Role Of GTP Hydrolysis Research Articles

Membrane fusion by Drosophila atlastin does not require GTP hydrolysis.

Atlastin (ATL) GTPases undergo trans dimerization and a power strokelike crossover conformational rearrangement to drive endoplasmic reticulum membrane fusion. Fusion depends on GTP, but the role of nucleotide hydrolysis has remained controversial. For instance, nonhydrolyzable GTP analogs block fusion altogether, suggesting a requirement for GTP hydrolysis in ATL dimerization and crossover, but this leaves unanswered the question of how the ATL dimer is disassembled after fusion. We recently used the truncated cytoplasmic domain of wild-type Drosophila ATL (DATL) and a novel hydrolysis-deficient D127N variant in single turnover assays to reveal that dimerization and crossover consistently precede GTP hydrolysis, with hydrolysis coinciding more closely with dimer disassembly. Moreover, while nonhydrolyzable analogs can bind the DATL G domain, they fail to fully recapitulate the GTP-bound state. This predicted that nucleotide hydrolysis would be dispensable for fusion. Here we report that the D127N variant of full-length DATL drives both outer and inner leaflet membrane fusion with little to no detectable hydrolysis of GTP. However, the trans dimer fails to disassemble and subsequent rounds of fusion fail to occur. Our findings confirm that ATL mediated fusion is driven in the GTP-bound state, with nucleotide hydrolysis serving to reset the fusion machinery for recycling.

Atypical Switch-I Arginine plays a catalytic role in GTP hydrolysis by Rab21 from Entamoeba histolytica

Entamoeba histolytica, the causative agent of amoebic dysentery, liver abscess and colitis, exploits its vesicular trafficking machinery for survival and virulence. Rab family of small GTPases play a key role in the vesicular transport by undergoing the GTP/GDP cycle which is central to the biological processes. Amoebic genome encodes several atypical Rab GTPases which are unique due to absence of conserved sequence motif(s) or atypical residues in their catalytic site [Saito-Nakano et al., 2005 ]. Previously, EhRab21 has been reported to involve in amoebic invasion and migration [Emmanuel et al., 2015 ]. The conserved Glutamine of switch-II region is universally accepted to be crucial for GTP hydrolysis. Mutations that reduce the sidechain polarity of Glutamine render the protein GTPase activity deficient [Krengel et al., 1990]. Here, we report a catalytic role of atypical switch-I Arginine (R36) in intrinsic GTP hydrolysis catalysed by EhRab21. Unlike the GTPase activity deficient QL mutants, the GTPase activity of EhRab21Q64L was found to be marginally enhanced compared to the wild-type protein. Although EhRab21R36L mutant showed normal GTPase activity, the double mutant (R36L/Q64L) was found to be GTPase deficient. Thus, EhRab21 is a unique member of small GTPase family in which an atypical switch-I Arginine is capable of driving GTP hydrolysis independent of the conserved switch-II Glutamine.

Dual use of GTP hydrolysis by elongation factor G on the ribosome.

Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor.

An MBoC Favorite: Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP

In celebration of MBoC’s first 20 years, members of the Editorial Board, members of the ASCB Council, and others comment on their favorite MBoC papers from the past two decades. Admirers of mitosis delight in viewing the dynamicity of microtubules during mitosis, so it is no surprise that the properties required for such dynamicity—termed “dynamic instability”—has been a major research focus since the mid-20th century. To study the role of GTP in the dynamic instability of microtubules, investigators have used different GTP analogues. In general, GTP that binds tubulin is stable in the microtubule lattice, but it becomes unstable when hydrolyzed to GDP-tubulin. Hyman et al. (1992) showed that the GTP analogue guanylyl(α,β)-methylene-diphosphonate (GMPCPP) was more stable than GTP and promoted polymerization, because it was hydrolyzed extremely slowly, making it almost equivalent to a nonhydrolyzable analogue. The authors concluded that GTP hydrolysis is not important for microtubule polymerization but is essential for microtubule depolymerization and, therefore, microtubule dynamics. They also noticed that GMPCPP hydrolysis was enhanced by the surrounding GTP lattice, identifying cooperativity as another key element in dynamic instability. Since the appearance of this article, our understanding of dynamic instability has continued to improve and evolve. A PDF file of the paper discussed above is attached to this article.

