a PCR-generated mutation that was isolated by chance
during the construction of the [Q71L]ARF3 plasmid and was found to
lie between switch I and II and so served as an additional control in
several studies. Residues 49 and 51 are each in switch I, whereas
residues 74-85 are in switch II.
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Table I
Interactions between switch I and switch II mutants and effectors
of ARF3
The interactions of mutants of ARF3 (listed in the leftmost column)
with different partners were tested. Diploid yeast strains, each
harboring a set of ARF3 binding domain and effector activating domain
plasmids, were assayed for -galactosidase activity after lysis on
nitrocellulose filters, as described under "Experimental
Procedures." Minus sign () represents white color indicating the
loss of interaction; /+ represents barely detectable blue color
development; + represents pale blue color indicating the retention of
some binding; ++ represents more binding; +++ represents full
interactions, as seen with the [Q71]ARF3 control. Color development
was detected by eye after 30 min.
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Although mutagenesis and reverse two-hybrid screening were not
saturated, the data in Table I offer an initial, low resolution map of
the sites of interactions between multiple ARF effectors and the
activated GTPase. For example, switch I and specifically residues 49 and 51 are likely important to the binding of MKLP1 and GGA1 but
perhaps not to that of POR1 or LTA1. POR1 binding to ARF3
appears to be very sensitive to any changes in the switch II domain as
all six changes described lead to loss of POR1 interaction in
two-hybrid assays. LTA1 may bind to ARF3 in a fashion quite distinct from these other effectors as only one mutant caused the loss
of LTA1 interaction. The reverse two-hybrid screening and
identification of residues involved in the binding of LTA1 to ARF3 are the subject of another
study5 so will not be
discussed further here. By scanning through the results in Table I it
appears clear that none of these four effectors shares identical
binding sites with ARF3. This serves to highlight the diversity in
effector-GTPase interactions and also supports the utility of this
approach in identifying specific loss-of-interaction mutations.
It is evident from the data in Table I that ARF effectors bind to
distinct but overlapping residues in switch II and probably switch I. It should also be clear that mutations, such as Y81C, in which the ARF3
has lost interactions with multiple effectors but retains full binding
to one, GGA1 in this case, will make potentially very powerful probes
for the in vivo roles of ARFs and their different effectors
in cell regulation of multiple pathways. A cautionary note is also
provided by the two mutations at residue 81. Changing this tyrosine to
cysteine or histidine yielded quite different spectra of binding
partners (see Table I), suggesting that not only the absence of the
wild type residue may be important but also the residue to which it is
being changed can have important consequences.
Purification and Characterization of Recombinant Human [Q71L]ARF3
Second Site Mutants--
Each of the switch I and II mutants was
expressed in bacteria and purified to allow biochemical analyses. The
proteins were also coexpressed in bacteria with an
N-myristoyltransferase to allow the production of
myristoylated ARF3 proteins. As described previously (28, 30), this
results in only partial N-myristoylation, and resolution of
the acylated and nonacylated forms cannot be achieved readily. We
typically obtain ARFs that are about 30% acylated, with a range of
20-50% myristoylated proteins for the mutants (see Table
II), as determined by reverse phase HPLC
(see "Experimental Procedures").
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Table II
Characterization of recombinant, myristoylated ARF protein preparations
and their ability to bind GTPS and stimulate recombinant PLD1
ARF proteins were prepared, the extent of N-myristoylation
determined by reverse phase HPLC, and ligand binding was determined, as
described under "Experimental Procedures." The amount of GTPS
bound after a 1-h incubation was normalized to the parent,
[Q71L]ARF3. The PLD activities were determined in duplicate, in the
presence of 0.1, 0.2, and 0.5 µM ARF preparation (see
Fig. 3) and are expressed relative to the activity of the parent,
[Q71L]ARF3. The data shown are the mean of these three
activities ± 1 S.D. The activity of the parental [Q71L]ARF3
assayed in the presence of GDPS is also shown.
