From the
Ran/TC4 is a ras-related GTP-binding protein predominantly
located in the nucleus. Ran/TC4 is essential for nuclear transport and
is involved in mitotic control. In Saccharomyces cerevisiae a
gene highly homologous to Ran/TC4 has been identified and named
GSP1. Like all ras-related GTP-binding proteins, Gsp1p
undergoes cycles of GTP hydrolysis and GDP/GTP exchange. The switching
between the two different nucleotide bound states regulates the
function of these GTP-binding proteins. Here we identify the product of
the yeast RNA1 gene as the GTPase-activating protein (GAP) of
Gsp1p. RNA1 belongs to a group of genes which are conserved in
a variety of different organisms. We have expressed and purified
recombinant Gsp1p and Rna1p from Escherichia coli. The GTPase
activity of Gsp1p is stimulated 10
Ran/TC4 (further referred to as Ran) was first cloned from a
human teratocarcinoma cDNA library
(1) . It is a GTP-binding
protein representing a distinct subfamily within the superfamily of
ras-related GTP-binding proteins. Ran is a very abundant protein
constituting about 0.36% of the total protein content in HeLa cells
(2) and it is localized mainly in the nucleus. Like all
ras-related GTP-binding proteins, Ran cycles between a GTP and a GDP
bound state. The conversion between these two states is very slow as
Ran binds guanine nucleotides very tightly and hydrolyses GTP very
slowly
(3) . The dissociation of Ran-bound GDP is increased
10
Genes homologous to Ran and RCC1 have been
identified in the fission yeast Schizosaccharomyces pombe as
spi1 and pim1(5) and in the budding yeast
Saccharomyces cerevisiae as GSP1/GSP2(14) (also isolated as CNR1/2(15) and
PRP20(16) , respectively). A comparison of the deduced
amino acid sequences revealed a similarity of about 88% between Ran,
spi1, and Gsp1/2p
(14) and functional homology was shown between
RCC1 and Prp20p
(17, 18) , suggesting that the functions
of these proteins have been conserved during evolution. In S.
cerevisiae the loss of PRP20 causes defects in RNA
processing and RNA export from the nucleus
(17, 19) .
While the exact function of Ran and RCC1 related proteins remains to be
shown, another class of proteins, known as GTPase-activating proteins
(GAPs), is certain to play an essential role in the regulation of Ran,
spi1, and Gsp1p. Recently the purification and characterization of a
GTPase-activating protein specific for Ran, named RanGAP1, has been
reported
(20) . Here we identify RNA1 as a gene encoding
GAP specific for the S. cerevisiae Ran homologue Gsp1p.
RNA1 has been previously identified as an important component
of the RNA export machinery from the nucleus
(21, 22, 23) and is highly conserved in different species. We have
expressed and purified recombinant Rna1p and Gsp1p and show that Rna1p
strongly increases the GTPase activity of Gsp1p. In addition, we show
that Gsp1p also interacts with human and S. pombe homologues
of Rna1p, indicating that this interaction has been conserved during
evolution. While this study was in progress RanGAP1 had been cloned and
found to be homologous to RNA1(24) .
The activity of the
purified protein was determined by its ability to bind GDP. Comparing
the measured protein concentration with the amount of protein able to
bind GDP in a nitrocellulose filter assay, we consistently found that
only 50-70% of purified Gsp1p was active. This phenomenon has
been observed with other small GTP-binding proteins assayed in filter
binding assays. In the case of Ran, fluorescence spectroscopy showed
that Ran forms a 1:1 complex with the guanine nucleotide although
filter binding tests indicated less nucleotide binding
(25) .
Testing the nucleotide-bound state of purified Gsp1p on high
performance liquid chromatography
(31) we found that >90% of
the protein was complexed with guanine nucleotide. Thus we conclude
that Gsp1p forms a tight equimolar complex with the nucleotide as has
been observed for all other ras-related GTP-binding proteins.