Biochemical Characterization of Ribosome Assembly GTPase RbgA in Bacillus subtilis

The ribosome biogenesis GTPase A protein RbgA is involved in the assembly of the large ribosomal subunit in Bacillus subtilis, and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45 S in sucrose density gradients. Here, we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow k(cat) (14 h(-1)) and has a high K(m) (90 μM). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K(+) ions for GTPase activity, which was confirmed experimentally. Interaction with 50 S subunits, but not 45 S intermediates, increased GTPase activity by ∼55-fold. Stable association with 50 S subunits and 45 S intermediates was nucleotide-dependent, and GDP did not support strong interaction with either of the subunits. GTP and guanosine 5'-(β,γ-imido)triphosphate (GMPPNP) were sufficient to promote association with the 45 S intermediate, whereas only GMPPNP was able to support binding to the 50 S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.

AbstractsAstrobiology Graduate Conference (AbGradCon) 2011HOSTED BYMontana State UniversityBozeman, Montana • June 5–8, 2011

AbstractsAstrobiology Graduate Conference (AbGradCon) 2011HOSTED BYMontana State UniversityBozeman, Montana • June 5–8, 2011

Transformation Efficiency of RasQ61 Mutants Linked to Structural Features of the Switch Regions in the Presence of Raf

Transformation efficiencies of Ras mutants at residue 61 range over three orders of magnitude, but the in vitro GTPase activity decreases 10-fold for all mutants. We show that Raf impairs the GTPase activity of RasQ61L, suggesting that the Ras/Raf complex differentially modulates transformation. Our crystal structures show that, in transforming mutants, switch II takes part in a network of hydrophobic interactions burying the nucleotide and precatalytic water molecule. Our results suggest that Y32 and a water molecule bridging it to the gamma-phosphate in the wild-type structure play a role in GTP hydrolysis in lieu of the Arg finger in the absence of GAP. The bridging water molecule is absent in the transforming mutants, contributing to the burying of the nucleotide. We propose a mechanism for intrinsic hydrolysis in Raf-bound Ras and elucidate structural features in the Q61 mutants that correlate with their potency to transform cells.

2P303 人工脂質二重膜を用いたCOPII小胞形成過程の可視化 : GTP加水分解の役割について(生体膜・人工膜,口頭発表,第45回日本生物物理学会年会)

2P303 人工脂質二重膜を用いたCOPII小胞形成過程の可視化 : GTP加水分解の役割について(生体膜・人工膜,口頭発表,第45回日本生物物理学会年会)

Analysis of GTPases carrying hydrophobic amino acid substitutions in lieu of the catalytic glutamine: Implications for GTP hydrolysis

Ras superfamily GTP-binding proteins regulate important signaling events in the cell. Ras, which often serves as a prototype, efficiently hydrolyzes GTP in conjunction with its regulator GAP. A conserved glutamine plays a vital role in GTP hydrolysis in most GTP-binding proteins. Mutating this glutamine in Ras has oncogenic effects, since it disrupts GTP hydrolysis. The analysis presented here is of GTP-binding proteins that are a paradox to oncogenic Ras, since they have the catalytic glutamine (Glncat) substituted by a hydrophobic amino acid, yet can hydrolyze GTP efficiently. We term these proteins HAS-GTPases. Analysis of the amino acid sequences of HAS-GTPases reveals prominent presence of insertions around the GTP-binding pocket. Homology modeling studies suggest an interesting means to achieve catalysis despite the drastic hydrophobic substitution replacing the key Glncat of Ras-like GTPases. The substituted hydrophobic residue adopts a "retracted conformation," where it is positioned away from the GTP, as its role in catalysis would be unproductive. This conformation is further stabilized by interactions with hydrophobic residues in its vicinity. These interacting residues are strongly conserved and hydrophobic in all HAS-GTPases, and correspond to residues Asp92 and Tyr96 of Ras. An experimental support for the "retracted conformation" of Switch II arises from the crystal structures of Ylqf and hGBP1. This conformation allows us to hypothesize that, unlike in classical GTPases, catalytic residues could be supplied by regions other than the Switch II (i.e., either the insertions or a neighboring domain).