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The rate and extent of binding of [35S]GTPS were
determined under our standard conditions (see Ref. 1 and
"Experimental Procedures") which include 3 mM
L--dimyristoylphosphatidylcholine and 2.5 mM
cholate. Also, the free magnesium concentration was held constant, in
the low micromolar range, by the inclusion of 1 mM EDTA and
0.5 mM MgCl2. As seen in Table II, all of the
mutants retained the ability to bind GTPS, though to somewhat
different extents. Although the rates of nucleotide exchange were
similar, reaching half-maximal binding in less than 5 min (see Fig.
1A), the binding
stoichiometries under our standard conditions varied from 49 to 140%
of the control, [Q71L]ARF3, which bound about 0.3 mol of GTPS/mol
of protein when N-myristoylated and about 0.05-0.1 mol of
GTPS/mol of protein when nonacylated.
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Fig. 1.
Time course of the binding of
GTPS to partially myristoylated ARF
proteins. Panel A, GTPS binding was determined at
30 °C under standard conditions, as described under "Experimental
Procedures," with 1 µM ARF and 10 µM
[35S]GTPS in a total volume of 100 µl. Panel
B, GTPS binding was performed at 37 °C with 4 µM ARF and 30 µM GTPS using conditions
optimal for assay of PLD activity, as described under "Experimental
Procedures," in a total volume of 150 µl. In each case, duplicate
samples (10 µl) were taken at each time point. The key
shows the proteins assayed, with ARF3Q the [Q71L]ARF3 protein; each
of the indicated mutations represents second site mutations present
with the Q71L change.
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Very similar relative binding levels were observed when the conditions
in the nucleotide binding assay were changed to those used in the assay
for PLD. This assay requires a different lipid mixture and higher
concentrations of free magnesium, resulting in slower off-rates for
GDP, and consequently on-rates for GTPS. Under these conditions, the
binding of GTPS was fairly linear for at least 1 h in each case
(Fig. 1B). Thus, all of the recombinant proteins retained
binding characteristics similar to those of the wild type and control,
[Q71L]ARF3, proteins with the main difference noted in the extent of
binding, ranging between 30 and 300% that of the [Q71L]ARF3 control.
The only mutant that behaved qualitatively differently in the two
nucleotide binding assays was Y81H, which was slightly lower than
controls in the standard assay (85%) but just over twice the level of
binding was observed in the PLD assay conditions (204%).
Identification of ARF3 Mutants That Lose the Ability to Activate
PLD1--
The purified, recombinant, N-myristoylated ARF3
mutant proteins were next assayed for the ability to activate
recombinant PLD1, using a modification of the method of Lopez et
al. (31). This assay uses exogenous 3H-labeled
phosphatidylcholine as substrate and measures the release of the
water-soluble [3H]choline. Human PLD1 was expressed in
insect cells as an NH2-terminal hexahistidine-tagged form
and partially purified by solubilization of Hi5 membranes in 1%
n-octyl--D-galactopyranoside and passing the
extract over a HiTrap column to enrich for ARF-stimulated PLD activity,
as described by Singer et al. (40).
As seen in Fig. 2, activation of PLD
activity in this assay is dose-dependent for added ARF and
GTPS. The activity of both ARF3 and [Q71L]ARF3 was increased by
N-myristoylation. Whereas the increased activity with
acylation shown is about 3-5-fold for the wild type protein and about
10-fold for the Q71L mutant, the specific activities are even greater
because the acylated proteins are only about 30% myristoylated. The
concentrations of ARF proteins needed to achieve half-maximal
activities were also different, with the nonmyristoylated forms
requiring around 2 µM ARF and the myristoylated proteins
less than 1 µM. The K1/2 for
N-myristoylated ARF as activator of PLD1 is seen more
clearly in Fig. 3 and estimated at
between 50 and 100 nM. Another difference seen between
these preparations is that the nonmyristoylated proteins reached a
plateau at around 4 µM ARF, and this held constant to at
least 16 µM ARF (data not shown). In contrast, the
acylated ARF preparations that stimulated PLD1 activity peaked with
around 2 µM ARF, and activities then decreased. This
effect is not totally understood but has been described previously and
was worse when we tried performing the assays with the PLD on Hi5
membranes. Solubilization in octyl glucoside and partial purification
made the PLD activity less sensitive to inhibition at high ARF
concentrations. We focused our analyses on the modified proteins as
N-myristoylation is a cotranslational event so only the
myristoylated forms are thought to be present in cells. However, as the
nonacylated proteins are more homogeneous preparations and assist in
the interpretation of results from preparations that include 30%
acylated and 70% nonacylated proteins, results from analyses with
nonmyristoylated protein preparations are also included. Typically, no
qualitative differences were noted when nonacylated ARF proteins were
compared.
