From the
During the 1980s, several cellular factors that enhanced cholera
toxin-catalyzed ADP-ribosylation of G
Because of limitations of
space, this Minireview deals only with the functions of ARF in
eukaryotic cells, where these proteins have a critical role in
vesicular membrane trafficking in both exocytic and endocytic pathways,
and the relationship of ARF structure to its biochemical actions.
Recent, more complete reviews (e.g. Ref. 4) consider also ARF
cDNA and genomic sequences, as well as data on distribution and
expression of ARF proteins and mRNAs.
ARFs are a multigene family, structurally related to ARF-like
proteins (ARLs) that do not activate cholera
toxin
(4, 5) . Based on size, amino acid sequences
(deduced from cDNA sequences), phylogenetic analysis, and gene
structure
(4) , mammalian ARFs (Fig. 1)
Other mutants expressed in HeLa cells
(10) were designed either to exist chiefly in the GDP-bound
state, ARF1(T31N), or to have an altered rate of nucleotide exchange
due to replacements in the third nucleotide-binding sequence,
ARF1(N126I) or ARF1(D129N). It was suggested that ARF1(T31N), which
preferentially binds GDP, prevents activation of endogenous ARF by
interacting with an ARF-GEP (guanine nucleotide-exchange protein),
effectively inactivating it
(10) . Myristoylation was again
required for this effect
(10) . Mutations in the third
nucleotide-binding sequence, which also result in preferential GDP
binding, similarly produced proteins with dominant negative activity,
although the effects on cell morphology differed from those of the T31N
mutant
(10) .
Myristoylation is clearly important for ARF
function, although non-myristoylated ARF proteins can be active in
several in vitro assays. Recently, improved conditions for
synthesis of myristoylated recombinant ARF1 in E. coli by
co-expression of yeast N-myristoyltransferase and its
purification to yield relatively homogeneous myr-rARF1 facilitated
evaluation of effects of myristoylation
(14) . Comparison of
rARF1 (non-myristoylated) and myr-rARF1 revealed that the modification
markedly affects guanine nucleotide exchange. It was concluded that
myr-rARF1-GDP associated with phospholipid more weakly than did
myr-rARF1-GTP because it interacted only via myristate, whereas
myr-rARF1-GTP interacted through both the fatty acid and the N-terminal
amphipathic helix
(14) .
To extend these studies, a peptide with the
sequence of ARF1 amino acids 2-17 was prepared
(15) . With
or without myristoylation, it inhibited ARF activation of cholera toxin
and Golgi transport
(15, 16, 17) as well as
endocytosis
(18) . It appears, however, that the effects of this
peptide may not be as specific as initially believed. The peptide and
mastoparan, which also forms an amphipathic helix, inhibited
phospholipase D and exocytosis, as well as a G protein-regulated
phospholipase C
Other mutations of the ARF1 N terminus yielded somewhat
different findings. Recombinant ARF1 lacking the first 13 amino acids
(r
It is
unclear just how GTP bound to recombinant ARF is hydrolyzed in E.
coli, as ARFs lack intrinsic GTPase activity
(23) and the
bacteria, which lack ARFs, presumably also lack ARF-GAP
(GTP-ase-activating protein). Perhaps GTP bound to the mutant ARF is
protected from bacterial enzymes that could otherwise degrade it. This
explanation was considered after the earlier isolation of a recombinant
ARF6 fusion protein in an active state with GTP bound
(24) . The
stability of the N-terminal deletion mutants to 7 M urea,
which partially inactivated rARF1, was consistent with a role for that
part of the molecule in overall structure or conformation
(21) .
Further study of r
Recently, Randazzo et al.(25) compared activities of r
Further comparisons of mutant and native proteins in specific assays
of individual interactions are needed to define more precisely the
effects of ARF molecular structure on function. Availability of the
recently published crystal structure of non-myristoylated ARF1-GDP
(13) should facilitate the design and interpretation of these
kinds of studies.
