(Received for publication, May 23, 1994; and in revised form, December 13, 1994)
From the
Biochemical, structural, and functional properties of Rab5
wild-type (WT) protein were compared with those of Q79L and N133I
mutants. The detergent
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
increased guanine nucleotide binding to Rab5 WT 10-fold. The
single-step catalytic rate of Rab5 WT exceeded that of Q79L 12.2-fold,
but the steady-state GTPase rate was only 2.8-fold greater because GDP
dissociation was rate-limiting and GDP dissociation was 3.6-fold slower
than for Q79L. In contrast, dissociation rates of GTP were
indistinguishable. Binding to Rab5 N133I was not detectable. GTP
protected Rab5 WT and Q79L from any apparent proteolysis by trypsin. A
20-kDa fragment was the major product of digestion in the presence of
GDP, and 12- and 8-kDa fragments were the major products in the absence
of added guanine nucleotides. Rab5 N133I underwent no apparent
proteolysis with 10 mM GTP or GDP, suggesting a
``triphosphate'' conformation may be induced in Rab5 N133I by
either GTP or GDP. Partially geranylgeranylated Rab5 WT stimulated
endosome fusion in vitro, whereas unmodified Rab5 WT did not.
Processed Rab5 Q79L failed to inhibit endosome fusion, and Rab5 N133I
could not be geranylgeranylated. These findings identify biochemical
and structural features of Rab5 proteins, providing data for the
interpretation of functional assays.
Eukaryotic cells maintain a highly compartmented organization, and are capable of ordered and specific transport among different intracellular compartments. A large number of Ras-related GTPases, termed Rabs, have been implicated in distinct steps of intercompartmental transport (for review, see (1) ). Regulatory GTPases shuttle between two activity states, which are determined by the phosphorylation status of bound guanine nucleotides(2) . It has been proposed that a cycle of regulated nucleotide exchange and GTP hydrolysis is superimposed on the cycling of Rab proteins between donor and acceptor compartments to ensure accurate and directional vesicle transport.
Among the known Rab GTPases, Rab5 is of great interest because it appears to be rate-influencing for receptor-mediated and fluid-phase endocytosis. Lateral fusion between endocytic vesicles is stimulated by Rab5 both in vitro and in vivo, and antibodies against Rab5 inhibit fusion in vitro(3, 4, 5) . Endosome fusion in vitro is inhibited by cytosol containing overexpressed mutant Rab5 N133I protein that has impaired guanine nucleotide binding (3) . In cells overexpressing Rab5 N133I, the rate of receptor-mediated and fluid-phase endocytosis is significantly decreased compared with control(4, 5) . The N133I mutant is believed to interfere with endocytosis by interacting nonproductively and competitively with some important component of the endocytic apparatus.
If Rabs must cycle between GDP- and GTP-bound forms in order to function, then a mutation reducing the GTPase activity (such as the cognate of H-Ras Q61L) is predicted to also be a dominant inhibitor. This is the case with Rab2, but not with Rab1(6) , and the Sec4 mutation has an intermediate phenotype(7) . Wild-type Rab25 actually contains Lys at the cognate position(8) . The molecular basis for the functional differences among these related proteins is not known.
Although
several in vivo and in vitro functional studies of
Rab5 have been published, this protein has not yet been subjected to a
detailed biochemical analysis. Correct interpretation of in vivo and in vitro experiments using WT ()and mutant
Rab5 proteins depends on thorough knowledge of the biochemistry of
these reagents. As a step toward understanding the role of Rab5 in
endosome fusion and endocytosis, we have expressed in Escherichia
coli Rab5 WT, its N133I mutant, and a putative GTPase defective
mutant (Q79L). The biochemical properties, particularly the kinetics of
nucleotide binding and GTPase activities, of purified recombinant
proteins were characterized; functional properties of the purified
proteins were studied using an in vitro endosome fusion assay
to verify predicted phenotypes; and nucleotide-dependent structural
properties of the proteins were analyzed by limited proteolysis.
