(Received for publication, August 29, 1995)
From the
Mg inhibits GDP release from Rab5
but not from Rab5
, a mutant lacking Ser
critical for Mg
coordination in the nucleotide
binding pocket. Thus, inhibition of GDP release is apparently exerted
via coordination of Mg
between Rab5 and GDP.
Mg
also induces conformational changes in
Rab5
, demonstrated by increased tryptophan fluorescence
intensity and a red shift in
for the GDP-bound
protein. Mg
-induced fluorescence changes are not
observed for Rab5
. The correlation between
Mg
effects on nucleotide exchange and the
fluorescence properties of Rab5 suggests that a conformation promoted
through Mg
coordination with Ser
also
contributes to inhibition of GDP release. The role of structural
changes in GDP release was investigated using C- and N-terminal
truncation mutants. Similar to Rab5
, Mg
inhibits GDP release and alters the fluorescence of
Rab5
but only partially inhibits release from
Rab5
and fails to induce changes in the
latter's fluorescence properties. Since Rab5
maintains Ser
necessary for Mg
coordination, the lack of Mg
-induced
fluorescence changes suggests a requirement for the N-terminal domain
to promote a conformation blocking GDP release. A model for mechanisms
of interaction between Ras-like proteins and their exchange factors is
proposed.
Rab proteins are a family of Ras-like small molecular weight
GTPases that are localized to distinct subcellular
compartments(1, 2) and believed to regulate specific
steps of intracellular membrane
trafficking(3, 4, 5, 6) . The
functional cycle of Rab proteins involves the delivery of the GDP-bound
forms to the target membrane by a GDP dissociation inhibitor
(GDI)()(7, 8, 9) , the exchange of
GDP for GTP at membrane surface catalyzed by a guanine nucleotide
exchange factor (GEF) (8, 9) and the retrieval of the
GDP-bound forms from the membrane by GDI after GTP hydrolysis and
membrane fusion(7) . Localized on plasma membrane,
clathrin-coated vesicles, and early endosomes(2) , Rab5 has
been shown to play an important role in early events of
endocytosis(4, 5) , although the exact mechanism of
its function remains to be determined.
It is known that
Mg is essential for GTPase function and structure.
Crystallographic studies of several GTP-binding proteins reveal a
single Mg
in the guanine nucleotide binding pocket,
coordinating between the protein and guanine nucleotide in both GDP-
and GTP analog-bound
conformations(10, 11, 12, 13, 14, 15) .
Effects of Mg
on guanine nucleotide binding, GTPase
activity, and the structural integrity of GTP-binding proteins have
been widely
documented(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) .
A key observation is that Mg
inhibits GDP release
from Ras-like GTP-binding proteins and therefore prevents binding of
GTP
S(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) .
However, the exact mechanism for this inhibitory effect and its
physiological significance remains unknown.
Important functional roles of the N-terminal domains of several Ras-like GTP-binding proteins also have been noted in studies of guanine nucleotide exchange(33, 34, 35, 36, 37, 38) . Myristoylation at the N terminus of ARF enhances its rate of GDP release(27) , and N-terminal truncation of ARF results in loss of function by reducing its affinity for GDP and permitting GDP/GTP exchange in the absence of phospholipids(33) . Moreover, deletion of the N terminus enables isolation of ARF in a nucleotide-free form(38) . Finally, deletion of the N-terminal domain of Rab5 results in a loss of function (34, 35, 36, 37) and interferes with the protein's post-translational processing(37) . These observations suggest that N-terminal domains of Ras-like GTP-binding proteins may participate in the regulation of guanine nucleotide exchange and represent crucial structural domains necessary for the function of the proteins.
We have investigated mechanisms through
which Mg and the N-terminal domain of Rab5
participate in its regulation of GDP release. Physiologic
concentrations of Mg
block GDP release from
Rab5
but not from Rab5
, a mutant lacking
Ser
critical for Mg
coordination.
