Dynamin GTPase Domain Mutants That Differentially Affect GTP Binding, GTP Hydrolysis, and Clathrin-mediated Endocytosis
2004; Elsevier BV; Volume: 279; Issue: 39 Linguagem: Inglês
10.1074/jbc.m407007200
ISSN1083-351X
AutoresByeong Doo Song, Marilyn Leonard, Sandra L. Schmid,
Tópico(s)Pancreatic function and diabetes
ResumoThe 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 Km for GTP. For some mutants and to a lesser extent for WT dynamin, self-assembly dramatically altered the Km 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. 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 Km for GTP. For some mutants and to a lesser extent for WT dynamin, self-assembly dramatically altered the Km 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. Dynamin is a large GTPase that is essential for clathrin-mediated endocytosis and synaptic vesicle recycling (1Conner S.D. Schmid S.L. Nature. 2003; 422: 37-44Crossref PubMed Scopus (3101) Google Scholar). Dynamin has biochemical properties that distinguish it from typical GTPases (2Song B.D. Schmid S.L. Biochemistry. 2003; 42: 1369-1376Crossref PubMed Scopus (102) Google Scholar). 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 (3Binns D.D. Helms M.K. Barylko B. Davis C.T. Jameson D.M. Albanesi J.P. Eccleston J.F. Biochemistry. 2000; 39: 7188-7196Crossref PubMed Scopus (46) Google Scholar, 4Neal S.E. Eccleston J.F. Hall A. Webb M.R. J. Biol. Chem. 1988; 263: 19718-19722Abstract Full Text PDF PubMed Google Scholar). Another distinguishing feature of dynamin is its ability to selfassemble to form higher order structures such as rings and spirals (5Hinshaw J.E. Schmid S.L. Nature. 1995; 374: 190-192Crossref PubMed Scopus (665) Google Scholar). Self-assembly can stimulate dynamin GTPase activity as much as 100-fold (6Stowell M.H.B. Marks B. Wigge P. McMahon H.T. Nat. Cell Biol. 1999; 1: 27-32Crossref PubMed Scopus (314) Google Scholar). 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 (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar) or clathrin (8Iversen T.-G. Skretting G. Sandvig K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5175-5180Crossref PubMed Scopus (66) Google Scholar) function. Recently, it has been shown that overexpression of an assembly-defective dynamin mutant blocks clathrin-mediated endocytosis (9Song B.D. Yarar D. Schmid S.L. Mol. Biol. Cell. 2004; 15: 2243-2252Crossref PubMed Scopus (67) Google Scholar), 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 β-sheet surrounded by five α-helices (10Niemann H.H. Knetsch M.L. Scherer A. Manstein D.J. Kull F.J. EMBO J. 2001; 20: 5813-5821Crossref PubMed Scopus (95) Google Scholar). Moreover, the dynamin GTPase domain contains four consensus motifs that are shared among all GTPases (11Wittinghofer A. Hall A. GTPases. 24. Oxford University Press, New York2000: 244-310Google Scholar) 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 α- and β-phosphates of the bound nucleotide (10Niemann H.H. Knetsch M.L. Scherer A. Manstein D.J. Kull F.J. EMBO J. 2001; 20: 5813-5821Crossref PubMed Scopus (95) Google Scholar); G2, comprising an invariant Thr residue (Thr-65 in dynamin) that coordinates Mg2+; G3, which encodes conserved residues that bind Mg2+ and the γ-phosphate; G4, which interacts through hydrogen-bonding with the guanine base. Residues around G2 and G3 that interact with and recognize the γ-phosphate of bound GTP constitute the switch 1 and 2 regions of GTPases, so-called because they are often disordered in the absence of bound GTP and switch to an ordered conformation upon GTP binding. As expected, these residues are not resolved in the structure of dynA GTPase domain in its unoccupied or GDP-bound states; however, given their positioning near the γ-phosphate, some are likely to play roles in nucleotide binding and GTP hydrolysis. 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 (10Niemann H.H. Knetsch M.L. Scherer A. Manstein D.J. Kull F.J. EMBO J. 2001; 20: 5813-5821Crossref PubMed Scopus (95) Google Scholar). Also unique for dynamin is the existence of a hydrophobic groove, formed by interactions between the N- and C-terminal α-helical extensions, that has been proposed to be the binding site for GED, 1The abbreviations used are: GED, GTPase effector domain; GTPγS, guanosine 5′-3-O-(thio)triphosphate; WT, wild type; sw, switch. the GTPase effector domain of dynamin (10Niemann H.H. Knetsch M.L. Scherer A. Manstein D.J. Kull F.J. EMBO J. 2001; 20: 5813-5821Crossref PubMed Scopus (95) Google Scholar). GED is required for self-assembly and for assembly-stimulated GTPase activity (12Sever S. Muhlberg A.B. Schmid S.L. Nature. 