It was in S. cerevisiae that ARF was first
implicated in Golgi function
(9) , and a large part of the
information on ARF function in cells is related to vesicular transport
in the Golgi
(29) , probably involving class I ARFs. ARF has been
implicated also in intravesicular acidification and fusion of
microsomal vesicles (30), endosome fusion
(18) , nuclear membrane
assembly
(31) , and formation of clathrin-coated
vesicles
(32) . In vesicular transport from endoplasmic reticulum
to Golgi, ARFs are required for binding of
Although ARF
The mechanism by which ARF
The demonstration that
coatomer
(44) , like analogous protein components of
clathrin-coated vesicles
(45) , can function as a K
ARF
activation of phospholipase D
(26, 27) provided the
basis for a new way of thinking about the function of ARF in vesicular
transport. Phospholipase D, of which there are several isoforms in
animal tissues, catalyzes the hydrolysis of phosphatidylcholine to
phosphatidic acid and choline. It also catalyzes a
transphosphatidylation reaction, e.g. with ethanol replacing
water to yield phosphatidylethanol instead of phosphatidic acid.
Stutchfield and Cockcroft
(49) had postulated a role for
phosphatidic acid in receptor-stimulated secretion based on inhibition
of that process by ethanol. Inhibition of intra-Golgi vesicular
transport by ethanol has also been reported
(50) , consistent
with the notion that phosphatidic acid has an important role in these
processes, whether through action in a signaling pathway or via its
well documented effects as a fusogenic lipid in model membranes.
Proteins required for GTP
Vesicular
transport (Fig. 2) appears to involve two seemingly very
different actions of ARF. First, ARF
It seems
not unlikely that the two different postulated functions of ARF in
vesicular transport, i.e. the initiation of vesicle formation
via coatomer binding and the initiation or facilitation of vesicle
fusion, are properties of two different parts of the ARF structure.
This is consistent with the recent demonstration of different
structural requirements for ARF activation of cholera toxin and
phospholipase D but remains to be established.
The contributions of cholera toxin to biomedical research (as
well as to disease) are well established. In addition to its use for
labeling and perturbing the activity of the G
We thank Dr. Vincent C. Manganiello for critical
review of this manuscript, Barbara Mihalko for expert secretarial
assistance, and the many past members of the Laboratory of Cellular
Metabolism as well as present members of the Pulmonary-Critical Care
Medicine Branch, including especially Dr. Su-Chen Tsai, who are
responsible for our ARF studies.
INTRODUCTION
ARF Structure
ARF Function in Vesicular Transport
Afterword
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
or activation of
adenylyl cyclase were described. Kahn and Gilman
(1) reported
the first purification of an
20-kDa membrane-associated protein
that enhanced ADP-ribosylation of G
and named it
ADP-ribosylation factor or ARF.
(
)
Two soluble
ARFs purified later stimulated toxin-catalyzed ADP-ribosylation of
G
and simple guanidino compounds (e.g. arginine) as well as toxin auto-ADP-ribosylation in a
GTP-dependent manner
(2) . It was shown in kinetic studies that
ARF interacted with the toxin catalytic subunit to lower the apparent
K
for both ADP-ribose donor (NAD) and
acceptor (agmatine)
(3) . Thus, ARF seemed to be an allosteric
activator of the toxin, active with GTP bound, and inactive with bound
GDP. Whether ARF has an effect on cholera toxin activity in intoxicated
cells remains unknown, but its more recently recognized physiological
actions are clearly quite different.
(
)
fall into three classes, ARF1, ARF2, and ARF3 in class I,
ARF4 and ARF5 in class II, and ARF6 in class III
(6) . In
vitro, mammalian ARFs from all three classes and ARFs from all
eukaryotic organisms activated cholera toxin, which has been, by
definition, a requirement for the designation ``ARF.'' Class
I ARFs are the best known and most closely resemble ARFs identified in
other species. Relatively recently, however, evidence for the presence
of class II and III ARFs in Drosophila melanogaster(7) and a possible class III ARF in Saccharomyces
cerevisiae(8) has been published.
