©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biochemical and Functional Characterization of a Recombinant GTPase, Rab5, and Two of Its Mutants (*)

(Received for publication, May 23, 1994; and in revised form, December 13, 1994)

Simon Hoffenberg (1)(§) Jack C. Sanford (3)(¶) Shaobin Liu (1) D. Sundarsingh Daniel (1) Michael Tuvin (1) Brian J. Knoll (1) (2) Marianne Wessling-Resnick (3)(**) Burton F. Dickey (1) (2) (4)

From the  (1)Departments of Medicine and (2)Cell Biology, Baylor College of Medicine, Houston, Texas 77030, the (3)Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115, and the (4)Division of Pulmonary and Critical Care, VA Medical Center, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 approx10-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.


INTRODUCTION

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 (^1)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.


MATERIALS AND METHODS

Construction of Rab5 Mutants

Recombinant plasmids with human Rab5 and Rab4 cDNAs were obtained from A. Tavitian(9) . The cDNA inserts were amplified by add-on polymerase chain reaction and cloned into M13mp18 for nucleotide sequencing(10) . DNA sequence analysis of Rab5 clones from several independent polymerase chain reactions identified three discrepancies from the published sequence(9) : Arg replacing Gly at position 81, Val replacing Ala-86, and Arg replacing Gly-197. The Gly to Arg conversions are identical to residues found in these positions (amino acids 81 and 197) in canine Rab5(11) . The remaining alteration, Val to Ala, is conservative and most likely does not affect the functional integrity of the protein; in fact, the product of this cDNA can bind and hydrolyze GTP, can be geranylgeranylated, and functions in an endosome fusion assay (see ``Results and Discussion''). Site-directed point mutagenesis of Rab5 was performed by the method of Kunkel et al.(12) . Two point mutants, Q79L and N133I, were generated using the oligonucleotides 5`-GGTATCGTTCTAGACCAGCTGTA-3` and 5`-AGGTCCGGCCTTGATTCCCGATAAAG-3`, respectively. Mutants were verified by nucleotide sequence analysis in both directions. WT and mutant Rab5 cDNAs and the Rab4 WT cDNA were cloned into the T7-polymerase expression plasmid pT7.7 (13) utilizing the NdeI cloning site to ensure the production of recombinant proteins with native amino termini.

Purification of Rab Proteins

E. coli BL21(DE3) cells were transformed with recombinant plasmids and grown in LB medium containing 50 µg/ml ampicillin. When the A was approx 1.0, isopropyl-1-thio-beta-D-galactopyranoside was added to a final concentration of 0.8 mM. Cells were harvested 3-4 h later by centrifugation at 1500 times g for 30 min. The cell pellet from a 2-liter culture was resuspended in Tris-buffered saline (20 mM Tris-HCl, pH 8.0, 150 mM NaCl), centrifuged, and then washed with the same buffer. The washed pellet was suspended in 10 ml of hypertonic buffer (2.4 M glucose, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA) and kept on ice for 30 min. One hundred ml of a lysozyme solution (0.5 mg/ml lysozyme, 0.02 mg/ml DNase, 40 mM Tris-HCl, pH 8.0, 5 mM MgCl(2), 1 mM EDTA) was then added, and the incubation was continued on ice for a further 15 min. One hundred ml of lysis buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 5 mM MgCl(2), 1 µM GDP, 2 mM beta-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride) was then added. The lysate was centrifuged in a GSA rotor (Sorvall) at 11,000 rpm for 30 min, and the supernatant was filtered through a glass fiber filter. CHAPS was added to a final concentration of 0.1% at this point, except where noted. This solution was applied to a 500-ml DEAE-Sepharose FF column equilibrated with several volumes of Buffer A (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM beta-mercaptoethanol, 5 mM MgCl(2), 1 µM GDP, 0.1% CHAPS). Protein that did not bind to the resin was collected and concentrated on an Amicon YM-10 membrane and then applied to a 1500-ml Sephacryl S-200 column equilibrated with Buffer A plus 150 mM NaCl. Rab proteins in eluting fractions were identified by SDS-PAGE, and concentrated by membrane filtration. The N133I mutant was insoluble when expressed in E. coli, similar to the cognate Ras mutant(14) . Therefore, the protein was extracted from the bacterial pellet with 8 M urea, which was removed during chromatography on the gel filtration column. Rab4 was purified by sequential chromatography as for Rab5 WT, except that Rab4 bound to DEAE-Sepharose and was eluted with a linear gradient of NaCl in Buffer A. Protein concentrations were determined with the Coomassie Plus assay (Pierce), using bovine serum albumin as a standard. Proteins were stored at 2-5 mg/ml in Buffer A at -80 °C.