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Fig. 2.
PLD1 activity is highly dependent on added
ARF proteins and activating guanine nucleotides. The PLD assay was
performed with different amounts of myristoylated or nonmyristoylated
ARF3 or [Q71L]ARF3 proteins in the presence of 30 µM
GTPS, GDPS, or no nucleotides. Data shown are the average of
duplicate samples with a difference of less than 5%.
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Fig. 3.
Mutations in switch I or II can result in
compromised abilities to stimulate PLD1 activity. PLD activity was
assayed with different amounts of myristoylated ARF3 proteins in the
presence of 30 µM GTPS. Two controls were included in
which the activity of [Q71L]ARF3 was determined with 30 µM GDPS or in the absence of guanine nucleotide.
Mutations labeled in the figure are mutations in addition to Q71L, in
human ARF3. Data shown are the average of duplicate samples with
differences less than 5%.
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Each of the switch I mutants (I49T and F51Y) had lost the ability to
activate PLD1, whereas only one of the switch II mutants (Y81C) had
compromised PLD-stimulating activity (see Table II or Fig. 3).
Differences between these three mutants and the other proteins were
even more dramatic when the nonacylated proteins were assayed (data not
shown). In that case, there was no PLD-stimulating activity for any of
these three proteins. Because the PLD assay appears most sensitive when
acylated ARFs are used, we focused our analysis on these preparations
(see Table II). Although the data shown in Fig. 3 suggest a
dose-dependent increase in PLD activity at the highest
concentrations of the I49T mutants, this was not always the case. The
lack of a clear dose dependence to the small activities seen and the
lack of statistical difference between stimulation by the I49T mutant
and that seen with the parental protein in the presence of GDPS is
highly suggestive of an inactive protein in our assay. The F51Y mutant
consistently lacked any discernible activity in this assay.
The other control, [S62G]ARF3, was equally active with controls, as
expected for this positive control. Although some differences in the
binding of GTPS were noted (see Fig. 1), these did not appear to be
a primary determinant of PLD1-stimulating activity. For example, the
F51Y and Y81C mutants each bound nucleotides to higher stoichiometries
than the others yet had among the lowest activities in the PLD assay.
Recruitment of COP-I to Golgi Membranes Is Also Impaired in Several
Mutants of ARF3--
The direct recruitment of COP-I has been proposed
as the mechanism of ARF action as regulator of vesicular traffic in
eukaryotes, and an in vitro assay has been developed to
monitor this activity. We used this assay to determine if any of the
switch mutants has lost the ability to recruit COP-I to Golgi membranes.
Bovine brain cytosol was resolved on a gel filtration column to
separate the high molecular weight, coatomer complex and the monomeric
ARF proteins to provide a source of COP-I that was free of ARFs.
Partially purified Golgi membranes from Chinese hamster ovary cells
were prepared as described (35) and further stripped of extrinsic
proteins by washing in 0.5 M KCl. The coatomer fraction, Golgi membranes, and myristoylated ARF proteins were incubated with
either GTPS or GDPS, as described under "Experimental
Procedures." The reaction was stopped by collecting the membranes by
centrifugation, and COP-I recruitment was detected by immunoblot
analysis using the -COP antiserum (33).
As reported previously (10) and shown in Fig.
4A, the binding of coatomer in
this assay was dependent on the addition of ARFs and the activating
guanine nucleotide. The nonmyristoylated ARF proteins did not promote
the recruitment of coatomer to membranes (data not shown). Most of the
myristoylated mutants retained the ability to recruit coatomer onto
Golgi membranes in a GTPS-dependent fashion (see Fig.
4B). However, the I74T, Y81C, and Y81H mutations resulted in
the loss of coatomer recruitment activity. The data in Fig.
