Mechanisms and functions of guanylate‐binding proteins and related interferon‐inducible GTPases: Roles in intracellular lysis of pathogens

Guanylate‐binding proteins (GBPs) are a group interferon‐inducible GTPases within the constellation of the dynamin GTPase superfamily. These proteins restrict the replication of intracellular pathogens in both immune and non‐immune cells. GBPs and their related family members immunity‐related GTPases target and lyse the membrane of the pathogen‐containing vacuole, destroying the residential niche of vacuolar protozoal and bacterial pathogens. They also prevent virion infectivity and target replication complexes of ribonucleic acid viruses. The exciting concept that GBPs and immunity‐related GTPases can directly target the membrane of bacteria and protozoa has emerged. Rupture and lysis of the pathogen membrane mediates liberation of concealed microbial ligands for activation of innate immune sensing pathways and the inflammasome. Further studies have demonstrated a capacity of GBPs to recruit additional antimicrobial factors, highlighting the complexity of the molecular mechanisms involved in pathogen killing. In this mini‐review, we discuss recent advances describing the localisation and functions of GBPs on the host and pathogen membrane. We also highlight unresolved questions related to the regulation of GBPs in cell‐autonomous immunity to intracellular pathogens.     

Interferon-induced guanylate-binding proteins lack an N(T)KXD consensus motif and bind GMP in addition to GDP and GTP.

The primary structures of interferon (IFN)-induced guanylate-binding proteins (GBPs) were deduced from cloned human and murine cDNAs. These proteins contained only two of the three sequence motifs typically found in GTP/GDP-binding proteins. The N(T)KXD motif, which is believed to confer guanine specificity in other nucleotide-binding proteins, was absent. Nevertheless, the IFN-induced GBPs exhibited a high degree of selectivity for binding to agarose-immobilized guanine nucleotides. An interesting feature of IFN-induced GBPs is that they strongly bound to GMP agarose in addition to GDP and GTP agaroses but failed to bind to ATP agarose and all other nucleotide agaroses tested. Both GTP and GMP, but not ATP, competed for binding of murine GBP-1 to agarose-immobilized GMP. The IFN-induced GBPs thus define a distinct novel family of proteins with GTP-binding activity. We further demonstrate that human and murine cells contain at least two genes encoding IFN-induced GBPs. The cloned murine cDNA codes for GBP-1, an IFN-induced protein previously shown to be absent from mice of Gbp-1b genotype.     

Immunity-related GTPase M (IRGM) proteins influence the localization of guanylate-binding protein 2 (GBP2) by modulating macroautophagy

The immunity-related GTPases (IRGs) are a family of proteins induced by interferon-γ that play a crucial role in innate resistance to intracellular pathogens. The M subfamily of IRG proteins (IRGM) plays a profound role in this context, in part because of the ability of its members to regulate the localization and expression of other IRG proteins. We present here evidence that IRGM proteins affect the localization of the guanylate-binding proteins (GBPs), a second family of interferon-induced GTP-binding proteins that also function in innate immunity. Absence of Irgm1 or Irgm3 led to accumulation of Gbp2 in intracellular compartments that were positive for both the macroautophagy (hereafter referred to as autophagy) marker LC3 and the autophagic adapter molecule p62/Sqstm1. Gbp2 was similarly relocalized in cells in which autophagy was impaired because of the absence of Atg5. Both in Atg5- and IRGM-deficient cells, the IRG protein Irga6 relocalized to the same compartments as Gbp2, raising the possibility of a common regulatory mechanism. However, other data indicated that Irga6, but not Gbp2, was ubiquitinated in IRGM-deficient cells. Similarly, coimmunoprecipitation studies indicated that although Irgm3 did interact directly with Irgb6, it did not interact with Gbp2. Collectively, these data suggest that IRGM proteins indirectly modulate the localization of GBPs through a distinct mechanism from that through which they regulate IRG protein localization. Further, these results suggest that a core function of IRGM proteins is to regulate autophagic flux, which influences the localization of GBPs and possibly other factors that instruct cell-autonomous immune resistance.     