Figure 1:
Purification of recombinant Rab5 WT. Lane1, molecular weight protein standards (M
10
); lane2, whole cell lysate of E. coli BL21(DE3)-pT7.7-Rab5 culture (60 µg); lane3, supernatant after 20,000
g spin and
filtration through a glass fiber filter (40 µg); lane4, DEAE-Sepharose FF flow-through pool (8 µg); lane5, Sephacryl S-200 HR peak pool (4 µg).
Proteins were resolved by electrophoresis through a 12% polyacrylamide
SDS gel.
Figure 2:
Binding of [P]GTP
to Western blotted proteins. Crude proteins (2 µg/lane)
were resolved by SDS-PAGE, electrotransferred to nitrocellulose, and
then incubated with (A) [
-
P]GTP or (B) [
-
P]GTP (both 10
cpm/pmol) for 1 h at room temperature. The nitrocellulose was
then washed for 1 h, dried, and exposed to x-ray film as
described(57) .
The
effect of 0.1% CHAPS on [S]GTP
S binding to
Rab5 was time-dependent as shown in Fig. 3A. To test
the hypothesis that the increase in [
S]GTP
S
binding induced by CHAPS might be due to acceleration of prebound
nucleotide dissociation, 100 nM [
H]GDP
was loaded onto Rab5 by transient magnesium chelation. However, the
dissociation of [
H]GDP from Rab5 in the presence
of 0.1% CHAPS was slower than in its absence (Fig. 3B, diamonds). When excess unlabeled GDP was not added after
loading [
H]GDP, binding remained stable in the
presence of CHAPS but declined rapidly in its absence (Fig. 3B, circles). These results suggest that
without added detergent, Rab5 quickly assumes a conformation that does
not bind guanine nucleotides with high affinity. This conclusion is
consistent with the decline in the small amount of
[
S]GTP
S initially bound to Rab5 in the
absence of CHAPS during the course of prolonged incubation (Fig. 3A, opencircles). It should be
noted that the recombinant protein is not post-translationally modified
when expressed in E. coli, and therefore lacks geranylgeranyl
groups that are attached to Rab proteins in
eukaryotes(29, 30) .
Figure 3:
Effects of CHAPS on guanine nucleotide
binding to Rab5. A, association of
[S]GTP
S. Recombinant Rab5 WT purified in
the absence of CHAPS was incubated with 500 nM [
S]GTP
S either in the absence or
presence of 0.1% CHAPS for 240 min at 30 °C or was incubated
initially in the absence of CHAPS for 90 min and then in the presence
of 0.1% CHAPS for the next 150 min. At the indicated times, aliquots
were removed and rapidly filtered through nitrocellulose. B,
dissociation of [
H]GDP. Rab5 WT was loaded with
[
H]GDP by transient magnesium chelation followed
immediately by the addition of binding buffer with or without 0.1
mM GDP and with or without 0.1% CHAPS (final concentrations).
Aliquots were removed at the indicated times and assayed by vacuum
filtration through nitrocellulose.
The findings with Rab5
contrast with the lack of effect of CHAPS on nucleotide binding to
unprocessed Rab6 (31) but are not unique to Rab5 in that
similar effects were observed by us with bacterial recombinant Rab4
(not shown). Of note, both nucleotide binding and GTPase activities of
Rab6 differ dramatically between the processed and unprocessed
forms(31) . Together with our results, this suggests that
conformational effects on the nucleotide binding and hydrolyzing site
of Rab proteins may be induced either by covalently attached prenyl
groups or by noncovalently associated detergents, and such effects may
vary from one Rab protein to another. Supporting the involvement of the
carboxyl terminus of Rab5 in guanine nucleotide binding was our finding
that Rab5 truncated after amino acid 184 (cognate of
H-Ras) was insoluble in E. coli (not shown),
similar to Rab5 N133I and to several Ras mutants that are both
defective in nucleotide binding and insoluble in E. coli(14) . In contrast, truncated H-Ras
is
soluble in E. coli and has guanine nucleotide binding and
hydrolyzing properties indistinguishable from those of the full-length
protein (32) . In addition, reciprocal interactions between
guanine nucleotide binding and guanine nucleotide dissociation
inhibitor binding to carboxyl-terminally prenylated Rabs have been
described for other Rab proteins(1) , suggesting an interaction
between the carboxyl terminus and the guanine nucleotide-binding
domains. Of interest, guanine nucleotide binding by ADP-ribosylation
factor, another small GTPase that regulates vesicular traffic, is
highly dependent on interactions with lipids and detergents when its
acylated amino terminus is intact (33) .
