GTP hydrolysis mechanism of Ras-like GTPases

The Ras-like GTPases regulate diverse cellular functions via the chemical cycle of binding and hydrolyzing GTP molecules. They alternate between GTP- and GDP-bound conformations. The GTP-bound conformation is biologically active and promotes a cellular function, such as signal transduction, cytoskeleton organization, protein synthesis/translocation, or a membrane budding/fusion event. GTP hydrolysis turns off the GTPase switch by converting it to the inactive GDP-bound conformation. The fundamental GTP hydrolysis mechanism by these GTPases has generated considerable interest over the last two decades but remained to be firmly established. This review provides an update on the catalytic mechanism with discussions on recent developments from kinetic, structural, and model studies in the context of the various GTP hydrolysis models proposed over the years.     

A GTPase-activating protein controls Rab5 function in endocytic trafficking

Rab-family GTPases are conserved regulators of membrane trafficking that cycle between inactive GDP-bound and activated GTP-bound states. A key determinant of Rab function is the lifetime of the GTP-bound state. As Rabs have a low intrinsic rate of GTP hydrolysis, this process is under the control of GTP-hydrolysis-activating proteins (GAPs). Due to the large number of Rabs and GAPs that are encoded by the human genome, it has proven difficult to assign specific functional relationships to these proteins. Here, we identify a Rab5-specific GAP (RabGAP-5), and show that RN-Tre (previously described as a Rab5 GAP) acts on Rab41. RabGAP-5 overexpression triggers a loss of the Rab5 effector EEA1 from endosomes and blocks endocytic trafficking. By contrast, depletion of RabGAP-5 results in increased endosome size, more endosome-associated EEA1, and disrupts the trafficking of EGF and LAMP1. RabGAP-5 therefore limits the amount of activated Rab5, and thereby regulates trafficking through endosomes.     

The significance of the free energy of hydrolysis of GTP for signal-transducing and regulatory GTPases

A large number of GTP/GDP binding proteins, which in general have intrinsic and/or stimulatable GTPase activity, have been identified in recent years and are involved in a wide range of cellular regulatory and signal transducing processes. A common property of these proteins is that they exist in what is generally described as an active form when GTP is bound and an inactive (resting) form when GDP is present. Thus, the intrinsic or stimulated GTPase activity of these ‘enzymes’ serves to turn off a signal or to terminate a regulated process. It has been suggested that these proteins, together with ATPases whose prime function is to convert the free energy of ATP hydrolysis into another form of energy or into energy-requiring chemical reactions should be grouped together under the heading of ‘energyases’. In this article, this suggestion is examined from the point of view of identifying the role of the free-energy of hydrolysis of GTP in the signal-transducing or regulatory process of the GTPases. It is concluded that there is a qualitative difference between ATPases and classical GTPases, in the sense that a quantitative relationship between the free-energy of GTP hydrolysis and the appearance of this energy in a different form cannot be directly defined. The significance of the high free energy of hydrolysis is that it allows efficient transition from the active to the inactive state of GTPases in spite of the tendency of the strong interaction of the GTP-bound form with a partner molecule (‘effector’), an essential feature of their mode of action, to stabilize the GTP-bound form.     

Characterization of a fast cycling ADP-ribosylation factor 6 mutant

Studies of GTPase function often employ expression of dominant negative or constitutively active mutants. Dominant negative mutants cannot bind GTP and thus cannot be activated. Constitutively active mutants cannot hydrolyze GTP and therefore accumulate a large pool of GTP-bound GTPase. These mutations block the normal cycle of GTP binding, hydrolysis, and release. Therefore, although the GTPase-deficient mutants are in the active conformation, they do not fully imitate all the actions of the GTPase. This is particularly true for the ADP-ribosylation factors (ARFs), GTPases that regulate vesicular trafficking events. In Ras and Rho GTPases replacement of phenylalanine 28 with a leucine residue produces a "fast cycling" mutant that can undergo spontaneous GTP-GDP exchange and retains the ability to hydrolyze GTP. Unfortunately this phenylalanine residue is not conserved in the ARF family of GTPases. Here we report the design and characterization of a novel activated mutant of ARF6, ARF6 T157A. In vitro studies show that ARF6 T157A can spontaneously bind and release GTP more quickly than the wild-type protein suggesting that it is a fast cycling mutant. This mutant has enhanced activity in vivo and induces cortical actin rearrangements in HeLa cells and enhanced motility in Madin-Darby canine kidney cells.     