Figure 1:
Deduced amino acid
sequences of human ARL1 and mammalian ARFs. Sources of ARF sequences
are in Ref. 4; human ARL1 (hARL1) sequence is in Ref. 28. The
amino acid number is above the hARL1 sequence. Amino acids
identical in hARL1 and hARF1 are indicated with a verticalline; those amino acids in other ARFs identical with
hARF1 are indicated with an asterisk. Four consensus sequences
(I-IV) believed to be involved in guanine nucleotide
binding and hydrolysis are shaded yellow; G2, the site of
N-terminal myristoylation after co-translational removal of methionine,
is shaded blue.
All ARFs contain
consensus amino acid sequences (Fig. 1) involved in GTP binding
and hydrolysis, critical functions that determine ARF activity. These
are identical in the mammalian ARFs and have very few, for the most
part conservative, differences in yeast or Giardia ARFs
(4) . It has often been noted that in these
``signature'' sequences, as well as in the myristoylation of
an N-terminal glycine, the ARFs are more similar to heterotrimeric G
protein subunits than they are to other 20-kDa Ras-like proteins
(9). It has also been suggested that ARF may be an ancestor of both of
those protein families, as it is present in the primitive parasite
Giardia lamblia that apparently lacks G
subunits
(4) .
Mutations in GTP-binding Sequences
Mutations in several
of the consensus sequences for GTP binding and hydrolysis have been
introduced to evaluate their effects on ARF functions. Replacement of
Gln-71 with Leu produces a mutant ARF1(Q71L) that lacks the ability to
hydrolyze GTP and is thus permanently active. Overexpression of
ARF1(Q71L) in HeLa
(10) or normal rat kidney
(11) cells
inhibited endoplasmic reticulum to Golgi and intra-Golgi transport,
respectively. In normal rat kidney cells, it also inhibited
endocytosis
(11) . Inhibition by the activated ARF mutant
presumably resulted from accumulation of vesicles unable to fuse with
target membranes, because the failure to inactivate ARF prevented
uncoating (Fig. 2). This was shown in vitro using Golgi
membranes and ARF1(Q71L) synthesized in Escherichia coli, as
well as in COS-1 cells expressing the mutant
(12) .
Myristoylation, believed necessary for membrane association, was
required for inhibition by the mutant; expression of a double mutant
that could not be myristoylated (ARF1(G2A,Q71L)) was without
effect
(10, 12) .
Figure 2:
Role of ARF in vesicular transport. This
simplified scheme is discussed, with references, in the text. Because
of space limitation, we have not considered the so-called SNARE
complexes (35), although it seems widely accepted that this sort of
molecular machinery, including the N-ethylmaleimide-sensitive
ATPase, functions in many types of vesicular transport systems.
Likewise omitted is discussion of the other 20-kDa GTP-binding
proteins (e.g. Rabs) and heterotrimeric G proteins (46), which
are almost surely involved in specific transport processes, as are
cytoskeletal elements (e.g. microtubules) and the obviously
necessary processes of retrograde transport for membrane retrieval.
PLD, phospholipase D; PC, phosphatidylcholine;
PIP, phosphatidylinositol 4-phosphate; PIPK,
phosphatidylinositol 4-phosphate 5-kinase; PA, phosphatidic
acid.
The mutant ARF1(Q71L) was prepared
because replacement of the apparently analogous Gln (in the sequence
DXXGQ) in Ras and G subunits yielded
constitutively activated proteins. Based on the crystal structure of
ARF1-GDP
(13) , however, Gln-71 does not correspond in a
three-dimensional position to Gln-61 of Ras, with which it is aligned
in the linear sequence. ARF-GDP, which crystallized as a dimer, differs
in this and several striking ways from the other
structures
(13) . It is clear that extrapolation of
structure-function relationships from Ras, for example, to ARF should
be approached with caution.