Mechanism of elongation factor G function in tRNA translocation on the ribosome.

The elongation cycle of ribosomal protein synthesis iscompleted by translocation, that is, the movement of thetRNA•mRNA complex on the ribosome. Translocation iscatalyzed by elongation factor G (EF-G) at the expense ofGTP. Early models of EF-G function and of the role ofGTP hydrolysis in translocation were based on two key results. One was that there is slow, spontaneous translocation, indicating that the reaction thermodynamically is exergonic without an additional energy source, such as GTPhydrolysis, and that the basic structural mechanism resides in the ribosome (Pestka 1969; Gavrilova and Spirin1972, 1974). Another was that the extent of EF-G-catalyzed translocation remained unchanged when GTP wasreplaced with nonhydrolyzable analogs, whereas the dissociation of EF-G from the ribosome was inhibited (Modolell et al. 1975; Belitsina et al. 1976). Thus, by analogywith the canonical GTPase switch mechanism, it was concluded that translocation is brought about by the bindingof EF-G•GTP to the ribosome, and that subsequent GTPhydrolysis is required for the dissociation of EF-G in theGDP-bound form (Kaziro 1978; Spirin 1985)...

Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis

Different models have been proposed that link the tubulin heterodimer nucleotide content and the role of GTP hydrolysis with microtubule assembly and dynamics. Here we compare the thermodynamics of microtubule assembly as a function of nucleotide content by van't Hoff analysis. The thermodynamic parameters of tubulin assembly in 30-100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM MgSO4, 2 mM EGTA, pH 6.9, in the presence of a weakly hydrolyzable analog, GMPCPP, the dinucleotide analog GMPCP plus 2 M glycerol, and GTP plus 2 M glycerol were obtained together with data for taxol-GTP/GDP tubulin assembly (GMPCPP and GMPCP are the GTP and GDP nucleotide analogs where the alpha beta oxygen has been replaced by a methylene, -CH2-). All of the processes studied are characterized by a positive enthalpy, a positive entropy, and a large, negative heat capacity change. GMPCP-induced assembly has the largest negative heat capacity change and GMPCPP has the second largest, whereas GTP/2 M glycerol- and taxol-induced assembly have more positive values, respectively. A large, negative heat capacity is most consistent with the burial of water-accessible hydrophobic surface area, which gives rise to the release of bound water. The heat capacity changes observed with GTP/2 M glycerol-induced and with taxol-induced assembly are very similar, -790 +/- 190 cal/mol/k, and correspond to the burial of 3330 +/- 820 A2 of nonpolar surface area. This value is shown to be very similar to an estimate of the buried nonpolar surface in a reconstructed microtubule lattice. Polymerization data from GMPCP- and GMPCPP-induced assembly are consistent with buried nonpolar surface areas that are 3 and 6 times larger. A linear enthalpy-entropy and enthalpy-free energy plot for tubulin polymerization reactions verifies that enthalpy-entropy compensation for this system is based upon true biochemical correlation, most likely corresponding to a dominant hydrophobic effect. Entropy analysis suggests that assembly with GTP/2 M glycerol and with taxol is consistent with conformational rearrangements in 3-6% of the total amino acids in the heterodimer. In addition, taxol binding contributes to the thermodynamics of the overall process by reducing the delta H degree and delta S degree for microtubule assembly. In the presence of GMPCPP or GMPCP, tubulin subunits associate with extensive conformational rearrangement, corresponding to 10% and 26% of the total amino acids in the heterodimer, respectively, which gives rise to a large loss of configurational entropy. An alternative, and probably preferable, interpretation of these data is that, especially with GMPCP-tubulin, additional isomerization or protonation events are induced by the presence of the methylene moiety and linked to microtubule assembly. Structural analysis shows that GTP hydrolysis is not required for sheet closure into a microtubule cylinder, but only increases the probability of this event occurring. Sheet extensions and sheet polymers appear to have a similar average length under various conditions, suggesting that the minimum cooperative unit for closure of sheets into a microtubule cylinder is approximately 400 nm long. Because of their low level of occurrence, sheets are not expected to significantly affect the thermodynamics of assembly.