Mg
also alters the intrinsic tryptophan fluorescence
properties of Rab5
but not Rab5
. While the
structure and function of Rab5
, a C-terminal
truncation mutant, is influenced by Mg
in the same
fashion as Rab5
, an N- and C-terminal truncation mutant,
Rab5
, is resistant to the cation's
effects. Thus, inhibition of GDP release by Mg
appears to be exerted via chemical constraints due to the
cation's coordination between GDP and Rab5, as well as
conformational restraints involving the protein's N-terminal
domain that are induced by Mg
coordination with
Ser
of Rab5. Based on the correlation between inhibition
of GDP release and conformational changes promoted by
Mg
, we propose that in vivo, Mg
prevents GDP dissociation from Rab5 until a guanine nucleotide
exchange factor acts to promote GDP/GTP exchange, perhaps through
interactions with the GTP-binding protein's N-terminal domain.
Figure 1:
Effects of
Mg on the rate of [
H]GDP
dissociation from Rab5
and Rab5
. Purified
Rab5
(panel A) and Rab5
(panel
B) (5 µM) were incubated at 37 °C for 30 min with
200 µM [
H]GDP (specific activity,
2.2
10
cpm/mol). Control reactions containing
the same amount of protein and [
H]GDP, but with
20 mM unlabeled GDP were incubated in parallel to measure
nonspecific binding. After equilibration at room temperature, an
aliquot of each sample was filtered to determine the amount of
[
H]GDP bound. Excess unlabeled GDP (20
mM) with 0.5 mM (triangle) or 5 mM (circle) or without (square) Mg
were then added, and GDP dissociation was monitored at room
temperature. An aliquot of each sample was filtered at indicated time
points, and the amount of [
H]GDP bound was
determined by scintillation counting. Specific binding was determined
as difference in cpm measured in samples incubated with or without
excess GDP. Shown is the mean of duplicate measurements plotted as a
function of ln (B/B
) against time (B
, initial specific binding; B, specific
binding at each time point). Identical results were obtained on three
separate occasions.
To investigate the mechanism through which Mg inhibits GDP release, the point mutant Rab5
was
constructed by site-directed mutagenesis. Ser
of Rab5 is
expected to coordinate with Mg
in the guanine
nucleotide binding pocket in both GDP- and GTP-bound states based on
crystallographic evidence obtained for other GTP-binding
proteins(10, 11, 12, 13, 14, 15) .
The cognate mutant of Ras at this position, Ras
,
displays an accelerated rate of GDP release that is insensitive to
Mg
(20) . Fig. 1B demonstrates that Rab5
also exhibits a rate of GDP
dissociation greater than Rab5
, but more importantly, the
release of GDP is no longer affected by Mg
. As
summarized in Table 1, this effect is reflected in the large
difference in k
determined in the presence of
high Mg
(0.245 versus 0.006 min
for Rab5
). Thus, it is likely that coordination of
Mg
with Ser
participates in a direct
chemical restraint preventing dissociation of the nucleotide. However,
it is also possible that Mg
could promote structural
rearrangements in Rab5 through interactions with Ser
,
resulting in a protein conformation that is unfavorable for GDP
release. These two mechanisms are not mutually exclusive, and both may
contribute to the observed inhibitory effect of Mg
.
Figure 2:
Effects of Mg on the
tryptophan fluorescence properties of Rab5
. Purified
Rab5
(400 nM) was incubated in buffer A at 37
°C for 30 min with 20 µM GDP and indicated amounts of
MgCl
. Fluorescence emission spectra were recorded between
300 and 400 nm for each sample at an excitation wavelength of 290 nm.
Shown is a representative results from at least three separate
experiments. The
values determined for Rab5
in the absence and presence of 5 mM Mg
were, respectively, 340-341 and 342-343
nm.