1999; 398: 481-486Crossref PubMed Scopus (317) Google Scholar). However, recent data suggest that GED can also influence GTP binding and hydrolysis in the unassembled state (9Song B.D. Yarar D. Schmid S.L. Mol. Biol. Cell. 2004; 15: 2243-2252Crossref PubMed Scopus (67) Google Scholar), consistent with cross-linking and limited proteolysis studies that reveal stable GED-GTPase/middle domain interactions in unassembled dynamin (13Muhlberg A.B. Warnock D.E. Schmid S.L. EMBO J. 1997; 16: 6676-6683Crossref PubMed Scopus (199) Google Scholar). Endocytosis is blocked in cells overexpressing dominant negative GTPase domain mutants of dynamin, suggesting that dynamin GTPase activity is required for clathrin-mediated endocytosis (14Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1046) Google Scholar). However, most of the mutants so far studied (K44A, S45N, T65F) are also defective in GTP binding (14Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1046) Google Scholar, 15Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (398) Google Scholar, 16Damke H. Binns D.D. Ueda H. Schmid S.L. Baba T. Mol. Biol. Cell. 2001; 12: 2578-2589Crossref PubMed Scopus (150) Google Scholar). One mutation, T65A, was suggested to significantly inhibit GTP hydrolysis without impairing GTP binding (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar). However, this assertion is inconsistent with the effects of this highly conserved, switch 1, Thr on GTP binding affinities in other GTPase family members (11Wittinghofer A. Hall A. GTPases. 24. Oxford University Press, New York2000: 244-310Google Scholar) and its role in coordinating Mg2+ for direct interaction with the γ-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. 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 ψ5 adenovirus DNA was then transfected into HEK293-cre4 cells as described (17Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar).Table IOligonucleotides used for point mutations in dynamin GTPase domainMutationSequenceS61A-f5′-CTTCTTGCCTCGAGGAGCTGGCATTGTCACC-3′S61A-r5′-GGTGACAATGCCAGCTCCTCGAGGCAAGAAG-3′S61D-f5′-CTTCTTGCCTCGAGGAGATGGCATTGTCACCCGAC-3′S61D-r5′-GTCGGGTGACAATGCCATCTCCTCGAGGCAAGAAG-3′T65A-f5′-GGATCTGGCATTGTCGCACGACGTCCCCTGGTC-3′T65A-r5′-GACCAGGGGACGTCGTGCGACAATGCCAGATCC-3′T65D-f5′-GGATCTGGCATTGTCGACCGACGTCCCCTGGTC-3′T65D-r5′-GACCAGGGGACGTCGGTCGACAATGCCAGATCC-3′T65H-f5′-GGATCTGGCATTGTCCACCGACGTCCCCTGGTC-3′T65H-r5′-GACCAGGGGACGTCGGTGGACAATGCCAGATCC-3′T141A-f5′-CCTGCCCGGAATGGCAAAGGTCCCGGTGG-3′T141A-r5′-CCACCGGGACCTTTGCCATTCCGGGCAGG-3′T141D-f5′-CCTGCCCGGAATGGACAAGGTCCCGGTGG-3′T141D-r5′-CCACCGGGACCTTGTCCATTCCGGGCAGG-3′ Open table in a new tab Proteins, GTPase, and Velocity Sedimentation Assays—Protein purification, dynamin GTPase, and velocity sedimentation assays were performed as described (9Song B.D. Yarar D. Schmid S.L. Mol. Biol. Cell. 2004; 15: 2243-2252Crossref PubMed Scopus (67) Google Scholar). All incubations were at 37 °C unless otherwise noted. Two different dynamin concentrations were used, 1.0 μm for basal and 0.1 μm for lipid tubule stimulated GTPase activity. Initial velocities for GTP hydrolysis were measured at varying GTP concentrations and plotted as a function of GTP concentration to obtain the values for kcat and Km based on the Michaelis-Menten equation. The values from repeated measurements (n = 3∼5) were summarized in Table II.Table IIKinetic constants for dynamin GTPase activityDynaminBasalLipid tubuleKmkcatKmkcatμmmin-1μmmin-1WT102 ± 352.60 ± 0.9837 ± 18105 ± 47S61A67 ± 101.94 ± 0.2441 ± 2286 ± 13S61D68 ± 110.43 ± 0.0256 ± 2226.3 ± 1.5T65A880 ± 3300.075 ± 0.0272115 ± 14131.22 ± 0.24T65D41 ± 150.016 ± 0.002513 ± 380.97 ± 0.20T65H149 ± 210.018 ± 0.002988 ± 2700.95 ± 0.06T141A174 ± 966.69 ± 0.7956 ± 20185 ± 55T141D763 ± 1200.15 ± 0.031940 ± 12001.50 ± 0.98 Open table in a new tab Filter Assay for GTP Binding—Dynamin (1.0 μm) was incubated