Insufficient recovery of radioactive nucleotide in the nitrocellulose
filter assay could either be attributed to insufficient binding of
Gsp1p itself or to limited protein denaturation which leads to
nucleotide release. In general we noticed that the nucleotide binding
activity of Gsp1p is very sensitive to changes in pH or ionic strength
of the solvent and to freezing/thawing procedures.
It was suggested that in order for Ran to facilitate import
and export of proteins and small nuclear RNP into and out of the
nucleus, Ran would have to shuttle from the cytoplasm into the nucleus
and back. For this mechanism it was suggested that two sets of GEFs and
GAPs specific for Ran would have to exist
(36) , each regulating
one cycle of GTP binding, GTP hydrolysis, and GDP dissociation specific
for one direction of this bidirectional transport. To investigate
whether more than one Gsp1p-specific GAP activity exists in yeast, we
fractionated a 100,000
In Caenorhabditis elegans the open reading frame coding for a gene homologous to RNA1 was identified during the genome sequencing project
(34) .
This DNA sequence exists as a tandem repeat, however, the functional
significance of this tandem repeat is unclear. Our biochemical analysis
suggests that Rna1p homologous proteins from different species are also
functionally conserved as they react with Ran homologous proteins from
different species. This finding is corroborated by the observation that
rna1 from S. pombe can functionally replace the
RNA1 gene from S. cerevisiae(29) .
The most
prominent features of Rna1p homologous proteins are the so called
leucine-rich repeats (LRR) constituting the major part of the protein
sequence. LRRs have been found in proteins with different functions and
intracellular localizations and participate in protein-protein
interaction
(37) . The three-dimensional crystal structure of one
of the LRR containing proteins, the porcine RNase inhibitor, has been
determined recently
(38) and shown to have a nonglobular
horseshoe-like structure where the LRR's form repeating
Comparing the primary structures of GAPs for Ran with those for Ras,
Rap, Rho, and Rab, these proteins show no apparent
similarity
(39) . It is striking that although GAPs specific for
different GTP-binding proteins share no apparent sequence homology, at
least Ran-GAP and Ras-GAP stimulate GTP hydrolysis at least
10
In addition to the yeast proteins,
mammalian Rna1p homologues contain an additional 260 amino acid
residues at the C-terminal end. As rna1p from S. pombe is able
to induce the GTPase activity of Ran, we conclude that these C-terminal
extensions are not involved in inducing GTP hydrolysis or providing
protein specificity. This observation that the GTP hydrolysis of human
Ran is not induced by Rna1p from S. cerevisiae is reminiscent
of the cross-reactivity of human H-Ras and yeast Ras1p with human
RasGAP and Ira1p from yeast. While the GTPase activity of Ras1p was
stimulated by Ira1p and RasGAP, the GTP hydrolysis of H-Ras was only
catalyzed by RasGAP but not by Ira1p
(41) .
Originally
RNA1 was identified in a genetic screen selecting for yeast
mutants defective in mRNA processing
(22) . Mutants of RNA1 were also defective in exporting mRNA from the nucleus (19, 42,
43). In S. cerevisiae, Rna1p is described as a cytosolic
protein apparently excluded from the nucleus
(44) . The
cytoplasmic localization of Rna1p was hard to reconcile with its
apparent functions inside the nucleus, i.e. mRNA processing
and export
(44) . As Gsp1p is localized mainly inside the
nucleus
(14) , the regulation of Gsp1p by Rna1p provides the
means for Rna1p to exert its function inside the nucleus. This could
explain why GSP1 and RNA1 mutants have similar
phenotypes
(15, 19) . If Rna1p and Gsp1p are localized in
different cellular compartments how do they effectively interact with
each other? The observation that a small fraction of rna1p in S.
pombe is localized to the nuclear envelope
(29) suggests
that the location of interaction could be the interface between the
nuclear matrix and cytoplasm, possibly the nuclear pore. As the nuclear
pore complex is crucial for both nuclear import and export it would
explain why mutants of Rna1p and Gsp1p effect both processes.