4B were generated using the same amounts of each ARF3 protein but do not take into account differences in GTP binding. When
this experiment was repeated after adjusting for steady-state binding
of GTPS so that equal amounts of activated ARF3 proteins were being
compared, the results were qualitatively the same as those shown in
Fig. 4B (data not shown). Thus, isoleucine 74 and tyrosine
81 are required for the recruitment of coatomer onto Golgi membranes by
ARF3. The two mutations in switch I, I49T and F51Y, also lead to a
substantial decrease in COP-I recruitment, although they consistently
promoted more GTPS-dependent COP-I binding than the
mutations in residue 74 or 81.
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Fig. 4.
ARF-promoted binding of COP-I to Golgi
membranes. The amount of coatomer that bound to enriched Golgi
membranes (10 µg) in the presence of different combinations of
N-myristoylated ARF proteins (4 µM; as labeled
in the figure) and GTPS or GDPS (25 µM) was
detected by immunoblotting with antibody (R-23000) to the -COP
subunit. Panel A, the binding of -COP to the Golgi
membrane is dependent on the addition of myristoylated ARF3 and GTPS
(compare lanes 3 with 6, and 5 with
6, respectively) but not on GDPS (lane 5). As
controls, Golgi membranes did not contain coatomer (lane 4),
and no coatomer was precipitated without membrane (lane 1).
Panel B, binding of -COP to the Golgi membrane with
different mutants of ARF3 (as labeled in the figure) in the presence of
GDPS (odd numbered lanes) and GTPS (even
numbered lanes). Each reaction contained enriched Golgi membranes
and ARF-free coatomer, prepared as described under "Experimental
Procedures." The mutations labeled in the figure were secondary
mutations on top of the [Q71L]ARF3. As seen in the control
(lane 2), the binding of -COP to Golgi membranes seen
with [Q71L]ARF3 (ARF3Q) is still dependent on the addition of
GTPS.
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Note that the ability of ARF mutants to recruit COP-I did not correlate
with their ability to activate PLD1. For example, I74T retained almost
80% of the stimulatory activity for PLD1 but could not recruit any
coatomer. In contrast, I49T and F51Y lost PLD activation but still
recruited coatomer to Golgi, although less well than ARF3 (data not
shown) or [Q71L]ARF3. Thus, these data reveal a clear biochemical
dissociation between the activation of PLD1 and recruitment of COP-I to
Golgi membranes.
Some Switch Mutants Have Lost the Ability to Vesiculate Golgi When
Expressed in NRK Cells--
Expression of the dominant activating
mutant, [Q71L]ARF1 (17) or [Q71L]ARF3,4 in cultured
mammalian cells leads to engorgement of the ER lumen and gross
enlargement and vesiculation of the Golgi stacks. These observations
are in contrast to predictions from the simple model for ARF proteins
as mediators of coatomer recruitment to the Golgi membranes yet remain
among the few phenotypes associated with the expression of activated
ARF in mammalian cells. Such studies have added value in not relying on
the use of nonspecific activators of GTPases, i.e.
nonhydrolyzable guanine nucleotide triphosphates.
The ability of the induced expression of [Q71L]ARFs in stably
transfected cell lines to alter Golgi morphology dramatically was used
and modified to develop a more rapid assay for ARF functions. Wild
type, [Q71L]ARF3, and the second site mutants were each subcloned into pcDNA3, which uses the strong, constitutive cytomegalovirus promoter, to direct expression in mammalian cells. NRK cells were microinjected with the pcDNA3-derived plasmids, and Golgi
morphology was monitored by indirect immunofluorescence using antisera
directed against mannosidase II, a protein that localizes to the lumen of predominantly medial Golgi. Injected cells were identified by the
coinjection of fluorescein isothiocyanate-dextran (molecular weight = 10,000).
Uninjected cells, or those injected with the empty vector, or with
pcDNA3 directing expression of wild type ARF3, reveal a tight
perinuclear staining of mannosidase II (Fig.
