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J. Biol. Chem., Vol. 279, Issue 39, 40431-40436, September 24, 2004

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Dynamin GTPase Domain Mutants That Differentially Affect GTP Binding, GTP Hydrolysis, and Clathrin-mediated Endocytosis^*

Byeong Doo Song, Marilyn Leonard, and Sandra L. Schmid

From the Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, June 23, 2004 , and in revised form, July 19, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The GTPase dynamin is essential for clathrin-mediated endocytosis. Unlike most GTPases, dynamin has a low affinity for nucleotide, a high rate of GTP hydrolysis, and can self-assemble, forming higher order structures such as rings and spirals that exhibit up to 100-fold stimulated GTPase activity. The role(s) of GTP binding and/or hydrolysis in endocytosis remain unclear because mutations in the GTPase domain so far studied impair both. We generated a new series of GTPase domain mutants to probe the mechanism of GTP hydrolysis and to further test the role of GTP binding and/or hydrolysis in endocytosis. Each of the mutations had parallel effects on assembly-stimulated and basal GTPase activities. In contrast to previous reports, we find that mutation of Thr-65 to Ala (or Asp or His) dramatically lowered both the rate of assembly-stimulated GTP hydrolysis and the affinity for GTP. The assemblystimulated rate of hydrolysis was lowered by the mutation of Ser-61 to Asp and increased by the mutation of Thr-141 to Ala without significantly altering the K_m for GTP. For some mutants and to a lesser extent for WT dynamin, self-assembly dramatically altered the K_m for GTP, suggesting that conformational changes in the active site accompany self-assembly. Analysis of transferrin endocytosis rates in cells overexpressing mutant dynamins revealed a stronger correlation with both the basal and assembly-stimulated rates of GTP hydrolysis than with the calculated ratio of dynamin-GTP/free dynamin, suggesting that GTP binding is not sufficient, and GTP hydrolysis is required for clathrin-mediated endocytosis in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Dynamin is a large GTPase that is essential for clathrin-mediated endocytosis and synaptic vesicle recycling (1). Dynamin has biochemical properties that distinguish it from typical GTPases (2). Specifically, it has a significantly lower affinity for nucleotide and a higher basal rate of GTP hydrolysis. Most notably, the rate of GTP dissociation from dynamin is 10⁴ times faster than that from, for example, Ras (3, 4). Another distinguishing feature of dynamin is its ability to selfassemble to form higher order structures such as rings and spirals (5). Self-assembly can stimulate dynamin GTPase activity as much as 100-fold (6). Dynamin spirals similar in dimensions to those formed in vitro can be detected as electron dense collars around the elongated necks of endocytic intermediates that accumulate in mammalian cells when clathrin-mediated endocytosis is inhibited by perturbing dynamin (7) or clathrin (8) function. Recently, it has been shown that overexpression of an assembly-defective dynamin mutant blocks clathrin-mediated endocytosis (9), confirming that dynamin self-assembly is required. However, dynamin collars have not been detected in unperturbed cells, suggesting that this intermediate is very short-lived.

Despite the biochemical differences, the three-dimensional structure of the GTPase domain of dynamin A, which is highly conserved among dynamin family members, reveals that the core GTPase domain adopts the common globular fold of Ras and other GTPases, consisting of a six-stranded -sheet surrounded by five -helices (10). Moreover, the dynamin GTPase domain contains four consensus motifs that are shared among all GTPases (11) and are involved in binding either to phosphate/Mg²⁺ ions or to the guanine base. As illustrated in Fig. 1, these include: G1, also called the P-loop, which in dynamin interacts with the - and -phosphates of the bound nucleotide (10); G2, comprising an invariant Thr residue (Thr-65 in dynamin) that coordinates Mg²⁺; G3, which encodes conserved residues that bind Mg²⁺ and the -phosphate; G4, which interacts through hydrogen-bonding with the guanine base.

View larger version (26K): [in this window] [in a new window]   FIG. 1. Dynamin domain structure and sequences of four conserved GTP binding elements in the dynamin GTPase domain. Sw1 and Sw2 regions associated with the -phosphate-sensing elements G2 and G3 are indicated. Point mutations were made in Sw1 and Sw2 residues at the indicated positions (arrows).

The larger size of the dynamin GTPase domain compared with Ras (30 versus 20 kDa) is due to two internal insertions of, as yet, undefined function and to smaller C- and N-terminal extensions (10). Also unique for dynamin is the existence of a hydrophobic groove, formed by interactions between the N- and C-terminal -helical extensions, that has been proposed to be the binding site for GED,¹ the GTPase effector domain of dynamin (10). GED is required for self-assembly and for assembly-stimulated GTPase activity (12). However, recent data suggest that GED can also influence GTP binding and hydrolysis in the unassembled state (9), consistent with cross-linking and limited proteolysis studies that reveal stable GED-GTPase/middle domain interactions in unassembled dynamin (13).