Using
in vitro nuclear protein import assays two cytosolic fractions
termed A and B have been characterized, each facilitating a specific
step of nuclear protein import
(45) . Fraction A promotes the
first step, the binding of nuclear localization sequence-tagged
proteins to the nuclear envelope. Importin was recently found to be
involved in this targeting
(46) . The second step is
ATP-dependent and facilitates the actual translocation of proteins into
the nucleus. Ran is the active component of the second step
(12, 13) and together with the newly identified importin is the only
protein necessary for complete nuclear protein import in
vitro(46) . For full import activity, a Ran interacting
protein of 10 kDa was found to be required
(49) . Importin
contains an 8-fold repeat of an hydrophobic motif, the so called arm
motif
(47) . Among other proteins the arm motif is found in
It is noteworthy that RCC1/Prp20 and RanGAP1/Rna1p have never been
implicated in nuclear protein import. Detailed analysis of the
interaction between Ran/Gsp1 and their interacting factors like GAPs,
GEFs, or the recently cloned Ran-binding
proteins
(48, 49, 50) will be necessary to show
how the cycle of GTP binding, GTP hydrolysis, and GTP/GTP exchange
regulates nuclear transport mechanisms or cell cycle events. The
multitude of different defects caused by mutations in Ran, RCC1,
RNA1, and their relatives from other organisms makes it
difficult to assign a unifying function for these proteins. The current
data is insufficient to decide whether these proteins have many
different functions or whether a defect in one key function is the
basis for the pleiotropic defects observed. At this point it is not
entirely unlikely that the nucleocytoplasmic transport could be this
principal function as the cell cycle regulation depends on the signal
transduction from the exterior through the cytoplasm into the nucleus.
We thank C. Koerner for excellent technical
assistance, I. Simon for providing purified Rab7 protein, C. Klebe for
providing purified Ran protein, and C. Herrmann and R. H. Cool for
discussions.
-fold by Rna1p. In
addition, we find that the previously identified human RanGAP1 and
rna1p from Schizosaccharomyces pombe are also able to induce
GTPase activity of Gsp1p. The GTP hydrolysis of Ran is induced by
RanGAP1 and rna1p but not by Rna1p. Implications for the suggested
functions of Ran/TC4/Gsp1p in nuclear transport and mitotic control are
discussed.
-fold by the gene product of RCC1
(
)
which represents the nucleotide exchange factor of
Ran
(4) . Mutants of RCC1 enter mitosis prematurely before
S-phase has been completed
(5, 6, 7) . As RCC1 is
involved in the checkpoint control of entry into mitosis, Ran is also
thought to be involved in cell cycle control. Ran has been reported to
regulate initiation of S-phase
(8) , entry into mitosis
(8-10), and exit from mitosis
(11) . Other groups have
shown that Ran is also an essential component of the nuclear transport
machinery (12, 13).
Cloning of Gsp1 and RNA1
GSP1 and
RNA1 were cloned by polymerase chain reaction using genomic
yeast DNA as a template. To isolate GSP1 the following primers
were used: GSP1-3, 5`-CTCATTTATATTATCCATGGCTGCCCCAGC-3`
(underlined is the restriction site NcoI); GSP1-2, 5`
GAAAGCTTGTTCTCGTTTGTCCC (underlined is the restriction site
HindIII). The resulting DNA fragment contained a 5`
NcoI and a 3` HindIII restriction site which
facilitated the cloning into pET3d-Ran
(25) . The protein coding
region of Ran in pET3d was removed as an NcoI/HindIII
fragment and GSP1 was inserted to yield pET3d-GSP1. To isolate
RNA1 the following primers were used: RNA1-1,
5`-GGGCCATGGCTACCTTGCACTTCGTTCC-3` (underlined is the restriction site
NcoI) and RNA1-2;
5`-GGATCCTAGCTATTTGATTTCAGTTTCAGCTAAACG-3` (underlined is the
restriction site BamHI). The polymerase chain reaction
generated a NcoI restriction site at the 5` end and a
BamHI restriction site at the 3` end of the resulting DNA
fragment. The polymerase chain reaction fragment was cloned into pET3d
as an NcoI/BamHI fragment to yield pET3d-RNA1. Both
expression plasmids were transformed into Escherichia coli strain BL21(DE3).