Figure 8:
Proteolysis of Rab5 proteins by trypsin.
Purified recombinant Rab5 WT (A), Q79L (B), or N133I (D), were preincubated in the absence or the presence of 10
mM nucleotides and 5 mM MgCl for 1 h at
30 °C. Proteins (2.5 µg) were then incubated for 1 h with or
without 0.25 µg of trypsin in the presence of the indicated
nucleotides in a total volume of 50 µl at 30 °C, as described
previously(42) , and boiled for 5 min in sample buffer, and the
resulting peptides were resolved by SDS-PAGE in a Tris-Tricine buffer
system(58) . PanelC depicts an identical
experiment to panelA, except that the concentration
of trypsin was reduced to 0.05 µg.
The
pseudo-first order association rate constant of
[S]GTP
S with Rab5 Q79L (0.013
s
) was 2.3-fold faster than that with WT (0.0057
s
) (Fig. 4). Since previously purified Ras
family proteins had GDP stoichiometrically bound due to their very high
affinities(17, 34, 35) , we hypothesized that
the difference in association rates was due to differences in
dissociation rates of GDP. As predicted, the rate of dissociation of
GDP from Rab5 Q79L exceeded that from WT 3.6-fold (Fig. 5A). This is similar to the 3.2-fold increase in
the k
of the cognate mutant of Rab3A (22) and the 6.7-fold increase in the Ram cognate(19) ,
but it contrasts with the 2-fold decrease in the k
of the cognate mutant of
G
(36) . Differences in the effects of the Gln
Leu mutation on nucleotide exchange rates among different
GTPases may account for some of the phenotypic differences
observed(6, 7, 36, 37) . In contrast
to GDP, the dissociation rates of GTP and GTP
S did not
significantly differ between Rab5 WT and Q79L (Fig. 5, B and C); such comparisons have not been reported for other
Rabs. The first-order dissociation rate constants for each nucleotide
are shown in Table 1and are intermediate among those of other
Rabs (summarized in (35) ). The ratio k
/k
is 0.93 for
Rab5 WT, similar to that of Ras and other Rabs with the exception of
Sec4, which is unique in having a ratio of
120(31, 35, 38) .
Figure 4:
Association of
[S]GTP
S with Rab5 WT and Q79L. Rab proteins
(50 nM) were incubated in the presence of 500 nM [
S]GTP
S at 30 °C for the indicated
periods of time. One hundred-µl aliquots were diluted with cold
washing buffer and filtered through
nitrocellulose.
Figure 5:
Dissociation of guanine nucleotides from
Rab5 WT and Q79L. Proteins were preincubated with 500 nM labeled nucleotide for 3 h and then chased with 1 mM cold
nucleotide at 30 °C. One hundred-µl aliquots were diluted with
cold washing buffer and filtered through nitrocellulose at the
indicated times. A, [H]GDP; B,
[
-
P]GTP; C,
[
S]GTP
S.
Figure 6:
Rab5 WT and Q79L GTPase activities. A, steady-state [-
P]GTPase
activities were measured over 4 h at 30 °C by the charcoal method. B, following rapid loading of Rab5 proteins with
[
-
P]GTP, pre-steady state GTPase rates were
measured at early times by the charcoal method. C, single-step
GTPase rates were measured by the filtration method. See
``Materials and Methods'' for experimental details and
calculations.