Binding of Nucleotides in Solution

Nucleotide binding was determined by a rapid filtration technique(15) . Binding buffer contained 20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 1 mM EDTA, 5 mM MgCl(2), 1 mM dithiothreitol, 0.1% CHAPS, 500 nM guanine nucleotide, and either [S]GTPS, [alpha-P]GTP, or [^3H]GDP (all 2,000 cpm/pmol). The protein concentration was 50 nM in a volume of 100 µl. After incubation at 30 °C for the indicated times, samples were diluted with 4 ml of iced buffer, filtered through BA85 nitrocellulose (Schleicher and Schuell), and then washed twice with 4 ml of iced buffer. Filters were then dried, immersed in scintillation fluid, and counted by scintillation counting. Data points in all figures represent the mean of triplicate determinations from a representative experiment that was repeated 2-4 times. Except where noted, maximum binding was determined by extrapolation from experimental data using the program Enzfitter (Biosoft, Ferguson, MO). In the presence of 0.1% CHAPS and 5 mM MgCl(2), maximum binding averaged 1.2 ± 0.2 mol of nucleotide/mol of Rab5.

Measurement of Dissociation Rates

These were measured as described previously(16) . Protein was preincubated for 3 h in the conditions described above for nucleotide binding, and then the bound labeled nucleotide was competed with 1 mM unlabeled nucleotide. At indicated times, samples were diluted with cold buffer, filtered, and counted, as above. In some experiments, Rab5 was rapidly loaded with radiolabeled guanine nucleotides by a modification of the procedure of Tucker et al.(17) as follows. Protein was incubated in the presence of 1.25 mM EGTA for 10 min at 30 °C to release prebound nucleotide and then radiolabeled nucleotide was added, followed by 5 mM MgCl(2).

GTPase Activity

Steady state GTPase activity was measured by the release of [P]P(i) using the ``charcoal assay'' as described(18) . Protein was incubated with 500 nM [-P]GTP (2,000 cpm/pmol) in the presence of 5 mM MgCl(2) at 30 °C, and at the indicated times aliquots were withdrawn and mixed with charcoal. Following centrifugation, the [P]P(i) in the supernatants was assayed by scintillation counting. Pre-steady state GTPase rates were estimated by the charcoal method at early times following rapid loading of Rab5 as above. The single-step GTPase rate was determined by a filtration assay following rapid nucleotide loading. This assay was performed essentially as described for the measurement of dissociation rates, with the exceptions that [-P]GTP was used, and the GTPase rate was calculated by subtracting the [S]GTPS dissociation rate from the apparent GTPase rate(19) . All rates were calculated using the program Enzfitter.

In Vitro Processing of Recombinant Rab5 Proteins

Post-translational modification of Rab proteins with geranylgeranyl was accomplished by an in vitro reaction supported by rabbit reticulocyte lysate (Promega, Madison, WI), as described(20) . Briefly, Rab proteins (final concentration, 0.1-0.8 mg/ml) were added to lysate together with 5 µM [^3H]geranylgeranyl pyrophosphate (33,300 dpm/pmol, American Radiolabeled Chemicals) in 10 mM Tris, pH 8.0, 0.1% CHAPS, 1 µM GDP, 5 mM MgCl(2), 1 mM EDTA, 2 mM beta-mercaptoethanol; the final mixture contained 40-60% reticulocyte lysate (v/v). The reaction mixtures were incubated at 37 °C for 3-4 h, after which time aliquots (3.4 µl) were removed for analysis by PAGE and fluorography. The amount of [^3H]geranylgeranyl incorporated into the proteins was determined by excising the bands from the dried gel, dissolving samples in 30% H(2)O(2) at 65 °C, and scintillation counting. After processing but prior to the addition to in vitro endosome fusion assays, the remaining reaction mixture was chromatographed over a Sephadex G25 spin column to remove excess radioactivity and to exchange the Rab protein into the appropriate buffer (20 mM Hepes, pH 7.4, 0.1 M KCl, 85 mM sucrose, 20 µM EGTA).