Expression of Gsp1p
BL21(DE3) cells containing
pET3d-GSP1 were diluted and spread on LB plates containing 200
µg/ml ampicillin to a density of 1000 colonies per plate and
incubated overnight. For 2 liters of LB medium containing 200 µg/ml
ampicillin, one plate of cells was washed off and incubated for about 2
h at 37 °C to yield an A of 0.5-0.9.
10 µM Isopropyl-1-thio-
-D-galactopyranoside
was added and cells were further incubated until they ceased to grow
after about 3 h. Cells from a total of 20 liters of medium were
harvested by centrifugation to yield about 80 g (wet weight). Cells
were resuspended at 3 ml/g cell paste in buffer A (20 mM
potassium phosphate, pH 6.0, 100 µM GDP, 10 mM
DTE, 2 mM MgCl
, 1 mM phenylmethylsulfonyl
fluoride). Cells were lysed by adding 1 mg/ml lysozyme and incubated 1
h at 4 °C while stirring. To decrease the viscosity of the lysate,
DNA was digested by adding DNase I (10 µg/ml). In order to
uniformly load Gsp1p with GDP, 5 mM EDTA was added and the
lysate incubated at 30 °C. After 30 min, 10 mM MgCl
was added to stabilize the Gsp1p
GDP complex. The lysate was
sonicated 5
2 min on ice at 50% (Branson) and cleared at 40,000
g for 1 h. The resulting supernatant was diluted 1:10
with buffer A to reduce the ionic strength and applied onto a
SP-Sepharose column (600 ml). A 3-liter gradient from 0 to 500
mM KCl was applied and Gsp1p eluted between 150 and 200
mM KCl. Pooled fractions were concentrated by ultrafiltration
and applied on a Superdex 200 (26/60) FPLC column equilibrated with
buffer B containing 20 mM potassium phosphate (pH 6.0), 2
mM DTE, 2 mM MgCl
. Peak fractions were
concentrated by ultrafiltration (Amicon), dialyzed against buffer B,
shock frozen in liquid nitrogen, and kept at -80 °C.
Expression of Rna1p
A 200-ml overnight culture of
BL21 cells containing pET3d-RNA1 was diluted into 20 liters of LB
medium containing 100 µg/ml ampicillin and grown at 37 °C to a
A of 0.1.
Isopropyl-1-thio-
-D-galactopyranoside was added to a
concentration of 10 µM and the culture was further
incubated for 5 h. Cells were harvested by centrifugation and stored
frozen at -20 °C. Cells were resuspended in buffer A lacking
GDP (3 ml/g cell paste) and lysed as described before. The cleared
lysate was applied directly onto a DEAE Sephadex column (600 ml) and
eluted with a 3-liter 0-1.5 M KCl gradient. Rna1p eluted
at about 600 mM salt. Fractions were treated as described
above including the purification on a Superdex 200 (26/60) FPLC column
(Pharmacia). The final protein solution was dialyzed against standard
buffer C (64 mM Tris Cl, pH 7.4, 2 mM DTE, 5
mM MgCl
).