Endocytosis is blocked in cells overexpressing dominant negative GTPase domain mutants of dynamin, suggesting that dynamin GTPase activity is required for clathrin-mediated endocytosis (14). However, most of the mutants so far studied (K44A, S45N, T65F) are also defective in GTP binding (14–16). One mutation, T65A, was suggested to significantly inhibit GTP hydrolysis without impairing GTP binding (7). However, this assertion is inconsistent with the effects of this highly conserved, switch 1, Thr on GTP binding affinities in other GTPase family members (11) and its role in coordinating Mg²⁺ for direct interaction with the -phosphate of bound GTP. Therefore, it remains uncertain as to whether endocytosis in vivo requires GTP binding and hydrolysis or only GTP binding.

To probe the mechanism of GTP hydrolysis by dynamin and to identify the residues that are critical for nucleotide binding and GTP hydrolysis, we have made a series of new mutations in the switch 1 and 2 regions of the dynamin GTPase domain. Analysis of the kinetic constants for the steady-state GTPase cycle revealed new mutants that differentially affect GTP binding and hydrolysis. The effects of overexpression of these mutants on endocytosis were examined so as to determine whether the rate of endocytosis correlated more closely with the ratio of dynamin-GTP/unoccupied dynamin or with the rate of GTP hydrolysis or both.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Recombinant Baculoviruses and Adenoviruses—Point mutations were generated in human dynamin 1 in a pBluescript vector by the QuikChange method (Stratagene) using the oligonucleotides (Operon) listed in Table I. The BamHI-KpnI fragment containing the entire gene was transferred to pVL1393 vector. The resulting plasmid together with linearized baculovirus DNA (BaculoGold, Pharmingen) was used to generate recombinant baculoviruses by following the manufacturer's instructions. To generate recombinant adenoviruses, the NdeI-NheI fragment from pBluescript was subcloned into pADTetT3T7 containing the gene for human dynamin 1 wild type, and the resulting plasmid together with 5 adenovirus DNA was then transfected into HEK293-cre4 cells as described (17).

Transferrin Internalization and Its Correlation with Dynamin GTPase Activity—The kinetics of internalization of biotinylated transferrin into an avidin-inaccessible compartment was determined in tTA-HeLa cells expressing dynamin mutants (n = 6, Fig. 2), exactly as described (18). To determine correlation coefficients, the extent of transferrin internalization at 10 min was plotted as a function of the ratio of dynamin-GTP/dynamin or the relative rate of GTP hydrolysis by dynamin, calculated as indicated below from Michaelis-Menten steady-state kinetics (Equation 1) using 100 µM as the intracellular concentration of GTP (19).

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

where K_m = (k_–1 + k_cat)k₁.

(Eq. 5)

View larger version (25K): [in this window] [in a new window]   FIG. 2. Velocity sedimentation. The ability of mutant dynamins to self-assemble on lipid tubules was followed by velocity sedimentation at a physiological salt concentration (150 mM KCl) in the presence of phosphatidylinositol 4,5-diphosphate-containing lipid tubules. The recovered pellets (P) and the supernatants (S) were analyzed by SDS-PAGE followed by Coomassie Blue staining. LT, lipid tubule.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Each of these mutations was generated in human dynamin-1, and the proteins were expressed and purified from insect cells infected with recombinant baculoviruses. To assess the role of each residue in GTP binding and hydrolysis, kinetic analyses were performed to measure both basal and assembly-stimulated GTPase activity of the individual mutant dynamins. Basal GTPase activity was measured at low concentrations of dynamin and at physiological salt concentrations to prevent dynamin self-assembly into higher order structures. Assembly-stimulated GTPase activity was determined using phosphatidylinositol 4,5-diphosphate-containing lipid nanotubules as a template for dynamin self-assembly. Under these conditions, the basal GTPase activity of wild type dynamin was stimulated 40–50-fold when measured in the presence of lipid nanotubules, consistent with previous reports (6, 9).