with 100 μm GTPγ35S (270 μCi/ml, Amersham Biosciences) in the GTPase assay buffer for 20 min at 37 °C or at room temperature in 6-well arrays of PCR tubes. Using a multichannel pipette, samples were applied to nitrocellulose (0.45 μm, BA85, Schleicher and Schuell) in a filter dot-bolt apparatus (Schleicher and Schuell, minifold I) under vacuum. The filter was rapidly washed once with 250 μl of cold buffer. The procedure was repeated, six samples at a time. Each sample was assayed five times. Rapid filtration and washing was necessary for reproducible results given the very high rate of dissociation of GTP from dynamin (3Binns D.D. Helms M.K. Barylko B. Davis C.T. Jameson D.M. Albanesi J.P. Eccleston J.F. Biochemistry. 2000; 39: 7188-7196Crossref PubMed Scopus (46) Google Scholar). The dried filter was imaged using an Amersham Biosciences PhosphorImager and quantified using ImageQuant Software. Values for individual mutant dynamins were normalized to wild type dynamin. 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 (18van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (591) Google Scholar). 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 (19Otero A.D. Biochem. Pharmacol. 1990; 39: 1399-1404Crossref PubMed Scopus (104) Google Scholar). Dyn+GTP⇄k−1k1Dyn-GTP→kcatDyn+GDP+Pi(Eq. 1) k1[Dyn][GTP]=(k−1+kcat)[Dyn-GTP](Eq. 2) [GTP](k−1+kcat)k1=[Dyn-GTP][Dyn](Eq. 3) [Dyn-GTP][Dyn]=[GTP]Km(Eq. 4) where Km = (k–1 + kcat)k1. Rate of GTP hydrolysis=kcatKm[Dyn][GTP](Eq. 5) We sought to identify critical residues in the dynamin GTPase domain that would exhibit differential effects on GTP binding and/or hydrolysis so as to test for differential requirements for these activities in endocytosis. For other GTPases, the switch (sw) 1 and 2 regions of the GTPase domain undergo major conformational changes upon GTP binding and are proposed to function in sensing the γ-phosphate of bound GTP (Fig. 1). Thus, we chose to mutate three amino acid residues in sw1 and 2 (Ser-61, Thr-65, Thr-141) to amino acids with different biochemical properties that could distinguish their roles in GTP binding and/or catalysis. Ser-61 is postulated to be located near the β-phosphate of bound GDP. To test whether its hydroxyl group might facilitate catalysis, Ser-61 was mutated to Ala, which would disrupt GTP hydrolysis, and to Asp, which might enhance GTP hydrolysis. The G2 motif (Thr-65) in sw1 is believed to coordinate the Mg2+ ion essential for nucleotide binding (11Wittinghofer A. Hall A. GTPases. 24. Oxford University Press, New York2000: 244-310Google Scholar); thus, it was surprising that its mutation to a hydrophobic residue (Ala) was reported to lower the rate of GTP hydrolysis without affecting GTP binding (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar). To determine whether the G2 motif of dynamin, in fact, plays a role distinct from the G2 motifs of other GTPases, Thr-65 was mutated to either Ala, Asp, or His. The T65A mutation was hypothesized to prevent nucleotide binding, since Ala, a hydrophobic residue, is unable to coordinate the Mg2+ ion required for nucleotide binding. On the other hand, mutation to Asp or His might increase nucleotide affinity if their greater ability to pull protons would correlate with their ability to coordinate Mg2+ ion. Finally, GTP hydrolysis requires the positioning and/or activation of a nucleophilic water molecule at the backside of the γ-phosphate of the bound GTP. In Ras-like GTPases, this is accomplished by a highly conserved glutamine residue within G3. This glutamine residue is not conserved in dynamin family members and corresponds to Met-140 in dynamin-1. Thus, to test the hypothesis that the neighboring Thr-141 in sw2 could instead play a role in positioning and/or activating the water molecule, it was mutated to Ala and Asp with the expectation that these mutations might reduce or increase the rate of GTP hydrolysis, respectively. 