Physical and functional connection between auxilin and dynamin during endocytosis

During clathrin-mediated endocytosis, the GTPase dynamin promotes formation of clathrin-coated vesicles, but its mode of action is unresolved. We provide evidence that a switch in three functional states of dynamin (dimers, tetramers, rings/spirals) coordinates its GTPase cycle. Dimers exhibit negative cooperativity whereas tetramers exhibit positive cooperativity with respect to GTP. Our study identifies tetramers as the kinetically most stable GTP-bound conformation of dynamin, which is required to promote further assembly into higher order structures such as rings or spirals. In addition, using fluorescence lifetime imaging microscopy, we show that interactions between dynamin and auxilin in cells are GTP-, endocytosis- and tetramer-dependent. Furthermore, we show that the cochaperone activity of auxilin is required for constriction of clathrin-coated pits, the same early step in endocytosis known to be regulated by the lifetime of dynamin:GTP. Together, our findings support the model that the GTP-bound conformation of dynamin tetramers stimulates formation of constricted coated pits at the plasma membrane by regulating the chaperone activity of hsc70/auxilin.     

GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37°C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-free YPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.     

Real-time in vitro measurement of GTP hydrolysis

Small GTPases require an active GTPase activity to function correctly in their cellular environment. Mutation of key residues involved in this activity renders the GTPase defective and the small G-protein constitutively active (GTP-locked). The GTPase activity is also a target for GTPase-activating proteins (GAPs) which act to attenuate GTPase signalling by accelerating the conversion of bound GTP to bound GDP. The measurement of GTP hydrolysis in vitro can therefore provide information on the intrinsic activity of the small GTPase (e.g., mutated GTPase activity) as well as help define GAP specificity. Current methods to measure GTP hydrolysis in vitro utilise either radioactivity-based filter-binding assays or measurements of GDP:GTP:P(i) ratios by high-performance liquid chromatography (HPLC). Both provide timed snapshots of the current GTP-bound state, can be prone to experimental errors, and do not provide a real-time observation of GTP hydrolysis. The method we describe here utilises a fluorescently labelled, phosphate-binding protein (PBP), which scavenges for free inorganic phosphate (P(i)). On binding of a single P(i), a change of protein conformation is coupled to a 7-fold increase in fluorescence of the fluorophore. This method therefore permits real-time monitoring of GTPase activity, through measurement of P(i) production. This review describes the process of preparing and labelling the PBP with the MDCC fluorophore, as well as an example of its use in measuring the GTPase activity of small GTPases. We also discuss the pros and cons, and implications of the technique in comparison to the radioactive and HPLC method of measuring the GTPase activity.     

Influencing cellular transformation by modulating the rates of GTP hydrolysis by Cdc42

The small GTPase Cdc42 has been implicated in a number of cellular responses ranging from the regulation of the actin cytoskeletal architecture to intracellular trafficking and cell cycle progression. Cdc42 mutants that constitutively exchange GDP for GTP but still hydrolyze GTP (called 'fast-cycling' mutants) promote cellular transformation, whereas Cdc42 mutants that are unable to hydrolyze GTP and are irreversibly trapped in the GTP-bound state often inhibit cell growth. In this work, we have set out to further establish that Cdc42 needs to cycle between its 'on' and 'off' states to stimulate cell growth, by examining the consequences of manipulating its GTP-binding/GTP hydrolytic cycle in two different ways. One approach was to examine whether substitutions that act in a manner opposite to the 'fast cyclers', and extend the lifetime of the activated GTP-bound state by slowing the GTP hydrolytic reaction (i.e., 'slow-cycling' mutations), positively influence cell growth. Indeed we show that one such slow-cycling mutant, Cdc42[Y32A], which is insensitive to Cdc42GAP but still exhibits a measurable intrinsic GTP hydrolytic activity, gives rise to increased levels of activated Cdc42 in NIH 3T3 cells. We go on to show that the Y32A mutant stimulates the actin cytoskeletal changes that lead to filopodia formation, confer growth advantages to fibroblasts under low serum conditions, and enable cells to grow to high densities when exposed to normal levels of serum. The second approach was to determine whether the transforming activity of the fast-cycling Cdc42[F28L] mutant can be reversed by compensating for its accelerated nucleotide exchange reaction through the expression of the GTPase-activating protein (Cdc42GAP) and the ensuing stimulation of GTP hydrolytic activity. We showed that expression of the limit functional domain of Cdc42GAP inhibited Cdc42[F28L]-induced transformation, as well as selectively reversed the transformed phenotypes caused by the hyperactivation of wild-type Cdc42 in cells expressing the oncogenic version of Dbl (for Diffuse B cell lymphoma), a guanine nucleotide exchange factor for Cdc42 and the related Rac and Rho GTPases. Overall, the results reported here establish the requirement for Cdc42 to cycle between its signaling-on and -off states in order to positively influence cell growth and highlight how the Cdc42GAP can play an important role in regulating cell proliferation.     