Mutations in Switch 1 Exhibit Differential Effects on GTP Binding and Hydrolysis—Using these assays, we next determined the Michaelis-Menten kinetic constants (k_cat and K_m) for basal and assembly-stimulated GTPase activities for WT dynamin and the individual mutants (Table II). We first examined the sw1 mutations and confirmed a role for sw1 residues in both GTP binding and hydrolysis. Although the S61A mutation did not significantly affect either GTP binding or hydrolysis, the S61D mutation significantly reduced the k_cat for both basal and assembly-stimulated GTPase activity without affecting the K_m (Table II). Importantly, in the presence of the lipid tubule assembly templates, the GTPase activity of dyn1-S61D was stimulated to the same extent (50-fold) as dyn1-WT, suggesting that this mutation does not affect dynamin self-assembly (see below). From these data we conclude that the Ser-61 hydroxyl residue is not directly involved in the catalysis. However, the fact that substitution of a carboxylate residue (Asp) at this position lowers the rate of GTP hydrolysis suggests that a negative charge may develop near Ser-61 in the transition state.

As previously reported (7, 16), we found that Thr-65 mutations (T65A, T65D, T65H) greatly reduced both basal and assembly-stimulated GTPase activity (36–100-fold), demonstrating that Thr-65 has an important role in catalysis (Table II). However, these mutations exhibited differential effects on the K_m for GTP. Contrary to previous observations (7), but as predicted for other GTPases (11), the T65A mutation lowered the apparent affinity for GTP by 8-fold for basal and by 50-fold for assembly-stimulated GTPase activity (Table II). Mutation of Thr-65 to a carboxylate residue, Asp, slightly increased the apparent GTP affinity of unassembled dynamin (2-fold) but significantly lowered that of assembled dynamin (14-fold). Similarly, although the T65H mutation showed a minimal effect on the K_m for GTP of unassembled dynamin (less than 2-fold), the K_m for assembled dynamin increased by 27-fold. The dramatic changes in the K_m for GTP observed in the presence of lipid tubules suggest that the GTP binding pocket can undergo significant conformational changes upon dynamin self-assembly. Consistent with this, the K_m of wild type dynamin-1 for GTP decreases >2-fold upon self-assembly.

Together, these data suggest that residues in switch 1 are involved in both GTP binding and hydrolysis and that they undergo assembly-dependent conformational changes. Interestingly, Ser-61 and Thr-65 are both located within the sequence ⁵⁷LPRGSGIVTR⁶⁶, which is highly conserved across the entire dynamin family and corresponds to a region previously identified in Mx1 as a self-assembly motif (20). Although the effect of self-assembly on GTP binding may in part be mediated through conformational changes in sw1, these sw1 mutations appear not to be defective in self-assembly or in assembly-stimulated GTP hydrolysis per se. Indeed, the degree of lipid tubule-dependent GTPase stimulation for most of these mutants, including S61A, S61D, T65D, T65H, was similar to or better than that observed for wild type dynamin (4461-fold compared with 40-fold). Moreover, velocity sedimentation demonstrated that these mutations are competent to self-assemble on lipid nanotubules (Fig. 2). The T65A mutation also showed significant (16-fold) assembly-dependent stimulation of GTPase activity and was unimpaired in its ability to self-assemble (Fig. 2) as reported by others (7).

A Mutation in Switch 2 Increases Dynamin Basal GTPase Activity—We next examined the effects of mutation of the switch 2 residue, Thr-141. Contrary to our hypothesis that Thr-141 could be involved in positioning and/or activating the nucleophilic water molecule, its change to a small hydrophobic residue (Ala) increased the k_cat by 2-fold for both basal and assembled GTPase activity with only small affects on K_m. Also unexpectedly, mutation to Asp, a potentially stronger activator of water, inhibited GTP binding and hydrolysis. Together, these results suggest that a hydrophobic environment is preferred in this region of the nucleotide binding pocket at the transition state and indicate that Thr-141 has roles in both GTP binding and hydrolysis, although the exact mechanism remains to be established. Although velocity sedimentation analysis established the ability of these mutants to self-assemble (Fig. 2), the degree of assembly-stimulated GTPase activity was reduced by mutations in this sw2 residue (10- and 27-fold stimulation for T141D and T141A, respectively). These data suggest that sw2 interactions also play a role in assembly-stimulated GTP hydrolysis.