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 (6Stowell M.H.B. Marks B. Wigge P. McMahon H.T. Nat. Cell Biol. 1999; 1: 27-32Crossref PubMed Scopus (314) Google Scholar, 9Song B.D. Yarar D. Schmid S.L. Mol. Biol. Cell. 2004; 15: 2243-2252Crossref PubMed Scopus (67) Google Scholar). Mutations in Switch 1 Exhibit Differential Effects on GTP Binding and Hydrolysis—Using these assays, we next determined the Michaelis-Menten kinetic constants (kcat and Km) 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 kcat for both basal and assembly-stimulated GTPase activity without affecting the Km (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 (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar, 16Damke H. Binns D.D. Ueda H. Schmid S.L. Baba T. Mol. Biol. Cell. 2001; 12: 2578-2589Crossref PubMed Scopus (150) Google Scholar), 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 Km for GTP. Contrary to previous observations (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar), but as predicted for other GTPases (11Wittinghofer A. Hall A. GTPases. 24. Oxford University Press, New York2000: 244-310Google Scholar), 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 Km for GTP of unassembled dynamin (less than 2-fold), the Km for assembled dynamin increased by 27-fold. The dramatic changes in the Km 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 Km 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 (20Nakayama M. Yazaki K. Kusano A. Nagata K. Hanai N. Ishihama A. J. Biol. Chem. 1993; 268: 15033-15038Abstract Full Text PDF PubMed Google Scholar). 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 (44∼61-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 (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar). 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 kcat by 2-fold for both basal and assembled GTPase activity with only small affects on Km. 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, Km, reflects the rate constants for association and dissociation of the substrate as well as for catalysis (Km = (k–1 + kcat)/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 Km data by directly measuring binding of the nonhydrolyzable analogue, GTPγ35S. Given the low affinity of dynamin for GTP and the rapid rate of dissociation (2 s–1) for bound GTP (3Binns D.D. Helms M.K. Barylko B. Davis C.T. Jameson D.M. Albanesi J.P. Eccleston J.F. Biochemistry. 2000; 39: 7188-7196Crossref PubMed Scopus (46) Google Scholar), it has been difficult to accurately measure GTP binding using conventional filter binding or UV cross-linking protocols (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar, 21Maeda K. Nakata T. Noda Y. Sato-Yoshitake R. Hirokawa N. Mol. Biol. Cell. 1992; 3: 1181-1194Crossref PubMed Scopus (92) Google Scholar). Therefore, we developed a rapid filtration protocol to measure binding of GTPγ35S 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 Km measurements. Thus, we find that GTPγ35S 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 Km data, mutation of the sw1 Thr-65 to Ala potently inhibited GTPγ35S binding. Surprisingly, GTPγ35S binding to the T65D mutant was also potently inhibited, in sharp contrast to the Km 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 GTPγS binds more weakly to dynamin than GTP (Ki for GTPγS = 340 μm), 2M. Leonard, unpublished data. 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 Km values obtained for assembly-stimulated GTPase activity, they may be influenced by dynamin-dynamin interactions that occur during filtration. Temperature-dependent Effects on GTP Binding and Hydrolysis—Contrary to previous reports (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar), 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 Km 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 (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar). Indeed, we find that both kinetic parameters for dynamin GTPase activity, Km and kcat, 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 (22Wolfenden R. Snider M. Ridgway C. Miller B. J. Am. Chem. Soc. 1999; 121: 7419-7420Crossref Scopus (110) Google Scholar). Similarly, when measured at 22 °C, the Km 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 Km 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. (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar). 