In Vitro Endosome Fusion Assay

Cell-free vesicle fusion assays were performed exactly as described previously(21, 22) . Postnuclear supernatant fractions were prepared from K562 cells that had endocytosed either biotinylated transferrin (BTf) or avidin-beta-galactosidase (AvbetaGal) at 20 °C for 45 min to load early endosomes. The postnuclear supernatant fractions were then dialyzed against 20 mM Hepes, pH 7.4, 0.1 M KCl, 85 mM sucrose, 20 µM EGTA, and frozen at -80 °C until use. Postnuclear supernatant fractions were rapidly thawed immediately prior to the assay, and 5-µl aliquots of each were added to a reaction mixture that contained 1 mM MgATP, 50 µg/ml creatine kinase, 8 mM phosphocreatine, 10 µg/ml biotin-insulin, 1 mM dithiothreitol, 400 µM GTP, with appropriate additions of Rab proteins and/or rabbit reticulocyte lysate as detailed in the legend to Table 1. Fusion activity was promoted by incubation of the reaction mixtures at 37 °C, while control reactions (4 °C) were held on ice. Specific vesicle fusion results in co-localization of AvbetaGal and BTf within fused vesicles, and the resulting avidin:biotin complex between the two probes was measured by a modified enzyme-linked immunosorbent assay using a fluorogenic beta-galactosidase substrate as detailed previously (21) . The signal in fluorescence units is directly proportional to the extent of endocytic vesicle fusion in the in vitro reaction. Samples (15-20 µl) of desalted prenylation reaction mixtures were tested in each assay.




RESULTS AND DISCUSSION

Expression of Rab5 and Two of Its Mutants

Induction of E. coli containing recombinant plasmids resulted in the accumulation of a major protein of 25 kDa in lysates as assessed by SDS-PAGE and Coomassie Blue staining (Fig. 1, lane2). Prior to purification, the predicted biochemical phenotypes of WT and mutant proteins were qualitatively confirmed by [P]GTP overlay assays. Lysates were Western blotted onto nitrocellulose filters and incubated with [alpha-P]GTP (Fig. 2A) or [-P]GTP (Fig. 2B). Autoradiography revealed that Western blotted Rab5 WT bound and hydrolyzed GTP, as indicated by reduced density of the 25-kDa band with -labeled GTP as compared with alpha-labeled GTP. In contrast, Rab5 Q79L showed a much smaller difference in band intensity between -labeled and alpha-labeled GTP, suggesting a reduced GTPase activity as expected from similar effects of the cognate mutation in Ras (23) and in other Rabs (7, 19, 24) . Rab5 N133I had a severe guanine nucleotide binding defect, as indicated by the complete absence of an autoradiographic signal with nucleotide labeled in either position, consistent with previous results (4, 5) . Cognate mutations in Ras (14, 25) and in other Rabs (6, 24, 26, 27) result in undetectable guanine nucleotide binding as assayed by rapid filtration or GTP overlay. This severe defect in nucleotide binding is not surprising in view of the central role of the highly conserved asparagine in linking three structural elements directly involved in nucleotide binding, as inferred from the crystal structures of Ras and EF-Tu(28) .


Figure 1: Purification of recombinant Rab5 WT. Lane1, molecular weight protein standards (M(r) times 10); lane2, whole cell lysate of E. coli BL21(DE3)-pT7.7-Rab5 culture (60 µg); lane3, supernatant after 20,000 times 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) [alpha-P]GTP or (B) [-P]GTP (both 10^6 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) .



Detergent Effects on Guanine Nucleotide Binding to Rab5

During our initial purification of Rab5, much of the partially fractionated protein precipitated. Efforts were therefore directed at stabilizing Rab5 in solution without losing [S]GTPS binding activity. Several nonionic and zwitterionic detergents caused a dramatic increase in [S]GTPS binding at a concentration of 0.05%, as did 0.01% bovine serum albumin, but there was no detectable binding in the presence of the ionic detergent SDS (not shown). When the concentration of the detergents was increased to 0.5%, only CHAPS still supported high [S]GTPS binding. Hence, subsequent protein purification and biochemical experiments were performed in buffers containing 0.1% CHAPS unless indicated.

The effect of 0.1% CHAPS on [S]GTPS binding to Rab5 was time-dependent as shown in Fig. 3A. To test the hypothesis that the increase in [S]GTPS binding induced by CHAPS might be due to acceleration of prebound nucleotide dissociation, 100 nM [^3H]GDP was loaded onto Rab5 by transient magnesium chelation. However, the dissociation of [^3H]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 [^3H]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]GTPS 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]GTPS. Recombinant Rab5 WT purified in the absence of CHAPS was incubated with 500 nM [S]GTPS 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 [^3H]GDP. Rab5 WT was loaded with [^3H]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) .