The net GTPase reaction may be schematized as in Fig. 7, in which the transit time is given by ,
Figure 7:
Rab5 GTPase cycle. A simplified scheme of
the GTPase cycle is illustrated, which does not resolve the chemical
step of GTP hydrolysis from P dissociation because these
are not readily analyzed independently and which does not show any
conformational states. Each step is numbered such that the forward and
reverse rate constants for Step i are k
and k
, but if the reverse reaction is
negligible, it is not included. Transit times for individual steps were
calculated as the inverse of the individual step net rate constants,
and the complete cycle transit time was calculated neglecting k
` due to its presumed negligible contribution to
the overall rate (see text for details).
where k` is the single net rate constant for each step (40) . If the rate of nucleotide binding to apo-Rab5 (k) is exceedingly fast compared with the rates of
GTP dissociation (k
), GTP hydrolysis (k
), and GDP dissociation (k
)
(for H-Ras, k
= 1.4
10
M
s
(41) , and for Rab9, k
= 1.2
10
M
s
(35) );
the hydrolysis of GTP is essentially irreversible under physiologic
conditions; and the association of GDP with apo-Rab5 may be ignored in
the presence of excess GTP, then reduces to
1/k
= 1/k
+
1/k
. This rearranges to k
= k
k
/k
+ k
. Calculated this way from the
measured first-order rate constants k
and k
, the turnover number (k
)
of both Rab5 WT and Q79L is 0.0037 s
(Table 2). The calculated and measured k
of Rab5 WT are nearly identical, but those of Q79L differ
approximately 3-fold (Table 2). Since measurement of the
first-order rate constants k
and k
by filtration does not depend on independent determination of the
number of active Rab5 molecules, these constants should be highly
accurate, and determination of k
by calculation
from single step rate constants may be preferable to direct measurement
of steady state kinetics by the charcoal method.
The rate for Rab5 WT is intermediate among the rates of other Rabs measured by charcoal sedimentation (summarized in (35) ). Since the hydrolytic step for Rab5 WT (transit time, 16 s) is considerably faster than the GDP dissociation step (transit time, 256 s), the overall rate constant approaches that of GDP dissociation. In contrast, due to the accelerated GDP dissociation rate and the retarded GTPase rate of Rab5 Q79L, both the hydrolytic step (transit time, 196 s) and the GDP dissociation step (transit time, 71 s) contribute substantially to the overall rate constant.
In the absence of added nucleotides, proteolysis of Rab5 WT yielded two major fragments of 12 and 8 kDa (Fig. 8A). It should be noted that Rab5 is not expected to be stoichiometrically free of bound guanine nucleotide in this circumstance because Ras and other Rabs are purified from E. coli with GDP bound(17, 34, 35) , and the concentration of GDP in solution was estimated at 30 nM after dilution of the protein as described under ``Materials and Methods.'' Nonetheless, the conditions of proteolysis were optimized to demonstrate the nucleotide-dependence of proteolysis. An ``empty state'' distinct from the GDP-bound state was effectively demonstrated since there was virtually none of the 20-kDa fragment, which was almost stoichiometrically generated in the presence of added GDP (Fig. 8A). It was not clear what the relationship is between the tryptic fragments observed by us and the one reported by Steele-Mortimer et al.(47) while this manuscript was in preparation. In that report, the major fragment appears by SDS-PAGE to be approximately 20 kDa, but amino-terminal sequencing indicates that only four amino acids had been digested.
The results for Rab5 Q79L were almost identical to those for WT (Fig. 8B), suggesting that the mutation did not result in major conformational changes. The only difference was that for Rab5 Q79L, GDP was less effective in preventing degradation to 12- and 8-kDa peptides, consistent with the accelerated dissociation of GDP from Rab5 Q79L compared with WT (Fig. 5A).