Protein Determination
Protein concentrations were
determined according to Bradford
(26) using bovine serum albumin
as a standard. The concentration of Gsp1p was defined as the amount of
purified Gsp1p that is able to bind GDP as measured by the
nitrocellulose filter binding test
(27) . 1 µM Gsp1p
was incubated for 30 min at 30 °C in the presence of 5 mM
EDTA, 20 mM potassium phosphate (pH 6.0), 2 mM DTE,
and 5 µM GDP and 43 nM
[H]GDP (11.5 Ci/mmol, Amersham). The
Gsp1p-nucleotide complex was stabilized by adding 10 mM
MgCl
. The amount of protein bound GDP was measured by
determining the filter bound radioactivity (BA85, Schleicher &
Schüll, Dassel).
GTPase Activity
To measure the GTPase reaction,
Gsp1p was first loaded with [-
P]GTP. 30
µM Gsp1p was incubated in the presence of 5 mM
EDTA, 20 mM potassium phosphate (pH 6.0), 2 mM DTE,
150 µM GTP, and 5 pM
[
-
P]GTP (6000 Ci/mmol) (ICN). The
Gsp1p-nucleotide complex was stabilized by adding 10 mM
MgCl
after 30 min. Unbound nucleotide was removed by gel
filtration (NAP-5, Pharmacia LKB Biotech). The rate of GTP hydrolysis
was measured at 30 °C by determining the filter bound radioactivity
after filtering aliquots of the GTPase reaction through nitrocellulose
filters. Data were fitted to a single-exponential function using the
computer program GraFit (Erithacus Software). Alternatively, the GTPase
reaction was followed by measuring the formation of inorganic phosphate
using a modified charcoal method
(28) . 50-µl aliquots of the
GTPase reaction were thoroughly mixed with 200 µl of 20 mM
phosphoric acid containing 5% (w/v) charcoal. This mixture was
centrifuged in a microcentrifuge at full speed for 10 min and the
formation of
-
P was determined by measuring 100
µl of the supernatant in a scintillation counter. The GTPase
activity of Ran, Rab7, and H-Ras was measured essentially as described
above except that standard buffer C was used as a reaction buffer.
Cell Fractionation
A wild type S. cerevisiae strain was grown in rich medium to late logarithmic growth phase
(A = 10), and harvested by centrifugation
at 1000
g for 5 min. The cells were washed in standard
buffer C containing 0.1 mM phenylmethylsulfonyl fluoride,
resuspended in 10 ml of lysis buffer, and lysed by vortexing for 5
1 min with an equal volume of glass beads (diameter 0.5 mm).
The crude extract was first centrifuged at 4 °C, 10,000
g for 30 min, and then at 4 °C, 100,000
g for 1 h. 30 mg of soluble protein was applied onto a 1-ml Resource
Q (Pharmacia) chromatography column and eluted with an linear gradient,
ranging from 0 to 1500 mM KCl. 1-ml fractions were collected,
and GTPase activity was measured using filter binding assays. 1
µM Gsp1p was incubated for 15 min at 30 °C with column
fractions diluted 1:100.
RESULTS
Analogous to mammalian cells where Ran is regulated by RCC1,
Gsp1p of S. cerevisiae is regulated by the gene product of
PRP20(14, 15) . Mutants of PRP20 are
defective in processing and exporting mRNA from the
nucleus
(17, 19) . A similar phenotype was observed in
yeasts carrying mutations in the RNA1 gene. Mutations in the
RNA1 gene disrupted the mRNA 3`-end formation and prevented
the transport of newly synthesized mRNA to the
cytoplasm
(19, 21, 22, 23) . In addition,
in yeast cells where the GSP1 gene was under control of a
GAL1 promotor, mRNA began to accumulate inside the nucleus 15
h after a switch to a glucose medium
(15) . The observation that
mutants of PRP20, RNA1, and GSP1 have
similar phenotypes suggested that their gene products might function in
a common pathway. Furthermore, immunofluorescence microscopy with
antiserum against rna1p, the Rna1p homologous protein of S.
pombe, showed a preferential staining of the nuclear periphery
(29). Rna1p is therefore positioned to play a potential role in nuclear
transport. Through these observations Rna1p, for which no biochemical
function had yet been identified, became a prime candidate as a protein
that could potentially interact with Gsp1p and that could possibly act
as a GTPase-activating protein for Gsp1p. To investigate the
interaction between Gsp1p and Rna1p we expressed and purified
recombinant Gsp1p and Rna1p. GSP1 and RNA1 genes were
cloned into pET3d and expressed in E. coli strain BL21(DE3).