Purification of Bacterial Recombinant Rab5 and Two of Its Mutants

After stabilization of Rab5 WT and Q79L in solution with 0.1% CHAPS, 1 µM GDP, and 5 mM MgCl(2), these proteins were readily purified from the supernatants of E. coli lysates. Rab5 WT (calculated pI, 7.05) did not bind to DEAE-Sepharose at pH 8.0 and was recovered in the flow-through, while the bulk of contaminating E. coli protein remained on the column (Fig. 1, lane4). Most of the residual contaminating protein was resolved by molecular sieve chromatography, yielding Rab5 WT of approximately 95% purity as estimated by Coomassie staining after SDS-PAGE (Fig. 1, lane5). A minor 20-kDa contaminant appeared to be a degradation product of Rab5 due to carboxyl-terminal proteolysis since it bound [P]GTP on a GTP overlay, it reacted with affinity-purified antiserum to Rab5 holoprotein, and the amino terminus was intact by Edman degradation (not shown). Rab5 Q79L was purified by an identical scheme and yielded protein of similar purity (see Fig. 8B). Rab5 N133I was insoluble in E. coli lysates and was extracted with 8 M urea. Urea was removed during the molecular sieve chromatography, but approximately 90% of the protein subsequently precipitated. The remaining Rab5 N133I (see Fig. 8D) was fairly stable in solution in Buffer A containing 1 mM GDP.


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(2) 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.



Kinetics of Binding of Guanine Nucleotides to Rab5 WT and Q79L

In preliminary studies, the binding of [S]GTPS to Rab5 N133I could not be detected by rapid filtration assay. This is consistent with the Western blot binding assay (Fig. 2) and with results from other Ras family proteins and was therefore probably not simply due to extraction in urea. Data acquired subsequently from proteolysis (see below) indicate that purified Rab5 N133I does effectively interact with guanine nucleotides but with an apparent affinity that is several orders of magnitude lower than those of Rab5 WT and Q79L. Therefore, binding and GTPase studies were performed only with Rab5 WT and Q79L.

The pseudo-first order association rate constant of [S]GTPS 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(s)alpha(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 GTPS 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]GTPS with Rab5 WT and Q79L. Rab proteins (50 nM) were incubated in the presence of 500 nM [S]GTPS 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, [^3H]GDP; B, [alpha-P]GTP; C, [S]GTPS.



GTPase Activities of Rab5 WT and Q79L

The GTPase activities of Western blotted Rab5 WT and Q79L appeared dramatically different (Fig. 2), but GTPase rates measured under steady-state conditions by charcoal sedimentation revealed only a modest 2.8-fold difference (Fig. 6A and Table 2). We hypothesized that in view of the relatively slow dissociation rate of GDP (Fig. 5A), a more rapid catalytic rate of Rab5 WT compared with Q79L might be masked by a slower GDP dissociation rate. To test this, pre-steady-state GTPase rates were measured by rapidly loading Rab5 proteins with GTP by magnesium chelation and then measuring P(i) release at early times by charcoal sedimentation (Fig. 6B). This yielded a rate for Rab5 WT (0.026 s), which exceeded that of Q79L (0.0038 s) by 6.8-fold, supporting the hypothesis. The single step first-order rate constant for GTP hydrolysis was then determined by rapid filtration (Fig. 6C) and was found to be 12.2-fold higher for Rab5 WT than for Q79L (Table 2). (During preparation of this manuscript, a single step rate constant of 0.05 min at 37 °C for Rab5 WT was reported, which agrees well with our data, but the rate for Rab5 Q79L was found to be about 100-fold slower(39) ).


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(i) 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(1) 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(1)` 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(1)) is exceedingly fast compared with the rates of GTP dissociation (k), GTP hydrolysis (k(2)), and GDP dissociation (k(3)) (for H-Ras, k = 1.4 times 10^8M s(41) , and for Rab9, k = 1.2 times 10^5M 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(2) + 1/k(3). This rearranges to k = k(2)bulletk(3)/k(2) + k(3). Calculated this way from the measured first-order rate constants k(2) and k(3), 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(2) and k(3) 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.