Rab5 N133I did not
undergo any observable shift in mobility by SDS-PAGE in the presence of
10 mM GDP (Fig. 8D) or GTP (not shown), but 10
mM GTPS conferred no protection (Fig. 8D). In contrast to Rab5 WT and Q79L, which were
highly protected by 100 µM guanine nucleotides, Rab5 N133I
was unprotected at this concentration (not shown). These results
confirm that Rab5 N133I has an extremely low affinity for guanine
nucleotides and suggest that its conformation with either GTP or GDP
bound is similar to the Rab5 WT
GTP conformation. This is
consistent with the transforming phenotype (implying an active GTP-like
conformation) of H-Ras N116I (14, 25) but raises the
question of whether the biochemical activities of Asn
Ile
mutants are dependent on the phosphorylation state of bound guanine
nucleotides. Similarly, it has been suggested that activation of the
downstream signaling pathway by H-Ras G12V may be less guanine
nucleotide-dependent than activation by H-Ras WT(48) .
Our
results also suggest that the mechanism of dominant inhibition of
endocytic function by Rab5 N133I (3, 4, 5) is
via nonproductive interaction with downstream (i.e. Rab5GTP-interactive) vesicle transfer regulatory components,
since the product of proteolytic protection of Rab5 N133I by native
guanine nucleotides was similar to that of Rab5 WT
GTP. The fact
that GTP
S offered no protection from proteolytic digestion (Fig. 8D) may simply reflect the relative inability of
GTP
S to induce a GTP-like conformation of Rab5 N133I, as was the
case for Rab5 WT (see above), or it may additionally reflect structural
instability of the N133I mutant with heightened sensitivity to subtle
differences among ligands.
Figure 9:
In vitro prenylation of Rab5
proteins. Prenylation of Rab5 WT, Q79L, and N133I proteins was
accomplished using rabbit reticulocyte lysate and
[H]geranylgeranyl pyrophosphate as described
under ``Materials and Methods.'' Proteins were resolved on a
urea (4-8 M)/acrylamide (10-15%) gel, and then
processed for fluorography.
While both Rab5 WT and Q79L become post-translationally modified in vitro, the extent of protein processing is limited (<2%). Other investigators have reported similar results in studies of the in vitro prenylation of recombinant Rab proteins isolated from E. coli(29, 30) . This is in contrast to modification of Rab proteins overexpressed in mammalian cells or expressed in a reticulocyte lysate, wherein such proteins are apparently processed quite efficiently(4, 5, 20, 39) . What is clear from our results is that post-translational modification of Rab5 is essential for its function in endosome fusion (see below), but what remains to be determined is in what ways the in vitro prenylation of the E. coli-expressed protein differs from that synthesized by mammalian systems. For example, when translated by the very same reticulocyte lysate used to support the modification of the recombinant proteins, newly synthesized Rab5 rapidly incorporates geranylgeranyl and is converted to a fully processed form within 3-4 h of incubation(20) . In contrast, similar amounts of bacterially expressed Rab5 proteins failed to become fully processed (Fig. 9), and it is possible that other events of co- or post-translational processing may occur in the reticulocyte lysate that are not supported by E. coli. Such is, in fact, the case for the post-translational modification of H-Ras, which is known to be palmitoylated on a cysteine residue upstream from the carboxyl-terminal site of isoprenylation(49) . In support of a similar situation for Rab5, there is at least one report of fatty acid acylation of Rab family members(50) , and it is possible that other protein processing steps can occur, including proteolysis and carboxymethylation at the carboxyl terminus. Another limiting factor for in vitro prenylation of recombinant Rab5 may result from the rather unique features of Rab geranylgeranyl transferase(51, 52) . A component of this enzyme, REP (Rab escort protein), continues to interact with Rab proteins after geranylgeranylation is complete(53) . The complex between REP and Rab proteins is thought to dissociate only upon interaction with other cellular factors, such as GDI guanine nucleotide dissociation inhibitor(54) . It is possible that for the E. coli-expressed protein, interactions with factors such as REP or GDI are somehow limited in the in vitro system, accounting for incomplete processing.