N-terminal Mutations
Sequence differences among
ARFs tend to be concentrated near the N terminus, and when sequences of
the six mammalian ARFs are aligned, ARF6 has a gap corresponding to
amino acids 8-11 in the other ARFs (Fig. 1). It has been
reported, however, that the ARF N terminus is critical for its activity
in vitro and probably in vivo(15) . A
recombinant myristoylated ARF1 mutant lacking the first 17 amino acids
failed to activate cholera toxin-catalyzed ADP-ribosylation of
G or to rescue the yeast
arf1
arf2
lethal double mutant;
it also differed from the native protein in guanine nucleotide-exchange
properties
(15) . A chimeric protein containing the first 18
amino acids of human ARF1 and amino acids 19-180 of
Drosophila ARL1 (ARF18/ARL) was reported to stimulate cholera
toxin-catalyzed modification of G
(15) . ARL1,
first identified in Drosophila by cDNA cloning, is an
essential protein of unknown function, very similar in structure to ARF
(Fig. 1) but possessing measurable GTPase activity and lacking
ARF activity
(5) .
(19) , and demonstrably damaged Golgi
membranes
(20) . Thus, conclusions about the function of the ARF
N terminus, based on experiments with the peptide, appear to be subject
to question.
13ARF1) and rPKA14ARF1 (in which the first 14 amino acids were
replaced by the first 7 amino acids of cAMP-dependent protein kinase
catalytic subunit) activated cholera toxin-catalyzed ADP-ribosylation
of agmatine as effectively as did rARF1
(21) . Unlike rARF1,
however, their activity was not dependent on added GTP; bound GDP and
GTP were identified, thus explaining the activity
(22) . After
prolonged dialysis against 7 M urea and renaturation, GTP
dependence was demonstrated if SDS was present in the
assay
(21) . In the absence of detergent, the nucleotide-free
r
13ARF1 was active, i.e. it appeared to assume an active
conformation in the absence of bound nucleotide
(22) .
13ARF1 provided evidence for an additional
effect of the N terminus on specificity of nucleotide
binding
(22) .
13ARF1 and r
17ARF1 in the
cholera toxin assay with G
as substrate and in their
responses to ARF-GAP, a partially purified protein
(23) that
accelerates hydrolysis of ARF-bound GTP. r
13ARF1, like
r
17ARF1, failed to activate the toxin in this assay, although it
did, in contrast to r
17ARF1, inhibit activation by rARF1. The
authors concluded that amino acids 14-17 are involved in binding
of ARF1 to the toxin and play a small but significant role in the
interaction of ARF1 with its GAP. They also reported that >80% of
the nucleotide bound to both mutant proteins was GTP; a marked effect
of the N terminus on GDP dissociation was noted
(25) .
Structural Requirements for Activation of Cholera Toxin
and Phospholipase D
After reports of ARF activation of
phospholipase D
(26, 27) , structural requirements for
activation of toxin and phospholipase D were compared using chimeric
proteins based on deduced amino acid sequences of ARF1 and ARL1
(Fig. 1). Recombinant proteins, in which the first 73 amino acids
of ARF1 and ARL1 were exchanged (rF73/L and rL73/F), were compared in
assays with cholera toxin and phospholipase D under conditions that
differed only in the identity of the radiolabel added
(28) . ARF1
activated both enzymes; ARL1 did not activate toxin and only slightly
stimulated phospholipase D. rF73/L, in which the N-terminal 73 amino
acids of ARF replaced those of ARL, failed to activate the toxin,
although it activated phospholipase D as effectively as rARF1.
Conversely, rL73/F activated phospholipase D only slightly but was much
more effective with cholera toxin. Thus, activation domains for
phospholipase D and toxin appear to lie, respectively, in the N- and
C-terminal portions of ARF. Effects of GTPS concentration on toxin
activation by rL73/F and rARF1 with different phospholipids were quite
different, perhaps related to the role of the N terminus in nucleotide
exchange and binding or in association with phospholipids
(28) .
