(Received for publication, July 21, 1994; and in revised form, October 31, 1994)
From the
Wild type and eight point mutants of Saccharomyces
cerevisiae ARF1 were expressed in yeast and bacteria to determine
the roles of specific residues in in vivo and in vitro activities. Mutations at either Gly or Asp
resulted in recessive loss of function. It was concluded that N-myristoylation is required for Arf action in cells but not
for either nucleotide exchange or cofactor activities in
vitro. Asp
(homologous to Gly
of
p21
) was essential for the binding of the
activating nucleotide, guanosine 5`-3-O-(thio)triphosphate.
This is in marked contrast to results obtained after mutagenesis of the
homologous residue in p21
or G
,
and suggests a fundamental difference in the guanine nucleotide binding
site of Arf with respect to these other GTP-binding proteins.
Two
dominant alleles were also identified, one activating dominant
([Q71L]Arf1) and the other ([N126I]) a negative
dominant. A conditional allele, [W66R]Arf1, was characterized
and shown to have 300-fold lower specific activity in an in
vitro Arf assay. Two high-copy suppressors of this conditional
phenotype were cloned and sequenced. One of these suppressors, SFS4, was found to be identical to PBS2/HOG4,
recently shown to encode a microtubule-associated protein kinase kinase
in yeast.
ADP-ribosylation factors (Arf) ()are 21-kDa
GTP-binding proteins, originally identified as the protein cofactor
required for efficient ADP-ribosylation of the stimulatory, regulatory
component of adenylate cyclase, G
, by cholera toxin ((1) ; for recent reviews, see (2) and (3) ).
This cofactor activity can be made linearly related to the formation of
the activated (GTP-bound) species and used as a quantitative assay of
Arf activity (1) . Arf proteins are ubiquitous in eukaryotes
and both structure and function have been very highly conserved
throughout eukaryotic evolution ((4) ; human and Xenopus
laevis, Schizosaccharomyces pombe, and Saccharomyces cerevisiae Arf1 are 99, 90, and 74% identical, respectively). ARF1 and ARF3
(96% identical) are usually the most abundantly expressed members of
the Arf family in mammalian cells and tissues.
A number of functions
have been ascribed to Arf proteins, particularly as components of
intracellular membrane traffic (for review, see (3) ). Since
its original purification, based on its activity as a cofactor for
cholera toxin(5) , Arf proteins have also been purified based
on their ability to inhibit intra-Golgi transport (6) and
nuclear vesicle fusion ()in the presence of GTP
S. In
addition, Arf was purified independently in two different laboratories
as the soluble factor required for the stimulation of phospholipase D
activity by GTP
S(7, 8) . Arf proteins have also
been identified as components of nonclathin-coated
vesicles(9) , on which much speculation regarding cellular
functions have focused.
One of the first indications that Arf proteins act in one or more steps of the protein secretory pathway came from studies in S. cerevisiae(10, 11) which have two ARF genes, ARF1 and ARF2. Deletion of both genes is lethal while deletion of ARF2 is without any defined effect. Disruption of ARF1 (arf1::URA3) was found to slow the rate of cell growth, cause a defect in the processing of invertase, and make cells supersensitive to fluoride ions(11) . Each of these phenotypes could be complemented by transformation with a centromere-containing plasmid carrying wild type ARF1 or ARF2. Thus, yeast offers an attractive system for genetic studies of the role(s) for Arf protein in eukaryotes as phenotypes have already been described, which result from mutation of ARF1.
Mutational studies, using both yeast and mammalian
proteins, have been used to define the role of specific residues in the
guanine nucleotide binding sites of a number of GTP-binding proteins,
including p21, G
, SEC4, YPT/RAB, and others. Some recent reports (12, 13, 14) have utilized homologous
mutations to study functions of mutant Arf proteins in mammalian cells.