Figure 3:
Effects
of Mg on the fluorescence intensities of Rab5
and Rab5
. Purified Rab5
(filled
squares) and Rab5
(open squares) (0.5
µM) were incubated in buffer A at 37 °C for 30 min
with 100 µM GDP and increasing amounts of
MgCl
. Fluorescence emission peak intensity and wavelength
was recorded for each sample at an excitation wavelength of 290 nm. The
fluorescence intensity of each sample was normalized against the
measurement of protein without Mg
and plotted as a
function of added Mg
concentration. Shown is the mean
of duplicate measurements. Similar results were obtained on at least
three separate occasions.
To evaluate the relative roles of
the N and the C termini as potential regulators of guanine nucleotide
exchange, three truncation mutants of Rab5 were characterized
(Rab5, Rab5
, and
Rab5
). Filter binding assays with
[
H]GDP and [
S]GTP
S
indicated that both Rab5
and
Rab5
bind guanine nucleotides in solution,
although Rab5
exhibits a reduced affinity for
both nucleotides (data not shown). Previous studies demonstrated that
Rab5
binds
[
H]GDP(38) . Further information on the
tertiary structure of these proteins was obtained from protease
protection assays(37) . As shown in Fig. 4, in vitro synthesized
S-labeled Rab5
migrates as a
27 kDa band on a urea/acrylamide gradient SDS gel. The synthetic
protein binds endogenous GDP(40) ; limited digestion of this
GDP-bound form with trypsin produces a single
S-labeled
fragment of 14 kDa. Addition of 30 mM EDTA to chelate
Mg
essential for guanine nucleotide binding markedly
reduces the amount of the latter tryptic peptide. However, incubation
with GTP
S prior to trypsinization results in protection of a
S-labeled 20-kDa peptide in addition to the 14-kDa
fragment, representing a ``core'' structure of Rab5 in the
GTP
S-bound conformation. Fig. 4further demonstrates that
protease protection profiles for Rab5
,
Rab5
, and Rab5
are all
similar to Rab5
, confirming that each of the truncation
mutants binds guanine nucleotides.
Figure 4:
Protease protection of N- and C-terminal
deletion mutants of Rab5 by guanine nucleotides. In vitro synthesized S-labeled Rab5
,
Rab5
, Rab5
, and
Rab5
were incubated at a final concentration of
3.5 nM for 45 min at 30 °C with or without 10 mM GTP
S or 30 mM EDTA as indicated. Proteolysis was
initiated by addition of 0.022 units of trypsin, and incubations were
continued for 45 min. Digestion was terminated with 2 µg of soybean
trypsin inhibitor at 20 °C for 5 min. Samples were diluted with 68
µl of Laemmli buffer, heated for 5 min at 100 °C, and
electrophoresed on urea/acrylamide gradient SDS gels. Shown is
fluorography of the dried gel (2-day exposure); tic marks indicate the migration of 20- and 14-kDa markers. Mass of
S-labeled tryptic fragments was determined as follows:
Rab5
, 14 and 20 kDa; Rab5
, 14 and 20
kDa; Rab5
, 14 and 18.3 kDa; and
Rab5
, 14 kDa and 18.3 kDa. The lowest molecular
weight species detected for Rab5
and
Rab5
is not a tryptic peptide fragment since it
appears in the(-) trypsin lanes.
By comparing the sizes of the
GDP- and GTPS-protected fragments of Rab5
and these
truncation mutants, a tryptic cleavage map can be predicted as shown in Fig. 5. Our assignments are made on the following premises.