Erratum: Role of GTP Hydrolysis in Fission of Caveolae Directly from Plasma Membranes

Figure 4B (p. 241) in the report "Role of GTP hydrolysis in fission of caveolae directly from plasma membranes" by J. E. Schnitzer et al. (11 Oct., p. 239) was printed incorrectly. The correct figure appears below. [See figure in the PDF file]

Erratum: Role of GTP Hydrolysis in Fission of Caveolae Directly from Plasma Membranes

Erratum: Role of GTP Hydrolysis in Fission of Caveolae Directly from Plasma Membranes

New Concept of Microtubule Dynamics and Microtubule Motor Movement and New Model of Chromosome Movement in Mitosis

In this study, microtubules are regarded as polymers with positive free surface energy. The dynamics of such polymers is complex, consisting of equilibrium, non-equilibrium, and one-dimensional processes. This view of microtubules enables us to propose a new model of microtubule dynamics both in vitroand in vivo, and to draw a new conclusion on the role of GTP hydrolysis and calcium cations in microtubule polymerization. The study also offers a new model of chromosome movement in mitosis and a new model of the movement of motor proteins along microtubules. In the conclusion the similarity and difference between microtubules and microfilaments is examined. We conclude that microfilaments, like microtubules, represent polymers with positive free surface energy and that the movement of myosins along microfilaments can be realized in the same manner as the movement of motor proteins along microtubules.

Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.

Evaluation of the Role of GTP Hydrolysis in the Interaction Between Mg2+and GTP in Regulating Agonist Binding to the Adenosine A1Receptor

Binding of agonists to adenosine receptors is reduced by GTP, whereas it is enhanced by Mg2+. The effect of GTP can be completely reversed by divalent cations, in contrast to the effect of the nonhydrolyzable analogue 5'-guanylylimidodiphosphate (GPPNHP). The present study addresses the role of divalent cation-stimulated specific and nonspecific GTP-ases in this reversal process. Under the conditions commonly employed in binding assays, almost all GTP is rapidly converted to GMP and Pi, indicating that maintenance of GTP levels is essential for the proper interpretation of results. A combination of a GTP-generating system and a competing substrate for high Km GTP-ases minimizes GTP breakdown. In the presence of these additions, the reversal of GTP effects is almost eliminated, and the inhibitory effects of both GTP and GPPNHP on agonist binding are reduced by divalent cations to a similar extent. Besides enhancing nonspecific GTP hydrolysis, Mg2+, but not Mn2+ or Ca2+, also stimulates specific agonist-dependent GTP-ase activity. Thus, it is evident that specific regulatory effects of Mg2+ and other divalent cations can only be identified when other, nonspecific, effects have been evaluated and controlled.

Role of GTP hydrolysis in microtubule polymerization: evidence for a coupled hydrolysis mechanism

The relationship between GTP hydrolysis and microtubule assembly has been investigated by using a rapid filtration method. Microtubules assembled from phosphocellulose-purified tubulin, double-labeled with [gamma-32P]- and [3H]GTP, were trapped and washed free of unbound nucleotide on glass fiber filters. The transient accumulation of microtubule-bound GTP predicted by uncoupled GTP hydrolysis models [Carlier & Pantaloni (1981) Biochemistry 20, 1918-1924; Carlier et al. (1987) Biochemistry 26, 4428-4437] during the rapid assembly of microtubules was not detectable under our experimental conditions. By calculating hypothetical time courses for the transient accumulation of microtubule-bound GTP, we demonstrate that microtubule-bound GTP would have been detectable even if the first-order rate constant for GTP hydrolysis were 4-5 times greater than the pseudo-first-order rate constant for tubulin subunit addition to microtubules. In a similar manner, we demonstrate that if GTP hydrolysis were uncoupled from microtubule assembly but were limited to the interface between GTP subunits and GDP subunits (uncoupled vectorial hydrolysis), then microtubule-bound GTP would have been detectable if GTP hydrolysis became uncoupled from microtubule assembly at less than 50 microM free tubulin, 5 times the steady-state tubulin concentration of our experimental conditions. In addition, during rapid microtubule assembly, we have not detected any microtubule-bound Pi, which has been proposed to form a stabilizing cap at the ends of microtubules [Carlier et al. (1988) Biochemistry 27, 3555-3559]. Also, several conditions that could be expected to increase the degree of potential uncoupling between GTP hydrolysis and microtubule assembly were examined, and no evidence of uncoupling was found. Our results are consistent with models that propose cooperative mechanisms that limit GTP hydrolysis to the terminal ring of tubulin subunits [e.g., O'Brien et al. (1987) Biochemistry 26, 4148-4156]. The results are also consistent with the hypothesis that a slow conformational change in tubulin subunits after GTP hydrolysis and Pi release occurs that results in destabilized microtubule ends when such subunits become exposed at the ends.