Mutations in the GTPase Domain of Dynamin Differentially Affect GTP Binding—The Michaelis-Menten constant, K_m, reflects the rate constants for association and dissociation of the substrate as well as for catalysis (K_m = (k_–1 + k_cat)/k₁); thus, it is an indirect measure of GTP binding affinity. Therefore, we sought to test our predictions regarding the relative GTP binding affinity of these mutations based on K_m data by directly measuring binding of the nonhydrolyzable analogue, GTP³⁵S. Given the low affinity of dynamin for GTP and the rapid rate of dissociation (2 s^–1) for bound GTP (3), it has been difficult to accurately measure GTP binding using conventional filter binding or UV cross-linking protocols (7, 21). Therefore, we developed a rapid filtration protocol to measure binding of GTP³⁵S directly (see "Experimental Procedures"). The results shown in Fig. 3 confirm most of our predictions regarding the relative GTP binding affinities of these mutants based on K_m measurements. Thus, we find that GTP³⁵S binding is unaffected by mutation of Ser-61 to either Ala or Asp and only slightly reduced by mutation of Thr-141 to Ala. Moreover, as predicted for other GTPases and consistent with our K_m data, mutation of the sw1 Thr-65 to Ala potently inhibited GTP³⁵S binding. Surprisingly, GTP³⁵S binding to the T65D mutant was also potently inhibited, in sharp contrast to the K_m measurements made under basal GTPase conditions. There are two possible explanations for this discrepancy. First, thio-substitution affects nucleotide binding. In fact, inhibition studies indicate that GTPS binds more weakly to dynamin than GTP (K_i for GTPS = 340 µM),² and it is possible that the T65D mutation is even more sensitive to structural differences in the bound nucleotide. Second, because the results obtained in the filter binding assay correspond more closely to K_m values obtained for assembly-stimulated GTPase activity, they may be influenced by dynamin-dynamin interactions that occur during filtration.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Filtration assay for GTP35S binding to wild type and mutant dynamins. Duplicate samples of wild type and mutant dynamins (1.0 µM) as indicated were incubated with 100 µM GTP35S for 20 min at room temperature and then subjected to rapid filtration and washing under vacuum, as described under "Experimental Procedures." Filter-bound dynamin-GTP35S was quantified by PhosphorImager analysis, and the resulting values were normalized to the amount bound to WT dynamin. The data shown are the averages ± S.E. for five such independent experiments. Indistinguishable results were obtained for incubations at 37 °C.

Temperature-dependent Effects on GTP Binding and Hydrolysis—Contrary to previous reports (7), our data establish that in addition to its reported effects on hydrolysis, mutation of Thr-65 to Ala substantially decreases GTP binding based on both K_m values and direct measurements. One possible explanation for this discrepancy is that we measure GTPase activity at the physiologic temperature of 37 °C, whereas the previous findings were based on measurements performed at room temperature (7). Indeed, we find that both kinetic parameters for dynamin GTPase activity, K_m and k_cat, are significantly affected by incubation at lower temperatures (Table III). Thus, both the basal and assembly-stimulated rates of GTP hydrolysis for wild type dynamin are 10-fold lower when assayed at 22 °C as compared with 37 °C, which is significantly greater than observed for many enzymes (22). Similarly, when measured at 22 °C, the K_m for GTP is 30-fold reduced for wild type dynamin measured under basal GTPase assay conditions and 6-fold reduced when measured in the presence of lipid tubules. The T65A mutant shows a pronounced defect in GTP hydrolysis at both temperatures. Strikingly, there is an 9-fold differential in K_m for GTP measured under basal conditions at the two temperatures and an 50-fold differential when measured in the presence of lipid tubules (Table III). Indeed, when measured at 22 °C, our findings correspond to those reported by Marks et al. (7). The relatively strong temperature dependence of these parameters of dynamin GTPase activity suggests that upon GTP binding and hydrolysis significant conformational changes may occur in the non-reacting parts of dynamin (23) in addition to the active site and explains previous discrepancies in reported k_cat and K_m values for both wild type and T65A mutant dynamins (6, 7, 12).

The Dependence of Endocytosis on GTP Binding or Hydrolysis—We have generated a new series of mutant dynamins that exhibit differential and graded effects on K_m and k_cat for GTP hydrolysis (Fig. 4A). With these mutants in hand we sought to determine whether endocytosis rates would correlate more directly with the ratio of GTP bound:unoccupied dynamin, the rate of GTP hydrolysis, or both. To this end, recombinant adenoviruses expressing wild type and mutant dynamins under control of a tetracycline-regulatable promoter were generated. Clathrin-mediated endocytosis was followed by measuring transferrin internalization in adenovirally infected tTA-HeLa cells overexpressing mutant dynamins (Fig. 4, B and C). Consistent with previous results (7, 16, 25), overexpression of either dyn1-T65A (open squares) or dyn1-T65D (black inverted triangles), which were severely defective in both GTP binding and hydrolysis, potently inhibited transferrin internalization. Also as expected, dyn1-S61A (black circles), which exhibited near wild type GTP binding and hydrolysis activities, had no effect on endocytosis as compared with dyn1-WT (open circles). In contrast, endocytosis was significantly inhibited by overexpression of dyn1-S61D (diamonds), which was defective in GTP hydrolysis, but exhibited normal GTP binding. Finally, endocytosis was, if anything, slightly stimulated by overexpression of dyn1-T141A (black triangles).