After induction with
isopropyl-1-thio--D-galactopyranoside, large amounts of
expressed proteins were visible in a Coomassie-stained SDS gel
(Fig. 1, A and B, lanes 2).
Figure 1:
Expression of Gsp1p
(A) and Rna1p (B) in E. coli. SDS-PAGE of
total bacterial lysates of cells containing pET3d (A and
B, lane 1), pET3d-GSP1 (A, lane 2), or
pET3d-RNA1 (B, lane 2). SDS-PAGE of pooled fractions after a
SP-Sepharose chromatography (A, lane 3) followed by Superdex
200 FPLC gel filtration (A, lane 4) or after DEAE-Sephadex
chromatography (B, lane 3) followed by Superdex 200 FPLC gel
filtration (B, lane 4).
Purification of Gsp1p
Bacterially expressed Gsp1p had an
apparent molecular mass of 24 kDa which is in good keeping with the
calculated molecular mass of 24,810 Da. It was purified in a two-step
purification procedure. First the soluble bacterial protein extract was
fractionated by gradient elution (0-500 mM KCl) from a
SP-Sepharose column at pH 6.0. Fractions containing Gsp1p were
identified by SDS-PAGE, pooled (Fig. 1A, lane 3), and
further purified on a Superdex 200 FPLC chromatography column at pH
6.0. A typical purification from 80 g of cells produced about 80 mg of
>90% pure protein as judged by SDS-PAGE (Fig. 1A, lane
4). The purified protein showed a minor second band when the SDS
gel was overloaded. Most likely the lower band is a degradation
product. A similar degradation band has been observed during the
purification of other small GTPases
(30) . Any attempts to purify
Gsp1p at a higher pH failed, as the protein did not bind to anion or to
cation exchange columns under these conditions. Attempts to neutralize
the buffer after the first purification step also failed since even the
most careful titration rendered the protein inactive, as measured by
its ability to bind GDP. Therefore all subsequent measurements
involving Gsp1p were performed at pH 6.0.
Purification of Rna1p
Bacterially expressed Rna1p
migrated with an apparent molecular mass of 46 kDa
(Fig. 1B) which correlates the calculated molecular mass
of 46,103 Da. Rna1p was purified in a two-step purification procedure
involving a gradient elution (0-1500 mM KCl) on a DEAE
Sephadex column at pH 6.0 and a Superdex 200 FPLC chromatography step
(Fig. 1B, lanes 3 and 4). A typical preparation
produced 50-60 mg of >95% pure protein per 80 g of cell paste
as judged by SDS-PAGE (Fig. 1B, lane 4). Unlike Gsp1p,
Rna1p was very stable tolerating changes in pH as well as changes in
the ionic strength of the solvent.