However, none of these previous reports characterized the biochemical
consequences of these mutations but instead relied upon the assumed
conservation of consequences between Arf and Ras or other GTP-binding
proteins. With the limited extent of sequence identity between ARF and
either the other members of the Ras superfamily or
subunits of
the heterotrimeric G proteins (
20-25% identity), it is risky
to assume that homologous mutations in one family will have the same
consequences on nucleotide handling or protein interactions in a member
of another family. Indeed, the solution of the crystal structure of
human ARF1, along with some of the data presented below, provide direct
demonstrations of the fact that the nucleotide binding site of Ras and
Arf have distinctive features.
The availability of a quantitative biochemical assay of Arf activity has proven of great value in the characterization of the protein, in spite of the unclear relevance of ADP-ribosylation to the in vivo action of Arf proteins in cells. Characterization of phenotypes of ARF mutants in yeast allowed the integration of biochemical information with functional characterization in cells.
Figure 1: Sites and cloning strategies for construction of plasmids directing the expression in yeast and bacteria of wild type and mutants of ARF1. The coding region of S. cerevisiae ARF1 is shown with the restrictions sites which were added on each end to facilitate subcloning into the different expression vectors. Details of the construction of mutants and subcloning are described under ``Materials and Methods.'' The different expression systems are indicated below, along with the promoters used in each and the sites used to clone into the parent plasmids.
Overexpression of ARF1 was previously shown to be harmful
or lethal to yeast cells(11) . This deleterious effect on
vegetative growth can be seen when overexpression is achieved either by
maintenance of an ARF1 gene on a high-copy, 2µ plasmid or
by expression from the stronger, GAL1 promoter. The
pGAL1-Arf-X plasmids were transformed into both a wild type
strain, PSY315, and an arf1 strain, TT104
(isogenic to PSY315 in all other respects), and plated on selective
medium using glucose as carbon source. Transformants were
replica-plated to selective plates, YPD, and YPGal plates and growth
was scored on days 2-4.
Sensitivity to fluoride was tested by
plating transformants on selective medium, YPD, and YPD plates
containing 40 mM sodium fluoride (YPD/F) and
scoring for growth after 2-4 days. YPD/F
plates were
used within 1 week after pouring as they become toxic to all yeast
strains after this time. The reason for this is not understood,
although it may involve chelation of one or more metals by these high
concentrations of fluoride ions.
Stoichiometry of GTPS
binding was determined by trapping bound nucleotide on BA85
nitrocellulose filters after 20 min at 30 °C in a binding reaction
that included 1 µM Arf, 25 mM HEPES, pH 7.4, 100
mM NaCl, 0.5 mM MgCl
, 1 mM EDTA,
1 mM dithiotheitol, 3 mM dimyristoyl-L-
-phosphatidylcholine, 0.1% sodium
cholate, and 10 µM [
S]GTP
S
(
10,000 cpm/pmol).
Dissociation rates of GDP were determined
after loading protein with [H]GDP. Arf1 (5
µM) was incubated with 5 µM [
H]GDP (
20,000 cpm/pmol) for 1 h at 30
°C in a reaction containing 25 mM HEPES, pH 7.4, 100
mM NaCl, 0.5 mM MgCl
, 1 mM EDTA,
3 mM dimyristoyl-L-
-phosphatidylcholine, and
0.1% sodium cholate. Protein was diluted 10-fold into the same buffer
but containing 0.5 mM unlabeled GDP and 50 µg/ml bovine
serum albumin. Duplicate 10-µl samples were taken at 10 time points
between 0 and 20 min by dilution into 2 ml of ice-cold 20 mM Tris-Cl, pH 8.0, 100 mM NaCl, 10 MgCl
, 1
mM dithiotheitol. Protein bound [
H]GDP
was determined after trapping on nitrocellulose as described previously (21) . Dissociation rates were calculated using the UltraFit
program (Biosoft, Cambridge, United Kingdom). Curves were well fitted
to equations for single exponential decay with an offset.