Rho GTPase activity zones and transient contractile arrays

The Rho GTPases-Rho, Rac and Cdc42-act as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state, to regulate the actin cytoskeleton. It has recently become apparent that the Rho GTPases can be activated in subcellular zones that appear semi-stable, yet are dynamically maintained. These Rho GTPase activity zones are associated with a variety of fundamental biological processes including symmetric and asymmetric cytokinesis and cellular wound repair. Here we review the basic features of Rho GTPase activity zones, suggest that these zones represent a fundamental signaling mechanism, and discuss the implications of zone properties from the perspective of both their function and how they are likely to be controlled.     

Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring

The GTPase dynamin plays an essential part in endocytosis by catalysing the fission of nascent clathrin-coated vesicles from the plasma membrane. Using preformed phosphatidylinositol-4,5-bisphosphate-containing lipid nanotubes as a membrane template for dynamin self-assembly, we investigate the conformational changes that arise during GTP hydrolysis by dynamin. Electron microscopy reveals that, in the GTP-bound state, dynamin rings appear to be tightly packed together. After GTP hydrolysis, the spacing between rings increases nearly twofold. When bound to the nanotubes, dynamin's GTPase activity is cooperative and is increased by three orders of magnitude compared with the activity of unbound dynamin. An increase in the Kcat (but not the K(m) of GTP hydrolysis accounts for the pronounced cooperativity. These data indicate that a novel, lengthwise ('spring-like') conformational change in a dynamin helix may participate in vesicle fission.     

The ribosome regulates the GTPase of the beta-subunit of the signal recognition particle receptor.

Protein targeting to the membrane of the ER is regulated by three GTPases, the 54-kD subunit of the signal recognition particle (SRP) and the alpha- and beta-subunit of the SRP receptor (SR). Here, we report on the GTPase cycle of the beta-subunits of the SR (SRbeta). We found that SRbeta binds GTP with high affinity and interacts with ribosomes in the GTP-bound state. Subsequently, the ribosome increases the GTPase activity of SRbeta and thus functions as a GTPase activating protein for SRbeta. Furthermore, the interaction between SRbeta and the ribosome leads to a reduction in the affinity of SRbeta for guanine nucleotides. We propose that SRbeta regulates the interaction of SR with the ribosome and thereby allows SRalpha to scan membrane-bound ribosomes for the presence of SRP. Interaction between SRP and SRalpha then leads to release of the signal sequence from SRP and insertion into the translocon. GTP hydrolysis then results in dissociation of SR from the ribosome, and SRP from the SR.     

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle

MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and P_i release can be uncoupled and that G-domain dissociation is directly responsible for the ‘ON’ state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and P_i. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.     

Proteomic identification and functional characterization of a novel ARF6 GTPase-activating protein, ACAP4