Interaction between Gsp1p and Rna1p
To measure the
effect of Rna1p on the GTPase activity of Gsp1p, 0.5 µM
Gsp1p was incubated with 0.1 nM Rna1p. The charcoal assay
demonstrated that 0.1 nM Rna1p increases GTP hydrolysis
30-fold over the intrinsic rate when initial rates were compared
(Fig. 2A). The rate of GTP hydrolysis is dependent on
the concentration of Rna1p in the concentration range of 0.05 to 10
µM Gsp1p (Fig. 2B). Treating the Rna1p
induced GTP hydrolysis as an enzymatic reaction, we attempted to
determine the Kand k
given that Ran and Gsp1p are 88% identical, we therefore expected
a K
value similar to that for RanGAP1
stimulated GTP-hydrolysis of Ran which is 0.43
µM(3) . Unexpectedly the K
of the Rna1p-induced GTP hydrolysis appears to be much higher
than that of RanGAP1-induced GTP hydrolysis of Ran. Due to protein
instability of Gsp1p at higher protein concentrations we were unable to
follow the rate of GTP hydrolysis at substrate concentrations higher
than 10 µM. However, our estimates indicate that
K
and V
must be at
least 30 µM and 300 s
, respectively
(Fig. 2B). Both values are almost 100-fold higher than
the K
and V
of the
RanGAP1-induced GTP hydrolysis of Ran. As the intrinsic GTP hydrolysis
of Gsp1p is 5
10
s
, we
calculated that the GTP hydrolysis of Gsp1p is stimulated at least
0.5-1
10
-fold. This increase in GTP
hydrolysis is significantly higher than that of other GTPase-activating
proteins, like RanGAP1 (10
-fold)
(3) and that of
RasGAP (2, 5
10
-fold)
(32, 33) .
Figure 2:
Rna1p activates the GTPase activity of
Gsp1p. 0.5 µM Gsp1p complexed with
[-
P]GTP was incubated with (
) or
without (
) 0.1 nM Rna1p for increasing amounts of time.
Liberated [
-
P] was determined in a charcoal
assay (27) (A). The rate increase of GTP hydrolysis is
dependent on the substrate (Gsp1p) concentration. 0.1 nM Rna1p
was incubated with increasing amounts of Gsp1p as indicated and the
initial rates of GTP hydrolysis are represented as a function of Gsp1p
concentration (B). Data were fitted non-linearly to a
hyperbolic function (the Michaelis-Menten equation) using the computer
program GraFit (Erithacus Software).
The ability of Rna1p to accelerate the GTP hydrolysis appears to be
specific for Gsp1p, as Rna1p is unable to increase the GTPase activity
of H-Ras or Rab7 even when incubated with 100-fold more Rna1p (10
nM) than used with Gsp1p (Fig. 3). Therefore, Rna1p
appears to be a GTPase-activating protein specific for Gsp1p.
Figure 3:
Rna1p is specific for Gsp1p. 1
µM Gsp1p (,
), H-Ras (
,
), and
rab7 protein (
,
) complexed with
[
-
P]GTP was incubated at 30 °C with
(open symbols) or without (closed symbols) 0.1
nM Rna1p (Gsp1p) or 10 nM Rna1p (H-Ras, Rab7). GTPase
activity was determined as the decrease of
[
-
P]GTP bound to nitrocellulose filters.
Data are presented as percent of the radioactivity bound to Gsp1p 30 s
after addition of Rna1p.
Interaction of Ran/Gsp1p with Rna1p
Homologues
Genes homologous to RNA1 have been found in
different organisms. Comparison of the deduced amino acid sequence of
RNA1 (S. cerevisiae)
(23) , rna1 (S. pombe) (29), CEC29E4 (C.
elegans)
(34) , fug1 (mouse) (35), and RanGAP1
(human)
(24) reveals that these proteins are highly
conserved
(24) . The conserved primary structure of the RNA1 related genes was also suggestive of a conserved function. We
therefore tested whether Rna1p homologues from different species could
activate GTP hydrolysis of Gsp1p. As shown in Fig. 4A,
0.1 nM RanGAP1 purified from HeLa cells and 0.1 nM
bacterially expressed rna1p were able to increase the GTPase activity
of Gsp1p. The observed rates of the induced GTPase activities are very
similar, suggesting that these GTPase-activating proteins are
functionally and structurally closely related.