5, panel B) in NRK cells. The
appearance of the Golgi is altered dramatically after expression of
[Q71L]ARF3 (see Fig. 5, panel D) as seen by the larger
area and more diffuse images of mannosidase II staining around the
nucleus which often can be seen to encircle the entire nucleus. Changes
in Golgi structure can be seen within 1-2 h after injection, and the
number of cells displaying the altered morphology usually peaks between
5 and 7 h and then decreases. This decrease may result from cell
lysis, as described for the inducible expression of activating ARF
mutants (17), but this question was not pursued further in these
studies. The percentage of injected cells that display vesiculated
Golgi varies between experiments and was between 20 and 60% when
assayed 7 h postinjection with the [Q71L]ARF3 plasmid. To allow
comparisons between experiments the [Q71L]ARF3 positive control was
always included and the percentage of injected cells with vesiculated
Golgi set to 100% when comparing other mutants.
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Fig. 5.
Expression of ARF3 mutants in NRK cells as an
in vivo assay for function at the Golgi. The
pcDNA3-derived plasmids that direct expression of the different
mutants of ARF3 (as labeled in the figure) were coinjected with
fluorescein isothiocyanate-dextran into NRK cells grown on coverslips.
The left panels show the injected cells, visualized by green
fluorescence of the fluorescein isothiocyanate-dextran. The right
panels show the immunofluorescence staining with mannosidase II
(Man II) antibody, visualized by Texas red-conjugated secondary
antibody. The expanded, diffuse, and perinuclear staining with
mannosidase II antibody is indicative of Golgi vesiculation
(panels D, F, J, L, and
N). As control, the mannosidase II staining showed tight
perinuclear staining that did not change with overexpression of wild
type (WT) ARF3 or when buffer alone was injected into cells.
The mutations indicated at the left of each set of panels
are the secondary mutations on top of [Q71L]ARF3 (ARF3Q).
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Each of the switch mutants of ARF3 was then tested in the Golgi
vesiculation assay. Because each of the expressed proteins contains the
Q71L mutation they should induce morphological changes unless the
second site mutation interferes with this activity. The results are
shown in Table III. Three different types
of responses were noted; wild type and the F51Y mutant completely
lacked the ability to induce changes in Golgi structure, I49T and R79G
had undiminished capacity for vesiculation, and the others (I74T and Y81C) has diminished but detectable activity in this assay. In other
experiments (not shown) the time after injection and the amount of
plasmid injected were varied, but the results were qualitatively the
same as those reported in Table III.
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Table III
Relative abilities of mutants of ARF3 to promote expansion and
vesiculation of Golgi in intact mammalian cells
NRK cells were injected with pcDNA3-derived plasmids containing
ARF3 or [Q71L]ARF3 or double mutants. The changes in Golgi morphology
of injected cells were visualized by indirect immunofluorescence
staining, using antibodies to mannosidase II 7 h after injection
with plasmids, as described under "Experimental Procedures." The
number of cells that showed the expanded, diffuse, perinuclear staining
indicative of Golgi vesiculation was scored. In different experiments,
the percentage of parental [Q71L]ARF3 injected cells exhibiting the
Golgi vesiculation varied from 20 to 60%. The relative percentage of
cells injected with different double mutants which exhibited the Golgi
vesiculation was normalized to the parent [Q71L]ARF3. Data in the
table are the average of three different experiments with at least 200 cells injected for each experiment.
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DISCUSSION |
We describe here the identification and characterization of nine
mutants in the switch I and II regions of human ARF3. Five different
assays were employed in the characterization of these proteins,
including reverse two-hybrid screens in which they were first
identified, the ability to bind guanine nucleotides with high affinity,
activation of PLD1, recruitment to Golgi membranes of COP-I, and the
ability to vesiculate the Golgi and enlarge the area occupied by this
organelle in intact cells. When each of these activities was assessed,
a range of deficiencies was noted among the mutants that allowed tests
of two current models for ARF action: (i) that activation of PLD1 by
Arf is a required step in the recruitment of coatomer and (ii) that
recruitment of coatomer, without the means to release it, results in
enlarged ER and Golgi lumen with concomitant vesiculation of Golgi in
intact cells expressing the dominant activating mutant, [Q71L]ARF.
Surprisingly, our data support neither of these models. Rather, on the
basis of these analyses we conclude that there exists a currently
unidentified effector for ARF action at the Golgi which is required for
the vesiculation and enlargement of Golgi structures in the presence of
an excess of activated ARF.