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 (23Chen Y.J. Zhang P. Egelman E.H. Hinshaw J.E. Nat. Struct. Mol. Biol. 2004; 11: 574-575Crossref PubMed Scopus (128) Google Scholar) in addition to the active site and explains previous discrepancies in reported kcat and Km values for both wild type and T65A mutant dynamins (6Stowell M.H.B. Marks B. Wigge P. McMahon H.T. Nat. Cell Biol. 1999; 1: 27-32Crossref PubMed Scopus (314) Google Scholar, 7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar, 12Sever S. Muhlberg A.B. Schmid S.L. Nature. 1999; 398: 481-486Crossref PubMed Scopus (317) Google Scholar).Table IIITemperature effects on dynamin GTPase activityLTaLipid tubule.TWT dynaminT65A dynaminKmkcatKmkcat°Cμmmin-1μmmin-1-223.4 ± 1.70.19 ± 0.0397 ± 280.017 ± 0.00237102 ± 352.60 ± 0.98880 ± 3300.075 ± 0.027+225.3 ± 2.57.6 ± 0.244 ± 8.20.062 ± 0.0043737 ± 18105 ± 472115 ± 14131.22 ± 0.24a Lipid tubule. Open table in a new tab A Search for Catalytic Residues in the GTPase Domain— Dynamin has a relatively high intrinsic rate of GTP hydrolysis, at least when measured at physiologically relevant temperatures, suggesting that residues essential for catalysis are intrinsic to the GTPase domain in the unassembled state. Thus, we propose that dynamin assembly-stimulated GTPase activity, mediated by the GTPase activating protein activity of GED, functions to better position intrinsic catalytic residues for more efficient hydrolysis of GTP. This situation is more akin to that of Gα subunits, whose intrinsic rate of GTP hydrolysis (1–2 min–1) is stimulated ∼50–100-fold by RGS-type GTPase activating proteins, which alter the conformation of active site residues in the switch regions of the Gα GTPase domain for more effective catalysis (11Wittinghofer A. Hall A. GTPases. 24. Oxford University Press, New York2000: 244-310Google Scholar, 24Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (937) Google Scholar). Among these is a highly conserved arginine residue in Gα subunits that is located in sw1, near the invariant G2 Thr, which functions to neutralize negative charges that develop in the transition state during catalysis (24Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (937) Google Scholar). Although we have established that mutation of the sw1 and sw2 residues, Ser-61 and Thr-141 alter catalysis, neither is essential for GTP hydrolysis. Similarly, although mutation of Thr-65 severely impairs GTP hydrolysis, it also severely compromises GTP binding, and thus, a direct role in catalysis cannot be determined. In an attempt to identify essential catalytic residues within the dynamin GTPase domain that are specifically required for GTP hydrolysis, we mutated three conserved Arg residues within the sw1 region, Arg-54, Arg-59, and Arg-67; however, neither of these mutations significantly perturbed basal GTPase activity. 3S. P. Sholly, M. Leonard, and S. Schmid, unpublished data. Others have established that the R66A mutation equally impairs both GTP binding and hydrolysis (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar). Thus, the catalytic Arg within the dynamin GTPase domain, if it exists, remains elusive. 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 Km and kcat 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 (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar, 16Damke H. Binns D.D. Ueda H. Schmid S.L. Baba T. Mol. Biol. Cell. 2001; 12: 2578-2589Crossref PubMed Scopus (150) Google Scholar, 25Hill E. van der Kaay J. Downes C.P. Smythe E. J. Cell Biol. 2001; 152: 309-324Crossref PubMed Scopus (110) Google Scholar), 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). For quantitative analysis, the extent of endocytosis in vivo at 10 min was plotted as a function of either the calculated ratio of dynamin-GTP/dynamin (Fig. 5, A and C) or as a function of their relative rates of GTP hydrolysis (Fig. 5, B and D), as determined in vitro based on Michaelis-Menten steady-state kinetics (see "Experimental Procedures"). We observed no apparent correlation between the rates of endocytosis and the degree of dynamin-GTP loading, calculated based on the basal GTPase properties of dynamin (Fig. 5A) and only a weak correlation when calculated based on assembly-stimulated GTPase activity (Fig. 5C). In particular, dyn1-S61D (Fig. 5, black diamonds), which exhibited near wild type GTP binding as determined either by Km measurements or direct filter binding assays, was a significant dominant-negative inhibitor of endocytosis. The