ARF6 GTPase is a conserved regulator of membrane trafficking and actin-based cytoskeleton dynamics at the leading edge of migrating cells. A key determinant of ARF6 function is the lifetime of the GTP-bound active state, which is orchestrated by GTPase-activating protein (GAP) and GTP-GDP exchanging factor. However, very little is known about the molecular mechanisms underlying ARF6-mediated cell migration. To systematically analyze proteins that regulate ARF6 activity during cell migration, we performed a proteomic analysis of proteins selectively bound to active ARF6 using mass spectrometry and identified a novel ARF6-specific GAP, ACAP4. ACAP4 encodes 903 amino acids and contains two coiled coils, one pleckstrin homology domain, one GAP motif, and two ankyrin repeats. Our biochemical characterization demonstrated that ACAP4 has a phosphatidylinositol 4,5-bisphosphate-dependent GAP activity specific for ARF6. The co-localization of ACAP4 with ARF6 occurred in ruffling membranes formed upon AIF(4) and epidermal growth factor stimulation. ACAP4 overexpression limited the recruitment of ARF6 to the membrane ruffles in the absence of epidermal growth factor stimulation. Expression of GTP hydrolysis-resistant ARF6(Q67L) resulted in accumulations of ACAP4 and ARF6 in the cytoplasmic membrane, suggesting that GTP hydrolysis is required for the ARF6-dependent membrane remodeling. Significantly the depletion of ACAP4 by small interfering RNA or inhibition of ARF6 GTP hydrolysis by overexpressing GAP-deficient ACAP4 suppressed ARF6-dependent cell migration in wound healing, demonstrating the importance of ACAP4 in cell migration. Thus, our study sheds new light on the biological function of ARF6-mediated cell migration.     

An Autoinhibited Noncanonical Mechanism of GTP Hydrolysis by Rheb Maintains mTORC1 Homeostasis

Rheb, an activator of mammalian target of rapamycin (mTOR), displays low intrinsic GTPase activity favoring the biologically activated, GTP-bound state. We identified a Rheb mutation (Y35A) that increases its intrinsic nucleotide hydrolysis activity ～10-fold, and solved structures of both its active and inactive forms, revealing an unexpected mechanism of GTP hydrolysis involving Asp65 in switch II and Thr38 in switch I. In the wild-type protein this noncanonical mechanism is markedly inhibited by Tyr35, which constrains the active site conformation, restricting the access of the catalytic Asp65 to the nucleotide-binding pocket. Rheb Y35A mimics the enthalpic and entropic changes associated with GTP hydrolysis elicited by the GTPase-activating protein (GAP) TSC2, and is insensitive to further TSC2 stimulation. Overexpression of Rheb Y35A impaired the regulation of mTORC1 signaling by growth factor availability. We demonstrate that the opposing functions of Tyr35 in the intrinsic and GAP-stimulated GTP catalysis are critical for optimal mTORC1 regulation.     

Synthesis of two fluorescent GTPγS molecules and their biological relevance

Fluorescent GTP analogues are utilized for an assortment of nucleic acid and protein characterization studies. Non-hydrolysable analogues such as GTPγS offer the advantage of keeping proteins in a GTP-bound conformation due to their resistance to hydrolysis into GDP. Two novel fluorescent GTPγS molecules were developed by linking fluorescein and tetramethylrhodamine to the γ-thiophosphate of GTPγS. Chemical and biological analysis of these two compounds revealed their successful synthesis and ability to bind to the nucleotide-binding site of tubulin. These two new fluorescent non-hydrolysable nucleotides offer new possibilities for biophysical and biochemical characterization of GTP-binding proteins.     

Rho family GTPases: more than simple switches

Rho family GTPases control a large variety of biological processes. Cycling of Rho proteins between the GDP-bound and the GTP-bound state is controlled by several classes of regulatory proteins. In this review, we discuss the signal-transduction mechanisms that control these regulators. We will emphasize the subcellular localization of Rho GTPases and their regulatory proteins and the role of GTP hydrolysis in signal transmission.     

Small GTP binding proteins and bacterial virulence

Many bacterial toxins and bacterial enzymes modify small GTPases. Toxins exhibit different enzymatic activities on either the switch 1 or switch 2 domains of these small GTPases leading to inactivation or activation of such intracellular timer molecules. In addition, some virulence factors of certain invasive bacteria such as Salmonella also modulate small GTP binding proteins either by mimicking GTPase exchange factors or GTPase activating proteins.     

The pre-hydrolysis state of p21(ras) in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins

BACKGROUND: In numerous biological events the hydrolysis of guanine triphosphate (GTP) is a trigger to switch from the active to the inactive protein form. In spite of the availability of several high-resolution crystal structures, the details of the mechanism of nucleotide hydrolysis by GTPases are still unclear. This is partly because the structures of the proteins in their active states had to be determined in the presence of non-hydrolyzable GTP analogues (e.g. GppNHp). Knowledge of the structure of the true Michaelis complex might provide additional insights into the intrinsic protein hydrolysis mechanism of GTP and related nucleotides. RESULTS: The structure of the complex formed between p21(ras) and GTP has been determined by X-ray diffraction at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing (100K). The structure of this complex differs from that of p21(ras)-GppNHp (determined at 277K) with respect to the degree of order and conformation of the catalytic loop (loop 4 of the switch II region) and the positioning of water molecules around the gamma-phosphate group. The changes in the arrangement of water molecules were induced by the cryo-temperature technique. CONCLUSIONS: The results shed light on the function of Gln61 in the intrinsic GTP hydrolysis reaction. Furthermore, the possibility of a proton shuffling mechanism between two attacking water molecules and an oxygen of the gamma-phosphate group can be proposed for the basal GTPase mechanism, but arguments are presented that render this protonation mechanism unlikely for the GTPase activating protein (GAP)-activated GTPase.     

The role of ADP-ribosylation factor and SAR1 in vesicular trafficking in plants

Ras-like small GTP binding proteins regulate a wide variety of intracellular signalling and vesicular trafficking pathways in eukaryotic cells including plant cells. They share a common structure that operates as a molecular switch by cycling between active GTP-bound and inactive GDP-bound conformational states. The active GTP-bound state is regulated by guanine nucleotide exchange factors (GEF), which promote the exchange of GDP for GTP. The inactive GDP-bound state is promoted by GTPase-activating proteins (GAPs) which accelerate GTP hydrolysis by orders of magnitude. Two types of small GTP-binding proteins, ADP-ribosylation factor (Arf) and secretion-associated and Ras-related (Sar), are major regulators of vesicle biogenesis in intracellular traffic and are founding members of a growing family that also includes Arf-related proteins (Arp) and Arf-like (Arl) proteins. The most widely involved small GTPase in vesicular trafficking is probably Arf1, which not only controls assembly of COPI- and AP1, AP3, and AP4/clathrin-coated vesicles but also recruits other proteins to membranes, including some that may be components of further coats. Recent molecular, structural and biochemical studies have provided a wealth of detail of the interactions between Arf and the proteins that regulate its activity as well as providing clues for the types of effector molecules which are controlled by Arf. Sar1 functions as a molecular switch to control the assembly of protein coats (COPII) that direct vesicle budding from ER. The crystallographic analysis of Sar1 reveals a number of structurally unique features that dictate its function in COPII vesicle formation. In this review, I will summarize the current knowledge of Arf and Sar regulation in vesicular trafficking in mammalian and yeast cells and will highlight recent advances in identifying the elements involved in vesicle formation in plant cells. Additionally, I will briefly discuss the similarities and dissimilarities of vesicle traffic in plant, mammalian and yeast cells.     

The role of ADP-ribosylation factor and SAR1 in vesicular trafficking in plants

Ras-like small GTP binding proteins regulate a wide variety of intracellular signalling and vesicular trafficking pathways in eukaryotic cells including plant cells. They share a common structure that operates as a molecular switch by cycling between active GTP-bound and inactive GDP-bound conformational states. The active GTP-bound state is regulated by guanine nucleotide exchange factors (GEF), which promote the exchange of GDP for GTP. The inactive GDP-bound state is promoted by GTPase-activating proteins (GAPs) which accelerate GTP hydrolysis by orders of magnitude. Two types of small GTP-binding proteins, ADP-ribosylation factor (Arf) and secretion-associated and Ras-related (Sar), are major regulators of vesicle biogenesis in intracellular traffic and are founding members of a growing family that also includes Arf-related proteins (Arp) and Arf-like (Arl) proteins. The most widely involved small GTPase in vesicular trafficking is probably Arf1, which not only controls assembly of COPI- and AP1, AP3, and AP4/clathrin-coated vesicles but also recruits other proteins to membranes, including some that may be components of further coats. Recent molecular, structural and biochemical studies have provided a wealth of detail of the interactions between Arf and the proteins that regulate its activity as well as providing clues for the types of effector molecules which are controlled by Arf. Sar1 functions as a molecular switch to control the assembly of protein coats (COPII) that direct vesicle budding from ER. The crystallographic analysis of Sar1 reveals a number of structurally unique features that dictate its function in COPII vesicle formation. In this review, I will summarize the current knowledge of Arf and Sar regulation in vesicular trafficking in mammalian and yeast cells and will highlight recent advances in identifying the elements involved in vesicle formation in plant cells. Additionally, I will briefly discuss the similarities and dissimilarities of vesicle traffic in plant, mammalian and yeast cells.