Screens for loss-of-interaction mutants allowed the unbiased
identification of specific residues critical to specific effector interactions with activated ARF. Not surprisingly, mutations in the
mobile and nucleotide-sensitive switch regions were prominent among the
mutations found. From the results shown above and by homology to
previous results with other GTPases, it is evident that different
effectors bind to distinct but overlapping regions of ARF3. It appears
from this limited sampling of mutants that specificity for effectors
was more likely derived from the interaction with the switch II region.
The ability to screen thousands of mutants for the loss and retention
of different combinations of effector interactions offers a low
resolution map of protein binding sites which is very useful in testing
models for integration of ARF signaling and should also aid parallel
efforts aimed at solving the more detailed structures by both NMR and
x-ray crystallography.
The three ARF-dependent activities used in these studies
each offer information that is crucial to the testing of current models
of ARF action at the Golgi. The COP-I recruitment assay was thought to
measure the rate-limiting step in the budding of nascent vesicles that
mediate intra-Golgi transport (7, 8). The activation of PLD1 by ARF is
clearly established as a direct G protein-effector system that has been
offered as an alternative hypothesis (15) to the COP-I recruitment
hypothesis; and the vesiculation of Golgi was described as the
consequence of the expression of the dominant activated mutant,
[Q71L], of ARF1 (17) or ARF3. This assay is particularly important as
it examines the consequences of ARF mutations in live cells and thus is
not dependent on the nonspecific GTPase activator, GTPS. It is worth
noting that the regulated expression of [Q71L]ARF1 was first used in stably transformed cells to test the COP-I recruitment hypothesis, and
the observation of large, heterogeneously sized vesicles runs counter
to the prediction made by that hypothesis (17). The cell-based assay
employed in these studies used the microinjection of plasmids and
represents a modification of the original assay that allowed more rapid
testing of ARF mutants and in an assay more easily controlled.
PLD1 has a number of features that make it a very attractive candidate
effector for the action(s) of ARF on membrane traffic at the Golgi. It
was purified as an ARF-sensitive activity that localizes to the Golgi
(15), and phospholipase activity was found to mimic several of the
previously described actions of ARFs in in vitro Golgi
transport assays (15, 38). Characterization of the PLD1 assay revealed
an activity that is highly dependent on ARF and GTP or GTPS. Other
protein activators of PLD1 (e.g. members of the RAC/RHO
family or PKC) were not tested in our assay but have been described
elsewhere (39-41). N-Myristoylation lowers the
Ka for ARF and gives about a doubling in the
maximal activity, compared with nonacylated ARF preparations (see Fig. 2). The partially (
30%) myristoylated ARF3 protein had a
Ka of 50-100 nM in our assay. This
is very close to that reported for a purified bovine brain ARF
preparation (20-30 nM; (42) and is indistinguishable if
the incompleteness of the acylation in our preparations is taken into
account. The presence of the Q71L mutation had a small effect but was
not required for activity (see Fig. 2). This is important as all other
mutations are being viewed within the context of this Q71L, activating mutation.
Each of the mutations in switch I, I49T or F51Y, resulted in the loss
of the ability to activate PLD1 in our assay (see Fig. 3 and Table II).
This increase in the Ka for PLD1 activation by each of these mutants was at least 20-fold, and no activity was observed at the highest concentrations of ARF used in this assay (16 µM). The Y81C mutant was found to be completely inactive
when assayed as the nonacylated protein but had up to 30% control
levels of PLD stimulating activity after N-myristoylation.
That interactions with at least one other partner in two-hybrid assays
and the ability to bind guanine nucleotides were conserved in each case
is evidence for a level of specificity in the loss of PLD1-stimulating
activity. We interpret these results as a reflection of the lowering of the Ka from N-myristoylation and a
more dramatic increase in Ka resulting from the
mutation. It is possible that each of these residues makes direct
contact with PLD1 in the activated heterodimer, and the mutations
disrupt the high affinity binding. For example, each of these residues
may be involved in hydrophobic interactions of the kind described by
Goldberg (22) between switch I of ARF1 and the Sec7 exchange factor
domain. If so, there must be specificity to this hydrophobic pocket
being involved with PLD1 as the binding to other effectors,
e.g. POR1 or LTA1 was not lost by the mutation of either Ile-49 or Phe-51.