Since both of the GTP
S-protected fragments for
Rab5
and Rab5
are
identical and approximately equal in size to undigested
Rab5
, a C-terminal proteolysis site must exist
upstream of, but close to, position 198. The only practical assignment
is Arg
. Trypsinization of the GDP-bound forms of all the
Rab5 molecules produces identical 14-kDa fragments, indicating that
cleavage sites must be downstream of position 23 and upstream of
position 198. The fact that this peptide is heavily radiolabeled
suggests that several methionine residues are present. Based on the
size of this tryptic fragment, Lys
and Arg
are the most likely tryptic sites since cleavage at these
residues would generate a 13.8-kDa fragment. Since the
GTP
S-protected fragments of Rab5
and
Rab5
migrate with a mass
1.7 kDa larger than
that of Rab5
on SDS-gels, if there is an
N-terminal tryptic site, it would be best positioned at Arg
or Arg
. However, because of the proximity of these
residues to the N terminus, and given the vagaries of peptide
mobilities on SDS gels, we cannot distinguish which Arg provides the
tryptic cleavage site nor can we confirm that proteolysis at the
extreme N terminus occurs. It should be noted that Steele-Mortimer and
co-workers (35) have previously assigned Arg
as a
tryptic site for Rab5 by N-terminal sequencing (35) . A key
prediction from the proposed tryptic map is that GTP
S-binding
protects against proteolytic cleavage at Lys
, supporting
the idea that the Rab5's N-terminal domain undergoes
conformational rearrangement upon GDP/GTP exchange.
Figure 5:
Schematic diagram of predicted sites of
trypsin proteolysis in the GDP- and GTPS-bound forms of Rab5.
Shown is a linear model of the Rab5 peptide backbone interrupted by solid bars depicting the putative guanine nucleotide binding
domains. Locations of all potential tryptic sites are indicated by short vertical bars. Locations of methionine residues in the
mature peptide are indicated by asterisks. Our proposed
assignment of cleavage sites (long vertical bars) in the GDP-
and GTP
S-bound forms of Rab5 was based on determination of the
mass of
S-labeled tryptic peptides as shown in Fig. 4and discussed in the text. Lys
and
Arg
are chosen as tryptic sites since cleavage would be
predicted to generate a 13.8-kDa fragment and Arg
is the
most logical assignment as a C-terminal proteolysis site in all of the
GTP
S-bound forms. Alternative assignments that cannot be
completely ruled out are at N-terminal residues
Arg
/Lys
and C-terminal residues
Lys
/Lys
/Lys
; however,
proteolytic cleavage at these sites would yield
15-kDa peptides.
It should be noted that this alternative assignment does not argue
against conformational changes protecting the N-terminal domain, it
simply indicates that the Arg
/Lys
is the
location of the protected cleavage site and that C-terminal
conformational changes also might occur upon guanine nucleotide
exchange.
In comparison
with Rab5, Rab5
produces a distinct
protease protection pattern (Fig. 6). In the absence of
exogenously added guanine nucleotide, Rab5
is completely
degraded by trypsin, unlike wild-type, which displays a 14-kDa fragment
protected by endogenous levels of GDP. The accelerated rate of GDP
release from the mutant (Fig. 1B) is most likely
responsible for this effect, making Rab5
more
susceptible to proteolysis. When saturating levels of GDP or GTP
analogs are added, a 14-kDa fragment of Rab5
is
protected. The absence of the 20-kDa core peptide in the GTP
analog-bound forms suggests that Rab5
resembles the
GDP-bound conformation of wild-type, consistent with previous reports
characterizing the cognate mutants of other Rab
proteins(48, 49, 50) . The failure of
GTP
S or Gpp(NH)p to protect the predicted Lys
cleavage site also suggests that Mg
coordination with Ser
is necessary to induce
conformational changes upon guanine nucleotide exchange.
Figure 6:
Protease protection of Rab5 and Rab5
. In vitro synthesized
S-labeled Rab5
and Rab5
were
incubated at a final concentration of 3.5 nM for 45 min at 30
°C with or without 10 mM guanine nucleotide analog
(GTP
S, Gpp(NH)p, or GDP
S) or 30 mM EDTA as
indicated. Proteolysis was carried out exactly as described for Fig. 4;
S-labeled tryptic fragments were 14 and 20
kDa for Rab5
and 14 kDa for
Rab5
.
Figure 7:
Effects of Mg on the
rate of [
H]GDP dissociation from
Rab5
and Rab5
.