-COP, a component of
the ``coatomers''
(33) that cover non-clathrin-coated
vesicles
(32) . In the formation of Golgi-derived clathrin-coated
vesicles, ARF is required for binding of the AP-1 adaptor protein
(
-adaptin), an analogue of
-COP
(32) . The report of
COP II-coated vesicles, in which ARF is replaced by Sar1
(34) ,
adds to the evidence that multiple pathways of intracellular vesicular
transport utilize analogous protein molecules and mechanisms. This is
emphasized in an excellent review by Rothman
(35) , whose
contributions, experimental and conceptual, to understanding these
processes have been major. We rely heavily on that, and several other
key articles from the Rothman laboratory, for the current view of
intracellular vesicular transport, which is summarized in a simplified
fashion in Fig. 2.
Vesicle Formation
Soluble ARF proteins are isolated with
tightly bound GDP that exchanges relatively slowly with GTP, even in
the presence of phospholipids or detergents that accelerate the process
when concentrations of Mg are submicromolar. ARF
binding to membranes is enhanced when bound GDP is exchanged for GTP.
In several systems, ARF binding to membranes is prevented by brefeldin
A, a fatty acid derivative that in intact cells causes apparent
disintegration of the Golgi (reversibly) and association of Golgi
enzymes with endoplasmic reticulum (36). Brefeldin A inhibition of a
Golgi-associated activity (37-39), termed here GEP for
guanine nucleotide-exchangeprotein, that
accelerates replacement of ARF-bound GDP with GTP has been reported.
GEP activity in brain cytosol
(40) , which was also inhibited by
brefeldin A, stimulated binding of ARF1 and ARF3, but not ARF5, to
crude Golgi membranes. It also increased binding of GTP
S to ARF1
and ARF3. With partial purification of this soluble GEP, its apparent
molecular size decreased from >600 to
60 kDa and susceptibility
to inhibition by brefeldin A was lost
(41) , consistent with the
conclusion that some component of the larger complex confers
sensitivity to brefeldin A.
GTP (but not
ARF
GDP) will bind to pure phospholipids as well as to cell
membranes, its physiological function presumably requires interaction
with a particular site at which vesicle formation will take place.
Whether this site involves an ARF ``receptor'' protein or is
defined in some other way has not been established. Interaction with a
specific Golgi protein was suggested after demonstration of a
saturable, liposome-resistant pool of bound ARF, in addition to a pool
of liposome-extractable ARF
(42) . A recent report of ARF binding
to G
(43) is consistent with a role for the
latter as a membrane receptor. The existence of ARF receptor proteins
could provide for specificity of binding of individual ARF proteins to
the ``correct'' sites for initiation of vesicle formation.
GTP bound to its putative receptor
causes binding of large protein complexes termed coatomers
(33) ,
whether by interacting with a coatomer protein or by an effect on
composition of the adjacent membrane, is unknown. ARF and coatomer
were, however, the only soluble components necessary for bud formation
on Golgi membranes (35); membrane bilayer fusion to close off the base
of the bud required only the addition of fatty acyl-CoA (Fig. 2).
The resulting vesicles were then capable of fusing with acceptor Golgi
in the presence of cytosol (to provide components of the fusion
machinery), ATP, and an ATP-regenerating system
(35) . Fusion did
not occur when vesicles had been prepared with GTP
S or with a
mutant ARF that does not hydrolyze GTP
(35) , consistent with an
earlier conclusion that uncoating of the vesicle, following conversion
of ARF
GTP to ARF
GDP and its release from the membrane, is a
prerequisite for fusion.
channel (whatever its relationship to ARF) is consistent with the
notion that this activity has a physiological role in vesicle
formation. Together with the observation that the channel activity can
be blocked specifically by certain inositol
polyphosphates
(44, 45) , this suggests a possible
mechanism for the inhibition of vesicular transport by fluoride plus
AlCl
. This effect has often been interpreted as evidence of
the participation of an AlF
-activated
heterotrimeric G protein in the transport process, which may, in fact,
be correct
(46) . The effect of fluoride could, however, result
from its demonstrated inhibition of the turnover of inositol
polyphosphates with consequent accumulation of pyrophosphate
derivatives of inositol pentakisphosphate and inositol
hexakisphosphate, as suggested by Menniti et al.(47) .