Eight point mutations were introduced into S. cerevisiae ARF1 and analyzed using a number of specific tests of in vivo and in vitro activities. The mutations and a brief indication of the rationale behind their construction appear in Fig. 2. More details of the rationale for each mutant appear in the relevant sections below. The strategy used in the construction of plasmids used to express wild type and each of the mutants in yeast, using either the ARF1 promoter or the inducible GAL1 promoter, and bacteria is shown in Fig. 1. Table 1lists each of these plasmids and the yeast strains used in the studies described below.
Figure 2: Point mutations introduced into Arf1. The consensus sequences for GTP-binding proteins are indicated in the first line, with those found in members of the Arf family on the line below. Each of the point mutation analyzed in this work are indicated, along with a brief description of the reason for its construction.
Two of the previously defined
phenotypes associated with ARF1 expression, the rescue of
supersensitivity to fluoride in arf1 cells
and inhibition of cell proliferation when overexpressed, were used as in vivo tests of Arf functions. The haploid arf1
strain, TT104, fails to grow on YPD in
the presence of 40 mM NaF (YPD/F
; Table 2; (11) ). When TT104 cells carried a copy of the ARF1 gene on a centromere-containing plasmid (strain RT135), growth on
YPD/F
was restored. In contrast, such a plasmid carrying
the ARF1 gene with the [G2A] mutation (RT136) failed
to allow growth of arf1
cells on
YPD/F
(see Table 2).
Overexpression of ARF1 is lethal to yeast cells (Table 2; (11) ). The inducible GAL1 promoter was used to express wild type or mutants of ARF1 to high levels. Cells (RT164) carrying a CEN plasmid, directing the expression of wild type ARF1 under control of the GAL1 promoter, grew well on S.D. (synthetic medium with dextrose) but not on SGal (synthetic medium with galactose), which induces the GAL1 promoter. In contrast, overexpression of the [G2A] mutant had no apparent effect on cell growth (Table 2). Immunoblots of cell lysates with an antibody directed against yeast Arf proteins confirmed that RT164 cells grown on SGal expressed much higher levels of Arf1 than either the RT152 control or any of the strains grown on glucose. These data confirmed the previous observations (11) that overexpression of wild type Arf1 was harmful to cells and sensitivity to 40 mM NaF resulted from loss of Arf1 function and indicate that the [G2A] mutant is a null allele of ARF1.
Yeast Arf
proteins were expressed in bacteria and several milligrams of each were
obtained, using previously established procedures(19) . N-Myristoylation of the yeast Arf1 in bacteria was achieved by
the co-expression of Arf with the yeast N-myristoyltransferase
gene, NMT1(22, 23) . The covalent
incorporation of myristic acid into Arf proteins expressed in bacteria
was monitored by inclusion of [H]myristic acid in
the culture medium. Although the Arf1 protein was efficiently
myristoylated in bacteria expressing N-myristoyltransferase,
the [G2A]Arf1 protein failed to incorporate any labeled
myristate (data not shown). Thus, the [G2A] point mutation
completely prevented N-myristoylation. Biochemical analyses of
non-myristoylated wild type and [G2A]Arf1 revealed no
differences in the rates of GDP dissociation, the steady-state level of
GTP
S binding, or specific activities in the cholera
toxin-dependent ADP-ribosylation of G
(Arf assay; see Table 3). Thus, the only apparent consequence of mutation of
Gly
to Ala
was to prevent N-myristoylation of Arf1. However, this single change was
responsible for the complete loss of in vivo activity.
For comparative purposes the non-myristoylated forms of each of the other mutant proteins were analyzed below, as the extent of myristoylation of recombinant proteins expressed in bacteria can vary dramatically.
Each of
the Asp mutants was expressed in and the proteins were
purified from bacteria, as described under ``Materials and
Methods.'' All three mutant proteins were found to have specific
activities in the Arf assay that were slightly higher than that of wild
type Arf1 (Table 3). The three mutants all behaved very similarly
and the range of activities were 200-500% that of wild type,
determined in at least four different experiments. It is important to
note that activity in the Arf assay is normalized to GTP
S binding
sites to determine the specific activities.