The presence of ARF on purified COP-I-coated vesicles and the in
vitro assay of the recruitment of coatomer to Golgi membranes in
an ARF-dependent manner are central to one model for ARF in vesicular traffic. The COP-I recruitment assay is thought to monitor the critical nucleating step in bud formation. Other data support this
model, e.g. electron microscopy data that correlate COP-I recruitment and bud emergence (7, 8) or genetic interactions between
ARF and components of secretory machinery (2), but there are also a
number of observations that appear inconsistent with the model. Most
notable in this regard are the apparent lack of requirement for ARF
(43-45) in any of the in vitro assays of membrane transport
in which ARF was shown to be responsible for the inhibition of
transport in the presence of GTPS (46-49) and the ability to form
apparently normal COP-I-coated vesicles in the absence of ARF (9).
When each of the switch mutants was assayed for the ability to recruit
coatomer to Golgi-enriched membranes, several were found to have lower
specific activities than the control, [Q71L]ARF3. The switch II
mutants I74T and each of the Y81 mutants appeared devoid of recruitment
activity in our assay. Like the activation of PLD1, we assume that
binding to COP-I involves multiple residues on the surface of each
protein, so the loss of any one results only in a decreased affinity,
but in this case it was decreased to the point that our assay could no
longer detect the interaction. That the most dramatic effects on COP-I
recruitment were found in switch II is surprising in that the results
reported by Goldberg (23) implicated switch I as the site of COP-I
binding. The binding of ARF GAP to switch II was shown by
crystallographic studies, and the enhancement of ARF GAP activity by
added COP-I was interpreted as evidence for its binding to switch I. Our findings that switch I mutants have decreased COP-I recruitment and
some switch II mutants are even more affected in this activity are not
necessarily at odds with the conclusions of Goldberg. It is possible
that COP-I is recruited to membranes by ARFs as a consequence of the binding to both switch regions but that the binding of COP-I to only
switch I may be sufficient to activate ARF GAP activity. It need not
even be the same domain or protein in the heptameric COP-I complex
which is responsible for these two activities. This speculation, and
our results implicating both switch regions in COP-I binding, are
consistent with the finding of cross-linking between switch I residues
(Ile-46 and Ile-49) to -COP (49) and a switch II residue (Phe-82) to
-COP (51).
All three of the mutants that lost the ability to recruit coatomer in
our assay (I74T, Y81H, and Y81C) had normal, or very nearly so, ability
to activate PLD1, and both of the mutants that lost detectable PLD1
activation (I49T and F51Y) retained the ability, though diminished, to
recruit coatomer. Comparison of other mutants in these two assays
reveals a general lack of correlation. These results are inconsistent
with the model for ARF action at Golgi in which the recruitment of
COP-I is dependent on the activation of PLD1 (15). This conclusion is
consistent with the data in Stamnes et al. (16) in which
there was poor correlation between the production of phosphatidic acid
(the product of PLD) and coatomer recruitment.
Engorgement of Golgi elements and their transformation into large
vesicles, diverse in size, with concomitant enlargement of the ER lumen
are phenotypes associated with the expression of the dominant
activating mutant, Q71L, of ARF1 (17), ARF3, or ARF4.4
These changes in morphology were described originally in NRK cells
stably transformed with ARF alleles under control of an inducible
promoter and analyzed using electron microscopy. We have adapted these
results into an assay that can be carried out over the course of a few
hours by microinjecting cells with plasmids directing the expression of
the ARFs and analyzing the morphology of the Golgi using specific
markers of that compartment, e.g. mannosidase II for the
lumen or -COP for the periphery. Although the mechanism by which
activated ARFs can cause the Golgi to change in this way is not clear,
it is clearly the result of increased ARF activity and not simply an
artifact from overexpression of the protein (17). The morphological
changes accompanying expression of [Q71L]ARF3 include a large
expansion in the area of mannosidase II staining around the nucleus
(see Fig. 5, panels D, F, J,
L, and N) and a more diffuse staining in the
cytosol which is apparent by microscopy but not readily captured in
pictures, such as those in Fig. 5.
Results from the cell-based Golgi expansion assay reveal a lack of
correlation among the assays for ARF function employed in this study.