dyn1-T65D mutant is also a significant outlier in this analysis, at least when based on basal GTPase properties (Fig. 5A); however, the disparity in apparent affinity for GTP as assessed by Km values compared with direct GTP binding assays renders results from this mutant difficult to interpret. Nonetheless, data represented in Fig. 5, A and C, argue that GTP binding is not sufficient for dynamin function in clathrin-mediated endocytosis but suggest that conformational changes induced by both GTP binding and self-assembly may be important for dynamin function in vivo. In contrast, we observed a strong correlation between the rate of endocytosis and the calculated rates for both basal and assembly-stimulated GTP hydrolysis (Fig. 5, B and D). Transferrin internalization was reduced in cells overexpressing mutant dynamins with lower rates of GTP hydrolysis. We (16Damke H. Binns D.D. Ueda H. Schmid S.L. Baba T. Mol. Biol. Cell. 2001; 12: 2578-2589Crossref PubMed Scopus (150) Google Scholar) and others (7Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (379) Google Scholar, 25Hill E. van der Kaay J. Downes C.P. Smythe E. J. Cell Biol. 2001; 152: 309-324Crossref PubMed Scopus (110) Google Scholar) report that GTPase-defective T65F and T65A mutants strongly inhibited transferrin internalization; however, because these mutations also severely impair GTP binding, the reasons for their inhibitory effects on endocytosis are difficult to interpret. Our observation that overexpression of dyn1-S61D (black diamonds), which has a 4–5-fold lower rate of GTP hydrolysis with little change in affinity for GTP, reduces the rate of transferrin internalization by ∼60% and is, by contrast, more informative. 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 (12Sever S. Muhlberg A.B. Schmid S.L. Nature. 1999; 398: 481-486Crossref PubMed Scopus (317) Google Scholar, 26Sever S. Damke H. Schmid S.L. J. Cell Biol. 2000; 150: 1137-1148Crossref PubMed Scopus (196) Google Scholar). These mutations, located in GED, did not significantly affect the basal rate of GTP hydrolysis (12Sever S. Muhlberg A.B. Schmid S.L. Nature. 1999; 398: 481-486Crossref PubMed Scopus (317) Google Scholar). 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 (27Evergren E. Tomilin N. Vasylieva E. Sergeeva V. Bloom O. Gad H. Capani F. Shupliakov O. J. Neurosci. Methods. 2004; 135: 169-174Crossref PubMed Scopus (30) Google Scholar) confirm our earlier observations in nonneuronal cells (14Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1046) Google Scholar) 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 (5Hinshaw J.E. Schmid S.L. Nature. 1995; 374: 190-192Crossref PubMed Scopus (665) Google Scholar, 6Stowell M.H.B. Marks B. Wigge P. McMahon H.T. Nat. Cell Biol. 1999; 1: 27-32Crossref PubMed Scopus (314) Google Scholar). 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 (21Maeda K. Nakata T. Noda Y. Sato-Yoshitake R. Hirokawa N. Mol. Biol. Cell. 1992; 3: 1181-1194Crossref PubMed Scopus (92) Google Scholar, 28Warnock D.E. Hinshaw J.E. Schmid S.L. J. Biol. Chem. 1996; 271: 22310-22314Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), 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 (29Miwako I. Schroter T. Schmid S.L. Traffic. 2003; 4: 376-389Crossref PubMed Scopus (86) Google Scholar). 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 (30Matsuoka K. Orci L. Amherdt M. Bednarek S.Y. Hamamoto S. Schekman R. Yeung T. Cell. 1998; 93: 263-275Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 31Kuge O. Dascher C. Orci L. Rowe T. Amherdt M. Plutner H. Ravazzola M. Tanigawa G. Rothman J.E. Balch W.E. J. Cell Biol. 1994; 125: 51-65Crossref PubMed Scopus (254) Google Scholar, 32Pepperkok R. Whitney J.A. Gomez M. Kreis T.E. J. Cell Sci. 2000; 113: 135-144PubMed Google Scholar, 33Legate K.R. Falcone D. Andrews D.W. J. Biol. Chem. 2000; 275: 27439-27446Abstract Full Text Full Text PDF PubMed Google Scholar, 34Schwartz T. Blobel G. Cell. 2003; 112: 793-803Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). 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. We are grateful to Alisa Jones and Tricia Glenn for technical assistance in protein expression and purification.
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