The binding of
radiolabeled GDP to the Asp mutants was also not much
different from that of wild type Arf1, although the GDP off-rate from
[D26V]Arf1 was found to be slower than wild type and the
other Asp
mutants (Table 3). However, the slowly
hydrolyzable analog of GTP, GTP
S, bound very poorly to each of
these three Asp
mutants ( Table 3and Fig. 3).
Thus, Asp
is critical for high affinity binding of the
activating nucleotide. Determination of specific activity in the Arf
assay takes into account this low level of binding of GTP
S as the
activity is normalized to GTP
S binding sites. It is the switch to
this active conformation that appears to be impaired by mutations at
position 26.
Figure 3:
Mutation of Asp results in
loss of GTP
S but not GDP binding. GDP (upper panel) and
GTP
S (lower panel) binding to Arf1 (circles),
[D26G]Arf1 (squares), [D26A]Arf1 (triangles), and [D26V]Arf1 (inverted
triangles) was determined as a function of time at 30 °C, as
described under ``Materials and Methods.'' The curves drawn are from first-order rate equations fit to the data by the
Marquardt algorithm.
Nucleotide exchange on [Q71L]Arf1 was
comparable to that of Arf1, with an apparent first-order GDP off-rate
of 0.535 min (see Table 3). The specific
activity in the Arf assay of [Q71L]Arf1 was also quite
similar to the wild type protein. It should be noted that hydrolysis of
GTP by Arf is not required for activity in the Arf assay(27) .
Thus, the dominant activation of the [Q71L]Arf1 in cells is
not readily explained by changes in the intrinsic nucleotide handling
of the purified protein. It was not possible to test for a decrease in
this rate, as has been found for homologous mutations in other
GTP-binding proteins, as Arf proteins, including yeast Arfs (data not
shown), have no intrinsic GTPase activity(27) .
It is worth noting that ARF, immunoprecipitated from cells labeled with radioactive inorganic phosphate, failed to incorporate any covalently attached phosphate. Thus, if Arf is a substrate for any yeast protein kinase the extent of modification was so low as to be undetectable, or the phosphate linkage was labile under the conditions used in these studies.
High level expression of [N126I]Arf1 in bacteria was readily achieved using the same expression system used for the other mutants. However, of the 10 alleles of S. cerevisiae ARF1 expressed in bacteria, this was the only one which was found to be insoluble in bacterial lysates. Solubilization was achieved with 7 M urea, but removal of chaotrope failed to allow refolding of protein into a conformation that bound radionucleotides. Such a procedure has been previously shown to allow renaturation of recombinant human/bovine Arf1(18) . Thus, we have no biochemical data on this mutant.
Overexpression of the [W66R]Arf1 protein was clearly
deleterious to PSY315 cells, although less so than was the wild type
protein (see Table 2). The centromere containing plasmid,
pJCY1-62, carrying this mutant behind the ARF1 promoter was
capable of restoring growth to TT104 cells plated on
YPD/F. However, wild type ARF1 rescued growth of
TT104 cells at both 30 and 37 °C, but the [W66R]ARF1 allele failed to allow growth on YPD/F
at the
elevated temperature.
The binding of both GDP and GTPS to
[W66R]Arf1 were similar to that of the wild type protein,
although the rate of dissociation of GDP from the mutant (0.86
min
) was almost twice that from the wild type
protein (0.48 min
) in submillimolar concentrations
of magnesium. A larger difference (
7-fold) in the rate of GDP
dissociation was observed in the presence of 2 mM Mg
; 0.0098 min
and 0.068
min
for wild type Arf1 and [W66R]Arf1,
respectively.