When the mutant ARFs were tested for the ability to vesiculate Golgi,
three different responses were observed: the control [Q71L]ARF3 and
two of the switch domain mutants, I49T and R79G, had equivalent
activities, some (I74T and Y81C) had decreased activity, and some (wild
type ARF3 or F51Y) had no activity. The loss of activity of the F51Y
mutant in this assay contrasts with its ability to bind GTPS and
retention of, though diminished, COP-I recruitment. Although this
mutant has also lost the ability to stimulate PLD1 activity, a further
comparison of activities of the I49T mutant, in which PLD1 activation
is lost but Golgi expansion is retained, led us to conclude that
neither PLD1 stimulation nor coatomer recruitment is required for Golgi
expansion in NRK cells. Because partial activity in a cell-based assay
is difficult to interpret we focused our discussion on the F51Y mutant,
but further comparisons of other mutants in the different assays
revealed an overall poor correlation, consistent with some level of
independence in mechanisms.
Finally, we address the possibility that two effectors, working
together, may result in vesiculation of Golgi in the presence of
[Q71L]ARF. We know of at least six different proteins, or complexes, that localize to the Golgi in an ARF-sensitive manner: PLD1, COP-I, AP-1, and three GGA proteins. We recently observed that the
overexpression of GGA1 actually prevents the vesiculation that results
from expression of [Q71L]ARF1, eliminating this group of proteins.
Although we did not find a second site mutation in [Q71L]ARF3 which
eliminated both PLD1 activation and COP-I recruitment with retention of
Golgi vesiculation, we conclude that the magnitude of the loss of
affinity of mutants for each of these effectors was sufficient to rule out the possibility that PLD1 activation and COP-I recruitment work
cooperatively to nucleate vesicle budding and formation, leading to
vesiculation in the presence of [Q71L]ARF3.
Recent results have turned the focus of COP-I action toward earlier
steps in the secretory pathway, e.g. actions at the
intermediate compartment (52) and its function primarily in retrograde
transport (53). It thus seems more likely that if COP-I is involved in defects in the secretory pathway, resulting from expression of an
activated ARF, it would be acting at an earlier step, e.g. expansion of the ER lumen, and not the changes in Golgi morphology. This leaves only AP-1, either acting alone or in concert with another
effector, to explain the changes to Golgi morphology. A role for ARF in
recruitment of AP-1 to the trans-Golgi network was first suggested by
Stamnes and Rothman (11) and characterized in detail by Traub and
colleagues (54). The recruitment of AP-1 by ARF to Golgi membranes
appears to be indirect as it occurs after GTP hydrolysis on ARF is
completed (55). Further, we found no evidence of AP-1 accumulation on
vesiculated Golgi of NRK cells expressing [Q71L]ARF1.4
Although we cannot rigorously exclude the possibility that some combination of these previously identified effectors is sufficient to
vesiculate Golgi membranes when ARF activity is in excess, we favor the
simpler explanation that there exists at least one more effector, not
yet identified, which is responsible for this activity.
Definitive conclusions regarding cellular mechanisms are difficult
because of the problems inherent in translating from reconstitution assays in vitro to functions in live cells. How much
residual PLD1 activity is enough to serve its role? How much COP-I
recruitment activity will suffice in a cell? By comparing effects of
specific mutations we have been able to uncouple clearly each of the
three different ARF-dependent activities: stimulation of
PLD1, COP-I recruitment, and Golgi vesiculation. We conclude that (i)
activation of PLD1 is not a required step in the recruitment of
coatomer; (ii) activation of PLD1 is not required for vesiculation of
Golgi in cells; and (iii) recruitment of COP-I by ARF is not required for vesiculation of Golgi. We further conclude that there must exist an
effector for ARF action at the Golgi, responsible in whole or part for
the vesiculation in the presence of excess activated ARF. Further
refinement of specific loss-of-interaction mutants will likely be the
best hope for further dissection of the complex signaling paths that
seem to converge at or near the Golgi but can impinge upon a diverse
array of cellular activities.
,
TOP
ABSTRACT
INTRODUCTION
Present address: Dept. of Biochemistry and Molecular Biology,
University of Minnesota Duluth, 10 University Dr., Duluth, MN 55812.