Inhibition of conversion of the pyrophosphates back to the parent
inositol polyphosphates, respectively, permitted recognition of the
rapid cycling of these parent compounds, previously judged to be rather
inert metabolically (47). Fluoride, by causing accumulation of inositol
polyphosphate pyrophosphates, which at submicromolar concentrations can
inhibit coatomer channel activity, could interfere with bud and/or
vesicle formation and thus inhibit vesicular transport, if operation of
the ion channel is involved in these processes
(44) .
Vesicle Fusion
Fusion with the target membrane
requires vesicle uncoating, which means release of coatomers, after
release of ARFGDP (Fig. 2). Other
20-kDa GTP-binding
proteins that have low GTPase activity interact with specific GAPs that
accelerate hydrolysis of the protein-bound GTP. ARF apparently lacks
GTPase activity
(23) , and it is presumed that conversion of
ARF
GTP to ARF
GDP occurs when ARF interacts with a GAP in
the target membrane. Description of a long sought ARF-GAP, present in
both soluble and particulate fractions of brain homogenate, was
published during the past year
(23) , and the complete
purification was reported more recently
(48) .
Phosphatidylinositol 4,5-bisphosphate (PIP
)
appeared to be a specific activator, and phosphatidic acid markedly
enhanced sensitivity of the impure GAP to PIP
.
S stimulation of membrane phospholipase D
activity were purified from brain cytosol and identified as ARF1 and/or
ARF3
(26, 27) . Brown et al.(26) partially purified ARF-stimulated phospholipase D from
HL-60 cell membranes and described the critical role for PIP
in its stimulation by ARF. Subsequently, ARF-stimulated
phospholipase D was separated from oleate-stimulated phospholipase D
after solubilization from brain membranes, and its activation by class
I, II, and III mammalian ARFs was demonstrated (51).
GTP, after activation by its
GEP, associates with the membrane, which is a prerequisite for binding
of coatomer and subsequent vesicle budding. Then, the action of ARF on
phospholipase D presumably takes place at the target membrane with
which the vesicle will fuse, although activation of phospholipase D
might occur at the donor membranes and be involved in vesicle release.
Based on the abilities of PIP
to activate phospholipase D
and of phosphatidic acid to activate phosphatidylinositol 4-phosphate
5-kinase, Liscovitch et al.(52) suggested that these
components could operate in a positive feedback loop to promote fusion
of vesicle and target membranes. Their hypothesis, shown partially in
Fig. 2
, is attractive in many ways. It includes a role for
ARF
GTP to initiate the loop by activating phospholipase D,
resulting in changes in membrane lipid composition with increased
phosphatidic acid that facilitates interaction of ARF
GTP with GAP
and activates phosphatidylinositol 4-phosphate 5-kinase in the target
membrane. With the generation and release of ARF
GDP followed by
coatomer, increased vesicle PIP
and phosphatidic acid could
also promote fusion with the target membrane. Based on experiments with
model membranes, it had earlier been suggested that fusion might be
regulated by ``local enzymatic generation of fusogenic lipids such
as diglycerides or phosphatidic acid''
(53) .
component of the adenylyl cyclase system, it led to the
recognition of numerous other bacterial ADP-ribosyltransferase toxins
that, for reasons still not clear, tend to use GTP-binding proteins as
substrates and sparked the discovery of analogous enzymes in animal
cells
(54) . Cholera toxin further led to the discovery of ARF,
another GTP-binding protein that can also be a substrate for the toxin.
The physiological function of ARF was a puzzle until the demonstration
that it is an essential protein in yeast
(9) . Since that time,
information about the structure and function of multiple ARFs has been
accumulated at an accelerating pace. We have tried to summarize some
parts of it and, we hope, to convey a sense of the many questions that
remain to be answered.
, phosphatidylinositol 4,5-bis-phosphate;
GTP
S, guanosine 5`-O-(thiotriphosphate).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.