Our cell-free system has
been previously shown to support fusion between early endocytic
vesicles(21) , an activity that is inhibited by the presence of
GTPS in vitro(22) . As demonstrated by the
results of Table 3, processed (geranylgeranylated) Rab5 WT
stimulated in vitro vesicle fusion when added to postnuclear
supernatant fractions containing endosomes that were not depleted of
any protein factors. This result suggests that Rab5 is rate-limiting
for endosome fusion, an idea consistent with observations reported for
transfected, Rab5-overexpressing
cells(4, 5, 39) . Unprocessed Rab5 either had
no activity in our assay or suppressed vesicle fusion slightly (Table 3). This result is consistent with in vivo deletion studies performed with overexpressed Rab5, which
indicated that removal of the terminal 4 amino acids from WT resulted
in nonfunctional protein(5) . Our observations with the
unprocessed, full-length Rab5 molecule support the idea that the latter
effect can be entirely attributed to the lack of post-translational
geranylgeranylation of the 2 cysteine residues contained within this
domain of the molecule.
As noted above, Rab5 N133I was a poor substrate for geranylgeranylation and progressively precipitated after purification. It was therefore not possible to determine whether its failure to significantly affect in vitro endosome fusion (not shown) was due to inadequate prenylation, structural instability, or an intrinsic property of the protein. Rab5 Q79L, in contrast, was processed to a similar extent to Rab5 WT and was stable in the presence of guanine nucleotides and magnesium. The modified Q79L mutant not only failed to inhibit fusion activity but actually stimulated fusion on every occasion tested, although the results for stimulation were not statistically different from control at 95% confidence intervals (p = 0.065). The stimulatory effect of Rab5 Q79L was never greater than or equal to that observed for WT in matched experiments, consistent with the results of Stenmark et al.(39) .
One inference that can be drawn from our results, assuming that the
Q79L protein is not functionally inactive despite its activity in the
above biochemical assays, is that Rab5 Q79L does not inhibit in
vitro endosome fusion. This is surprising given that the
equivalent GTPase-defective mutant of Ras is a dominant promoter of
transformation(23) , and Rab cognates could be expected to be
dominant inhibitors of vesicle transfer. However, in vivo studies of cells overexpressing Rab5 Q79L document that this
mutant instead acts as does Rab5 WT to stimulate
endocytosis(5, 39) . The cognate GTPase defective
mutant of Rab1B also fails to interfere with in vivo ER to
Golgi transport, although Rab2 Q65L is a potent inhibitor of this
transport step when overexpressed(6) . One explanation for
these apparently conflicting results might be the near-normal k for Rab5 Q79L despite a 12-fold reduction in
the single-step GTPase rate constant (Fig. 7). However, by
analogy with other well-studied GTPases(2) , each step of the
Rab5 GTPase cycle is likely to be regulated by interactive proteins in vivo, and a more likely explanation for the apparently
discrepant results among various Gln
Leu mutants might lie in
differences in their interactions with endogenous target proteins and
GAPs (GTPase activating proteins). The transforming potential of H-Ras
Q61L is primarily due to an alteration in the protein's ability
to interact with GAPs (p120-GAP and neurofibromin) such that the mutant
is predominantly found in the GTP-bound state(55) . The
corresponding point mutants of several Rab proteins result in defective
intrinsic GTPase activities, yet the Rabs are still capable of
interacting with GAPs such that the hydrolysis of GTP is not impaired in vivo relative to WT. Thus, even though Rab3A Q81L displays
a defective GTPase activity, it remains sensitive to the action of
Rab3A GAP to stimulate hydrolysis, the ratio of GDP/GTP bound in
vivo is the same as for WT, and its action in cells is similar to
that of WT (24, 56) . Likewise, Sec4 Q79L displays
defective GTPase activity, yet hydrolysis can be stimulated by a
GAP(7) . These examples suggest that Rab5 Q79L might
effectively interact with a target protein and act as WT in the fusion
process. This is also consistent with our proteolysis data, which
suggest that the guanine nucleotide-dependent conformations of Rab5
Q79L are similar to those of Rab5 WT. It remains to be determined in
future studies exactly how Rab5's function in endocytosis relies
on interactions with GAPs and other proteins and precisely what role
the cycling between GTP- and GDP-bound forms of Rab5 plays in endosome
fusion.