Comparison of the specific activities in the Arf assay
revealed this mutant to be more than 300-fold less active as cofactor
in the cholera toxin assay when compared to the Arf1 (see Table 3). As the stoichiometry of binding of GTPS to
[W66R]Arf1 was similar to wild type, the low specific
activity reflects a loss of productive protein:protein interaction and
not decreased ability to bind the activating guanine nucleotide.
High-copy suppressors of arf1-2 were cloned by
transforming RT269 with a YEp24 based (2µ genomic S.
cerevisiae) library marked with the URA3 gene, after
replica plating transformants onto YPD/F at 37 °C. ARF2 and two extragenic suppressors were isolated from this
screen, termed suppressors of fluoride sensitivity (SFS3 and SFS4), are described. Each
restored growth to RT269 cells grown at 37 °C on
YPD/F
. Neither suppressor was able to suppress temperature
sensitivity to fluoride when present on a low-copy, centromere
containing plasmid. Suppression was tightly linked to the SFS genes as sensitivity was restored when library plasmids were lost,
by selection on 5-fluoroorotic acid. The level of expression of ARF2 was also examined to determine if suppression resulted
from increased expression of that gene. No differences were observed in
the level of ARF2 expression in strains carrying the SFS genes on 2µ plasmids.
The smallest genomic fragment with SFS3 activity was a 3765-bp PvuII-SalI fragment that mapped to chomosome XV. The only open reading frame greater than 400 bp in length encodes a predicted protein of 444 amino acids. A search of the Swiss-Prot data bank with FASTA revealed that this open reading frame was identical in predicted protein sequence to that of STD1, a high-copy suppressor of a negative dominant mutation in the TATA-binding protein and a putative transcription factor(31) .
The other high-copy suppressor of arf1-2, termed SFS4, was located on a 4500-bp SstI-BamHI fragment and mapped to chomosome VII.
Translation of this fragment in all six reading frames revealed a
single open reading frame of greater than 100 amino acids. An open
reading frame of 2004 bp was present, which encodes a predicted protein
of 668 amino acids. A search of SWISS-PROT revealed near identity ()with the predicted sequence of PBS2/HOG4,
originally identified as a high-copy suppressor of sensitivity to the
polyamine antibiotic polymyxin B(32) . More recently, this
protein has been identified as a member of the microtubule-associated
protein kinase kinase family involved in a signal transduction pathway
that is sensitive to changes in the osmolarity of the extracellular
environment(33) .
S. cerevisiae Arf1 binds guanine nucleotides
more rapidly and with higher stoichiometry than mammalian Arf
proteins.The purified recombinant yeast Arf1 protein
has previously been shown to bind guanine nucleotides with high
affinity and have activity in the (cholera toxin) Arf
assay(4) . Further characterization of the nucleotide exchange
rate revealed that the yeast protein has a much higher rate of GDP
dissociation (k = 0.48
min
) than does human Arf1 (k
= 0.023 min
). This decreased affinity
for GDP likely contributes to the higher stoichiometries of GTP
S
binding observed for the yeast Arf1 protein (typically 20-60%)
compared to the human Arf1 protein (typically 1-5%).
Expression and functional analyses of point mutants of Arf1 were undertaken to analyze the properties of an ARF family member both in vivo and in vitro. These analyses revealed the essential nature of N-myristoylation of Arf proteins to their functions in cells. Mutational analysis of the guanine nucleotide binding site revealed some similarities to other GTP-binding proteins but also some differences. A conditional allele, arf1-2, was found which allowed suppressor analysis. Genetic evidence suggesting a common signaling pathway which includes both an Arf and a microtubule-associated protein kinase kinase are intriguing in light of the recent demonstrations of the role Ras proteins play in similar kinase cascades(35, 36) .
N-Myristoylation
of Arf was shown to be an essential processing step and required for
activity in vivo. In contrast, N-myristoylation is
required for only some of the activities of Arf proteins in in
vitro assays. The need for N-myristoylation of ARF1 to
regulate membrane traffic has previously been described after analyses
of transient overexpression of the [G2A]ARF1 mutant in HeLa
cells(12) . The ability of Arf to serve as cofactor in the
cholera toxin assay and dependence of GTP binding on phospholipids are
observed for both the acylated and unmodified proteins(18) .