Role of GTP hydrolysis in microtubule treadmilling and assembly.

GTP hydrolysis accompanies addition of tubulin to microtubules. I find that hydrolysis is a requirement for the opposite-end assembly/disassembly of microtubules and consequent subunit treadmilling from one end to the other of the polymer. Neither GDP nor guanosine 5'-[beta, gamma-imido]triphosphate allows or participates in the treadmilling reaction. Therefore, there is a requirement for hydrolysis in the addition of subunits to the favored assembly end of the microtubule. Podophyllotoxin, an assembly inhibitory drug, "caps" the microtubule assembly end, preventing subunit loss from that site to equilibrium. Continued hydrolysis of GTP is required to maintain the podophyllotoxin cap. A corollary of this finding is that GTP hydrolysis is required for cap formation. Microtubules assembled in GTP enter a metastable state when all remaining GTP is hydrolyzed. This state is characterized by its ability to maintain indefinitely a subunit/polymer distribution ratio that is arbitrary and that can be altered at will by brief chilling or by addition of small amounts of GTP. This metastable state is labile to podophyllotoxin. Use of podophyllotoxin allows measurement of the microtubule treadmilling rate; use of podophyllotoxin in the absence of GTP allows measurement of the overall rate of dimer dissociation from the microtubule. Measurement of these rates has permitted determination of the efficiency with which adding dimers incorporate into the microtubule treadmill and are not lost to assembly end equilibrium. The efficiency varies with GTP concentration for unknown reasons, being high at 0.1 mM GTP and low at higher GTP concentrations.

Stoichiometry and role of GTP hydrolysis in bovine neurotubule assembly

A method is given for preparing tubulin with 1 mol of exchangeably bound [gamma-32P]GTP/mol of 6 S dimer. Bovine tubulin is shown to hydrolyze 1 mol of GTP/mol of 6 S dimer added to assembling microtubules at 37 degrees. Hydrolysis and assembly occur at the same rate and to the same extent. When microtubule-associated proteins (MAPs) are removed, both hydrolysis and assembly fail to occur. Readdition of the MAPs restores both activities. Tubulin with exchangeable GDP will co-assemble with GTP.tubulin even at equimolar levels. Exchangeability is demonstrated by pulse-chase experiments with GDP or GTP. GDP is also a potent inhibitor of assembly under these conditions, and the rate of assembly is reduced by 50% at 10 micron GDP. One mole of inorganic phosphate is released to the solvent per mole of exchangeable GTP hydrolyzed. An assembly mechanism is proposed in which exchangeable GTP is hydrolyzed without intermediate transphosphorylation of nonexchangeable GDP.

Competitive binding of EF1 and EF2 by mammalian ribosomes: Role of GTP hydrolysis in overcoming inhibition by EF2 of aminoacyl-tRNA binding

Preincubation of mammalian ribosomes with EF2 retards subsequent binding of EF1-leucyl tRNA when GTP hydrolysis does not occur. This indicates that both the elongation factors have a common binding site on the ribosome. Since the conditions permitting hydrolysis of GTP overcome this inhibition, the possible regulatory role of GTP at the common site on the ribosome is discussed.