Figure 4:
The GTPase activity of Gsp1p is activated
by S. pombe rna1p and human RanGAP1 while the GTPase activity
of Ran is induced by RanGAP1 and rna1p but not by Rna1p. 1
µM Gsp1p (A) or Ran (B) complexed with
[-
P]GTP was incubated at 30 °C with 0.1
nMS. cerevisiae Rna1p (
) or 0.1 nMS. pombe rna1p (
), or 0.1 nM human-RanGAP1
(▾) or with buffer (
). GTPase activity was determined as the
decrease of [
-
P]GTP bound to nitrocellulose
filters. Data are presented as the percentage of the amount of
radioactivity bound to Ran/Gsp1p 30 s after addition of the respective
GAP (``zero time point'').
Testing whether the
GTPase activity of human Ran could also be enhanced by S.
cerevisiae Rna1p and S. pombe rna1p, we found that Rna1p
was unable to increase the GTP hydrolysis of Ran while rna1p could
(Fig. 4B). The observation that Gsp1p is able to
interact with all three GAPs investigated while Ran is not indicates
that the specificity of the interaction rests mainly with the
nucleotide-binding protein rather than with the GTPase-activating
protein.
g supernatant of a protein
extract from S. cerevisiae on a Resource Q column
(Fig. 5). We detected only one discrete peak of GAP activity
specific for Gsp1p eluting at about 550 mM salt. Purified
Rna1p eluted at the same salt concentration under these conditions.
Therefore, we did not find any evidence for more than one GAP activity
specific for Gsp1p, although we cannot exclude the possibility of
overlapping activities.
Figure 5:
GTPase activating activity specific for
Gsp1p in a cell extract from S. cerevisiae. A 100,000
g supernatant of a crude cell lysate was eluted from a
Resource Q FPLC column using a 0-1500 mM KCl gradient.
1-ml fractions were analyzed for protein concentration (
) and
GTPase activity of Gsp1p (hatched bars). GTPase activity was
determined in filter binding assays. The hatched bar on the
left represents 100% GTP hydrolysis and the solid bar represents the intrinsic amount of GTP hydrolysis by
Gsp1p.
DISCUSSION
We have demonstrated here that Rna1p from S. cerevisiae is a GTPase-activating protein for Gsp1p. Sequence analysis
reveals that RNA1 belongs to a family of conserved genes,
including human (RanGAP1), mouse (fug1), S. pombe (rna1), S. cerevisiae (RNA1), and C.
elegans(24) . When compared to members of the RasGAP
family, Rna1p homologues are relatively small and significantly
stronger conserved. Furthermore, the conserved regions extend over the
entire protein, while in RasGAPs only a small region which possesses
catalytic activity is conserved.
-
structural units. The
-strands form a curved
-sheet constituting the inner circumference, while the
-helices align on the outer circumference
(38) . In addition
to the conserved leucines the LRR-containing proteins contain a
conserved Asn, Cys, or Thr at a defined position of the repeat, but the
functional and evolutionary significance of the various consensus
residues is as yet unknown. Rna1p homologous proteins have a conserved
asparagine at this position. Furthermore, the RNase inhibitor contains
14 repeats while Rna1p contains only 8, two of which appear to be
separated by intervening sequences
(29) . Although it is likely
that the LRRs of Rna1p homologues form similar characteristic
-
structural units it will be interesting to see the
structural and functional difference between these proteins.
-fold (this paper, Refs. 3 and 32). In the case of the
GTPase activity of Ras it has been argued that residues of Ras-GAP
contribute to the active site to stabilize the reaction intermediate
and/or transition state of the reaction
(40) . Potential residues
for such stabilization, i.e. arginines, have been identified
as totally conserved residues in Ras-GAP and their mutagenesis reduces
k
approximately 50-fold.
(
)
It is interesting that similar residues are also totally
conserved in Ran-GAP's.
-catenin, plakoglobin, adenomatosis polyposis coli, p120, and
smgGDS
(47) . The arm motif is believed to function in
protein-protein interactions and although it remains to be determined
whether Ran interacts directly with importin it is interesting that one
of the arm motif containing proteins is a GTPase-regulatory protein.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.