However, activation of phospholipase D activity(7) , guanine
nucleotide regulated binding of Arf to membranes and lipid
micelles(37) , and GTPS dependent inhibition of ER-Golgi (38) and intra-Golgi transport(39) , and endosome
fusion (40) assays are all highly dependent on myristoylation
of Arf. As the nucleotide-regulated binding of Arf to micelles and
membranes requires N-myristoylation, we conclude that
reversible membrane association, regulated by guanine nucleotide
binding, plays an essential role in the action of Arf in cells.
What
is the role of the covalently attached myristate in Arf action? In the
GDP-bound state, it is likely that the myristate moiety is not exposed
to solvent as we have been unable to resolve the processed and
unprocessed forms of Arf on a variety of chomatography resins. ()Such a structural role for myristate has previously been
demonstrated for the catalytic subunit of the cAMP-dependent protein
kinase, in which the fatty acid is buried inside the protein, resulting
in increased thermal stability and ordering of the amino
terminus(41, 42) . N-Myristoylation of
G
was found to increase its affinity for
G
(43) . Upon binding GTP the Arf protein acquires
increased affinity for micelles and membranes, in a process that is
highly dependent on N-myristoylation. Thus, it is reasonable
to conclude that the myristate becomes much more exposed and may be
directly involved in the membrane association of the active protein.
Limiting the protein to the two-dimensional space of the bilayer may be
sufficient to increase protein:protein interaction and result in an
apparent increase in affinity of Arf for its effector. There is
currently no evidence that the myristate is directly involved in the
interaction of Arf with any protein effector, receptor, or modulator; e.g. Arf GAP.
Functional effects of mutation of Asp were quite different from predictions, based on homologous
mutations made in p21
or
subunits of G proteins.
The [D26G/A/V]Arf1 proteins display unaltered binding of GDP
but a dramatically decreased affinity for GTP
S. However, the
specific activities of the Asp
mutants in the Arf assay
are comparable to wild type. Thus, the mutant proteins are less able to
bind GTP but once activated, appear to have full activity. In contrast,
mutations in the homologous residue of Ras (Gly
) increase
transforming activity, likely as a result of decreased intrinsic GTPase
activity(44) . This allele of Ras binds both guanine
nucleotides and GAP with similar affinity to the wild type protein.
Similar to Ras, a homologous mutant of the
subunit of G
([G49V]) is a potent activator of adenylate cyclase
with a 5-fold lower intrinsic GTPase rate and a 7-fold increase in
fractional occupancy by GTP; while the GDP dissociation rate is
unchanged(24) . Interestingly, bacterial EF-Tu, like Arf, lacks
a glycine at the corresponding position (Val
) in the first
consensus GTP binding domain. However, the [V20G] mutant of
EF-Tu has dramatic consequences on nucleotide binding and GTPase rates
quite different from what was seen with [D26G/A/V]ARF1. This
mutant EF-Tu has 20- and 10-fold increases in GDP dissociation and
association rates, respectively, which leads to a higher affinity for
GTP, in comparison to wild type(45) . In addition, the
intrinsic GTPase activity of the mutant is reduced about 5-fold. Arf1
differs from these other GTP-binding proteins in having severely
reduced affinity for GTP after mutation of this residue. The absolute
conservation of Asp
in this concensus GTP binding domain
of members of the Arf family further suggests that this unique property
is important to the actions of this subfamily of regulatory proteins.
Changing the conserved glutamine in the consensus GTP binding
domain, DXXGQ, to leucine is a dominant activating mutation
among most GTP-binding proteins, including Ras (Gln) and
G
(Gln
), but not SEC4(30) . The
homologous mutation in Arf1 is a dominant lethal allele which leads to
an accumulation of the active form of the protein in yeast cells. The
biochemical properties of the purified recombinant [Q71L]Arf1
protein are not much different from wild type. This is similar to what
has been observed for the homologous mutation in Ras and suggests that
the most important consequence of this point mutation may be increased
affinity for, but decreased sensitivity to, Arf GAP(46) .