View larger version (30K): [in this window] [in a new window]   FIG. 4. Transferrin internalization in cells overexpressing mutant dynamins. A, summary of the data in Table II on the effects of mutations in the GTPase domain on Km and kcat for both basal and assembly-stimulated GTPase activities. B, Western blot showing the protein expression of mutant dynamins in cells where transferrin internalization was followed. C, internalization of biotinylated transferrin was followed in tTA-HeLa cells infected for 16 + 2 h with recombinant adenovirus encoding human dynamin 1; , wild type; , S61A; , S61D; , T65A; , T65D; , T141A. Results are the average ± S.D. of five experiments. LT, lipid tubule.

View larger version (34K): [in this window] [in a new window]   FIG. 5. Correlation of endocytosis with dynamin GTPase activity. The extent of transferrin internalization was plotted as a function of [dynamin-GTP]/[dynamin]([GTP]/Km) or the relative rate of GTP hydrolysis (kcat/Km), was which calculated from the kinetic parameters obtained for both basal (A and B) and assembly-stimulated (C and D) GTPase activity of individual mutant dynamins: , wild type; , S61A; , S61D; , T65A; , T65D; , T141A. Results shown are the average ± S.D. derived from five experiments.

We have previously shown that overexpression of dynamin mutants specifically defective in self-assembly and thereby assembly-stimulated GTPase activity can accelerate a rate-limiting step in clathrin-mediated endocytosis (12, 26). These mutations, located in GED, did not significantly affect the basal rate of GTP hydrolysis (12). In contrast, each of the mutations studied here have equal effects on both basal and assembly-stimulated rates of GTPase activity. Thus, the tight correlation we see here between basal GTPase activity and endocytosis rates may reflect a specific role for dynamin basal rate of GTP hydrolysis in clathrin-mediated endocytosis. Recent studies at the synapse (27) confirm our earlier observations in nonneuronal cells (14) that unassembled dynamin is present on coated pits from their earliest stages of assembly and maturation. Dynamin interacts with multiple SH3 domain-containing effector molecules, and these interactions are likely to occur from the outset of dynamin association with the emerging coated pit. Thus, basal GTPase activity may play a role in regulating dynamin interactions with itself and with partner proteins during earlier stages of coated pit formation and maturation. Further studies will be needed to assess whether these effector interactions are regulated by and/or regulate the dynamin cycle of GTP binding and hydrolysis.

There are also several not mutually exclusive possibilities for the role of dynamin assembly-stimulated GTPase activity in clathrin-mediated endocytosis. Assembly-stimulated GTP hydrolysis may be directly involved in driving conformational changes in assembled dynamin that mediate membrane fission (5, 6). Alternatively, GTP hydrolysis by assembled dynamin may function to dismantle the fission machinery for recycling in vivo. Indeed, GTP hydrolysis in vitro has been shown to trigger the rapid disassembly of dynamin (21, 28), and this interpretation would be consistent with recent observations that GTP hydrolysis by dynamin may not be required for a single round of vesicle formation in vitro (29). There is precedence for this model in that GTP hydrolysis is required for the in vivo function and recycling of other GTPases such as Sar, Arf, SRP54, and SRP receptor, although GTPase defective mutants of each of these proteins are able to support single rounds of activity in vitro (30–34). Analysis of the effects of acute inhibition of dynamin GTPase activity in vivo, as opposed to long term overexpression experiments, will be needed to resolve these issues. In addition, the generation and analysis of new classes of mutants that selectively impair either assembly-stimulated or basal GTPase activities would be informative.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant GM42455 (to S. L. S.). This is The Scripps Research Institute manuscript number 16557-CB. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 858-784-2311; Fax: 858-784-2345; E-mail: slschmid{at}scripps.edu.

¹ The abbreviations used are: GED, GTPase effector domain; GTPS, guanosine 5'-3-O-(thio)triphosphate; WT, wild type; sw, switch.

² M. Leonard, unpublished data.

³ S. P. Sholly, M. Leonard, and S. Schmid, unpublished data.

	ACKNOWLEDGMENTS

 
We are grateful to Alisa Jones and Tricia Glenn for technical assistance in protein expression and purification.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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