[
H]GDP dissociation of Rab5
(panel A) and Rab5
(panel
B) were measured in the absence (square) and presence of
0.5 mM (triangle) or 5 mM (circle)
added MgCl
as described in Fig. 1. Each data point
is the mean of duplicate measurements. Identical results were obtained
on three separate occasions.
To test this
possibility, the influence of Mg on the fluorescence
properties of Rab5
and Rab5
was also compared (Fig. 8). Like Rab5
the
intrinsic fluorescence intensity of Rab5
is
increased by Mg
. In contrast to Rab5
but
similar to Rab5
, the fluorescence properties of
Rab5
do not change in response to increasing
[Mg
]. Thus, in addition to chemical
constraints exerted by Mg
coordination within the
guanine nucleotide binding pocket, the Mg
-Ser
link imposes structural restraints that involve the N-terminal
domain and contribute to inhibition of GDP release.
Figure 8:
Effects of Mg on the
fluorescence intensities of Rab5
and
Rab5
. Effects of Mg
on the
fluorescence properties of Rab5
(filled
square) and Rab5
(open square)
were measured as described in Fig. 2. Normalized fluorescence
intensities were plotted against added Mg
concentration as shown in Fig. 1. Similar results were
obtained on at least three separate
occasions.
Studies on crystallized Ras(10) , EF-Tu(11) ,
G(12) , ARF(13) , and Ran (14) in their GDP-bound forms have revealed identical
Mg
coordination within the guanine nucleotide binding
pocket. The cation interacts directly with the
-phosphate of GDP
and a highly conserved Ser/Thr near the N terminus of these proteins;
indirect interactions are mediated through four associated water
molecules. In the GTP analog-bound forms of Ras(10) ,
EF-Tu(1) , G
(12) , and
G
(15) , Mg
coordination with
two of these water molecules is replaced by a direct coordination with
the
-phosphate of the nucleotide and another highly conserved Thr.
Inhibitory effects of Mg
on GDP release have been
documented for a wide variety of small GTP-binding
proteins(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) ,
and our results extend this general observation to include Rab5.
Mutation of Ser
in Rab5 results in an increased rate of
GDP release, which is no longer affected by Mg
, a
phenomenon also observed for the cognate mutants of other small
GTP-binding proteins, including Ras(20) , Rab1(48) ,
Rab9 (49) , and Rab3(50) .
In vivo, the
release of GDP from Rab5, and most likely other small GTP-binding
proteins, would be inhibited by intracellular free
Mg. Thus, a common mechanism to regulate guanine
nucleotide exchange may be adopted by this family of proteins. In order
to facilitate GDP/GTP exchange, a GEF must overcome the inhibitory
restraints imposed by intracellular Mg
levels.
Through transient disruption of the Mg
-Ser/Thr
coordination, GEF could be envisioned to induce structural changes such
that the GTP-binding protein adopts a conformation similar to its Ser
Asn cognate mutant, thereby promoting GDP dissociation.
Several lines of evidence support this idea. Our fluorescence
studies demonstrate that Rab5 adopts a
Mg
-insensitive conformation that is distinct from the
Mg
-sensitive conformation of Rab5
.
Although the strong dominant inhibitory effects of cognate mutants are
generally attributed to their preferential affinity for GDP over
GTP(6, 48, 49, 52, 53, 54, 55, 56, 57, 58, 59, 60) ,
Wittinghofer and co-workers (20, 63) have argued that
the distinct structures of the Ser
Asn cognate mutants are most
likely responsible for disruption of wild-type protein function through
competition for GEF interactions. The fact that Ras
has
an even greater affinity for GDP than Ras
but is not a
suppressor of Ras function supports this idea(20) . Moreover,
Rab3A
(50) , Ras
(51) , and
Ran
(52) have been shown to have a much higher
affinity for their respective GEFs than wild-type, further suggesting
that complexes formed between the Ser
Asn cognate mutants and
their corresponding GEFs are thermodynamically more favorable. It has
been proposed that the yeast GEF Cdc25p promotes exchange by
stabilizing Ras in a nucleotide-free state(53) ; thus, it seems
likely that Ser
Asn cognate mutants of Ras-like factors are
analogous to the nucleotide-free conformations of these proteins. The
fact that exogenously added GDP and GTP analogs produce the same
protease protection profile indicates that Rab5
must
accommodate both nucleotides in its binding pocket but fails to undergo
molecular rearrangements associated with guanine nucleotide exchange.