Preliminary evidence supports this conclusion as human
[Q71L]Arf3 is a potent inhibitor of partially purified bovine
brain Arf GAP. (
)Thus, the dominant nature of this mutant
may be explained by the activity of the mutant protein, by the
increased activity of wild type proteins which share a common GAP (and
which would become activated as a result of the inhibition of that GAP
by the mutant), or by some combination of these two events.
The
asparagine in the conserved NKXD motif is known to be involved in
binding the guanine base in other GTP-binding proteins, including Ras
and G. This is likely true of Arf1 as well, as the
homologous Arf mutant was expressed at high levels but unable to bind
nucleotides. The [N126I]Arf1 mutant appears to be a partial
dominant allele in yeast as it is harmful to the vegetative growth of
both wild type and arf1
cells. Either
increased or decreased expression of Arf is deleterious to the growth
of yeast cells. However, as the effect of this mutant appears more
marked in the Arf1 deficient cells, it is a negative dominant allele.
Our working hypothesis is that the decreased affinity for guanine
nucleotides results in an accumulation of the apo-protein. The
nucleotide-free form of other GTP-binding proteins has been proposed to
have high affinity for guanine nucleotide exchange
factors(47, 48) . Thus, the negative dominance may
result from the decrease in activated Arf proteins, resulting from the
inhibition of Arf exchange factor(s).
The [W66R]Arf1
mutant was found to have decreased affinity for GDP and
Mg, resulting in higher stoichiometries of GTP
S
binding. Surprisingly, the specific activity of this protein was
300-fold lower than that of the wild type protein. Thus, in
contrast to the Asp
mutants which bind GTP poorly but
retain wild type specific activities, this mutant binds GTP better than
wild type yet remains inactive. Possible explanations for these
observations include an essential role for Trp
in the
effector binding site or in interactions with other parts of the
protein that are required for formation of the active conformation. The
importance of Trp
in the activation process makes it a
likely major contributor to the large change in intrinsic fluoresence
that has previously been shown to accompany the binding of
GTP(27) . The ability to separate GTP binding from formation of
the active conformer may prove useful in the further dissection of the
molecular mechanisms of Arf regulation.
Suppressor analysis of arf1-2 led to the isolation of genes that permitted
growth of RT269 cells on YPD/F at 37 °C. The
identification of SFS4 as PBS2/HOG2, a
microtubule-associated protein kinase kinase, is particularly
intriguing in light of the recent work documenting the role of Ras in
triggering the microtubule-associated protein kinase cascade by
localizing the kinases to the plasma membrane in higher eukaryotes.
Curiously, the roles for RAS genes in S. cerevisiae has been shown to be intimately associated with the regulation of
adenylate cyclase and not protein kinase activation. It will likely
prove interesting to determine if other similarities exist between the
Arf and Ras signaling pathways in mammals and yeast.
The identification of SFS3 as identical to STD1 suggests that this putative transcription factor may regulate the expression of one or more of the components in an Arf signaling pathway. The immunoblotting to test for the level of expression of ARF2 in strains carrying SFS genes is probably not sensitive enough to rule out ARF2 as a possible target for SFS3/STD1. Alternatively, it might prove fruitful to identify other genes whose expression is affected by SFS3/STD1 and test them as potential modulators of Arf action.
Results from these studies further
reinforce the conclusion that the Arf proteins have distinctive
structural and functional features compared to other members of the RAS
superfamily of low molecular weight GTP-binding proteins. The crystal
structure of human Arf1/GDP has recently been solved ()(49)
and will clearly provide a wealth of new information and suggest
hypotheses which may be tested using variations of the protocols
described in these studies.