Nucleotide-dependent Proteolysis

The structures of Rab5 proteins in solution were assessed by limited tryptic digestion. The products of degradation were strongly dependent on the presence of nucleotides and magnesium, similar to what has been observed for other GTPases(36, 37, 42, 43) . In the absence of added nucleotides, magnesium chelation with 1 mM EDTA resulted in the progression of proteolysis such that no peptide fragments were visible by SDS-PAGE with Coomassie staining (not shown). The addition of 10 mM GDP, GTP, or GTPS in the presence of 5 mM MgCl(2) resulted in the appearance of a proteolytic doublet of approximately 20 kDa (Fig. 8A). However, protection by these different guanine nucleotides was not comparable, since GDP consistently permitted the appearance of faint 12- and 8-kDa fragments, characteristic of the empty state (see below). GTP allowed almost no appearance of the 12- and 8-kDa peptides under the conditions used, and GTPS showed an intermediate pattern. Furthermore, when the trypsin/Rab ratio was increased from 1:10 to 1:50 (w:w), a band indistinguishable from the 25-kDa holoprotein was observed in the presence of GTP but not in the presence of GDP or GTPS (Fig. 8C). At even higher ratios, the protection of the 25-kDa protein was almost complete (not shown). These results suggest that limited proteolysis may be used to distinguish the Rab5bulletGTP conformation from the Rab5bulletGDP conformation and that GTPS does not efficiently induce a conformation comparable with Rab5bulletGTP. The latter is distinct from the situation with trimeric G-proteins in which GTPS promotes greater functional activation and confers greater protection from trypsin than GTP(36, 37) . This difference between trimeric G-proteins and a Ras-related protein to a ligand that differs only at the -phosphate position is reminiscent of the sensitivity of G-proteins but not small GTPases to aluminum fluoride(44) . The conformational differences between Rab5bulletGTP and Rab5bulletGTPS detected by limited proteolysis raise the question as to whether the inhibitory function of GTPS in endosome fusion reactions (22, 45, 46) is due to the failure of GTPS to induce a GTP conformation in Rab5 rather than to the inhibition of the GTPase activity of Rab5 or some other GTPase.

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 WTbulletGTP 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. Rab5bulletGTP-interactive) vesicle transfer regulatory components, since the product of proteolytic protection of Rab5 N133I by native guanine nucleotides was similar to that of Rab5 WTbulletGTP. The fact that GTPS offered no protection from proteolytic digestion (Fig. 8D) may simply reflect the relative inability of GTPS 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.

In Vitro Prenylation of Recombinant Rab5 Proteins

Prenylation of purified Rab5 proteins was supported by rabbit reticulocyte lysate upon incubation for 3 h in the presence of [^3H]geranylgeranyl pyrophosphate, and the extent of processing was assessed by SDS-PAGE and fluorography (Fig. 9). The incorporation of [^3H]geranylgeranyl into each protein was as follows: Rab5 WT, 7,366 cpm, or 1.2% of the protein modified; Rab5 Q79L, 7,816 cpm, or 1.3% of the protein modified; Rab5 N133I, 2,793 cpm, or less than 0.4% of the protein modified. Previous studies have established that co-translational prenylation of Rab5 is dependent on guanine nucleotide binding and that Rab5 N133I is a very poor substrate for this reaction (20) .


Figure 9: In vitro prenylation of Rab5 proteins. Prenylation of Rab5 WT, Q79L, and N133I proteins was accomplished using rabbit reticulocyte lysate and [^3H]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.

Stimulation of in Vitro Endosome Fusion by Recombinant Rab5 Protein and Its Mutants

In order to characterize the recombinant WT and mutant Rab5 proteins functionally, their activity in regulating endosome fusion in vitro was assessed in a well-established assay(21) . Previous studies have documented the ability of Rab5 to stimulate endocytosis when overexpressed in vivo(4, 5, 39) , and in vitro studies utilizing cytosol from transfected, Rab5-overexpressing cells have demonstrated the capacity of this preparation to stimulate endosome fusion(3, 39) . Our investigation of the functional properties of Rab5 is unique in that the recombinant protein was highly purified, it was not modified by fusion to another polypeptide such as glutathione S-transferase, and the exact role of post-translational processing of the cognate protein could be assessed. Using this approach, it was also possible, for the first time, to directly compare the properties of Rab5 WT with the Q79L mutant within the same experimental system.

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.


FOOTNOTES

*
This work was supported by United States Public Health Service Award HL43161 and a Veterans Administration Merit Review Award (to B. F. D.), a grant-in-aid from the American Heart Association, Texas Affiliate (to B. J. K), and Grants 3381 from the Council for Tobacco Research U. S. A., and CB-15 from the American Cancer Society (to M. W. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Div. of Pulmonary and Critical Care Medicine, VA Medical Center, Bldg. 109, Rm. 106, 2002 Holcombe Blvd., Houston, TX 77030. Tel.: 713-794-7794; Fax: 713-794-7853.

A fellow of the American Heart Association (Massachusetts Affiliate).

**
Recipient of a Junior Faculty Award from the American Cancer Society.

(^1)
The abbreviations used are: WT, wild-type; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PAGE, polyacrylamide gel electrophoresis; GTPS, guanosine 5`-O-(3-thiotriphosphate); GAP, GTPase activating protein.


ACKNOWLEDGEMENTS

We thank Dr. Ted Wensel and Dr. Lutz Birnbaumer for helpful discussions.


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