This evidence supports the view that the mutant adopts a structure
intermediate between GDP- and GTP-bound states. Ser
is
therefore predicted to be a critical residue involved in the
conformational switch during guanine nucleotide exchange.
Our study
demonstrates that Mg-Ser
coordination
between GDP and Rab5 not only provides a chemical constraint, but also
promotes conformational changes involving the N terminus, which impose
additional structural restraints against GDP dissociation. The markedly
reduced inhibition of GDP release by Mg
and the lack
of Mg
-induced fluorescence changes in
Rab5
indicate that the N-terminal domain of Rab5
must play a key role in maintaining a conformation that blocks GDP
release. It should be noted that the first 23 amino acids of Rab5
correspond to a rather small domain comprising only 4 residues in Ras
based on pattern-induced multiple alignment analysis(37) . As
previously noted in a comparison with Ras(36) , the N-terminal
domain of Rab5 is one of five regions of this molecule predicted to
impart functional specificity. Thus, although Ras
(20) behaves in a fashion similar to Rab5
,
whether Mg
-induced N-terminal conformational changes
contribute to inhibition of GDP release from Ras is uncertain.
Comparison of Ras crystal structures in the GDP and GTP analog-bound
states reveals no obvious conformational changes in its N-terminal
domain(10) , but this does not preclude the possibility of
structural rearrangement during the guanine nucleotide exchange
process. Since the N terminus of ARF also has been reported to
participate in guanine nucleotide exchange regulation, it is possible
that structural features of this domain may be shared among other
Ras-like GTP-binding proteins. We speculate that GEF directly interacts
with the N-terminal domain of Rab5 to interrupt Mg
coordination with Ser
, thereby promoting a transient
conformation that facilitates GDP release. In support of this idea,
truncation of the N-terminal domain abolishes the function of
Rab5(34, 35) .
Since our data show that cellular
levels of Mg are sufficient to block GDP release,
what is the role of the guanine nucleotide dissociation inhibitor GDI?
Even though this factor was purified based on its ability to inhibit
GDP dissociation(64, 65) , under physiologic
Mg
concentrations its relative activity is only
marginal(65, 66, 67) . Except for Rac (68, 69) and Rho(70) , similar factors have
yet to be identified for other Ras family members. This suggests that a
GDI per se is not required to prevent GDP dissociation from
small GTP-binding proteins since cellular Mg
levels
are sufficient to provide this function. In fact, upon delivery of Rab
proteins to membranes, exchange for GTP is not immediate after GDI
dissociation(8, 9) . Our study indicates that during
this time period, Mg
most likely prevents GDP
dissociation. Thus, we speculate that the true function of the Rab GDI
may be one of a molecular escort protein or chaperone. In this
capacity, GDI may stabilize the Rab
Mg
GDP
complex, blocking interaction sites for GEF until the appropriate
membrane target is reached. This function is comparable with that of
the
subunit complex of heterotrimeric G proteins, which also
retards GDP release and targets
subunits to their appropriate
membrane receptors. Indeed, our recent studies revealed
structure-function similarities between Rab5 and the
subunits of
GTP-binding proteins (39) . However, GDP release from most
G
subunits is unaffected by Mg
. In
fact, the G
subunits contain a unique
-helical
domain absent from Ras-like GTP-binding proteins that may block GDP
release(12, 13) . The N-terminal domain of Rab5 may
substitute, in part, for this structural element.