The antiviral dynamin family member, MxA, tubulates lipids and localizes to the smooth endoplasmic reticulum

Mx proteins are induced by type I interferon and inhibit a broad range of viruses by undefined mechanisms. They are included within the dynamin family of large GTPases, which are involved in vesicle trafficking and share common biophysical features. These properties include the propensity to self-assemble, an affinity for lipids, and the ability to tubulate membranes. In this report we establish that human MxA, despite sharing only 30% homology with conventional dynamin, possesses many of these properties. We demonstrate for the first time that MxA self-assembles into rings that tubulate lipids in vitro, and associates with a specific membrane compartment in cells, the smooth endoplasmic reticulum.     

Three-dimensional reconstruction of dynamin in the constricted state

Members of the dynamin family of GTPases have unique structural properties that might reveal a general mechanochemical basis for membrane constriction. Receptor-mediated endocytosis, caveolae internalization and certain trafficking events in the Golgi all require dynamin for vesiculation. The dynamin-related protein Drp1 (Dlp1) has been implicated in mitochondria fission and a plant dynamin-like protein phragmoplastin is involved in the vesicular events leading to cell wall formation. A common theme among these proteins is their ability to self-assemble into spirals and their localization to areas of membrane fission. Here we present the first three-dimensional structure of dynamin at a resolution of approximately 20 A, determined from cryo-electron micrographs of tubular crystals in the constricted state. The map reveals a T-shaped dimer consisting of three prominent densities: leg, stalk and head. The structure suggests that the dense stalk and head regions rearrange when GTP is added, a rearrangement that generates a force on the underlying lipid bilayer and thereby leads to membrane constriction. These results indicate that dynamin is a force-generating 'contrictase'.     

Mitochondria-specific function of the dynamin family protein DLP1 is mediated by its C-terminal domains

The dynamin superfamily of large GTPases has been implicated in a variety of distinct intracellular membrane remodeling events. One of these family members, DLP1/Drp1, is similar to conventional dynamins as it contains an N-terminal GTPase domain followed by a middle region (MID), an unconserved region (UC), and a coiled-coil (CC) domain. DLP1 has been shown to function in membrane-based processes distinct from conventional dynamin, most notably mitochondrial fission. In this study, we tested whether the functional specificities of DLP1 and dynamin stems from differences in the individual domains of these proteins by generating dynamin/DLP1 chimeras in which correlate domains had been interchanged. Here we report that three consecutive C-terminal domains of DLP1 (MID-UC-CC) contain information necessary for DLP1-specific function and removing any one of these domains results in a loss of DLP1 function. Importantly, the coiled-coil (CC) domain of DLP1 alone targets specifically and exclusively to mitochondria, implicating its involvement in localizing DLP1 to this organelle in vivo. The mitochondrial targeting information within the DLP1 CC domain is not sufficient to retarget dynamin to mitochondria but is still able to adequately function as an assembly domain in a dynamin background. These data suggest that whereas the GTPase domain of DLP1 provides an enzymatic function, other domains contain information for intermolecular assembly and mitochondrial targeting.     

Garrotes, springs, ratchets, and whips: putting dynamin models to the test

The GTPase dynamin is essential for clathrin-mediated endocytosis. Numerous new and exciting discoveries regarding dynamin function in vivo and in vitro have led to various models in which dynamin functions directly in membrane fission and the release of clathrin-coated vesicles from the plasma membrane. This would make dynamin unique among GTPases in its ability to act as a mechanochemical enzyme. Here we review the various models and their supporting data. We then discuss new findings that raise doubts as to whether dynamin breaks the paradigm that governs regulatory GTPases.     

Dynamin II is involved in endocytosis but not in the formation of transport vesicles from the trans-Golgi network

Dynamins are a family of approximately 100-kDa GTPases that are thought to play a pivotal role in the formation of endocytic coated vesicles. There are three dynamin genes in mammals: dynamin I is neuron-specific, dynamin II shows ubiquitous expression, and dynamin III is expressed in testis, brain, and lung. However, most studies on the functions of dynamins to date have been restricted to dynamin I. In the present study, we show that, like dynamin I, dynamin II is involved in receptor-mediated endocytosis. While this study was in progress, Jones et al. [Jones, S.M., Howell, K.E., Henley, J.R., Cao, H., and McNiven, M.A. (1998) Science 279, 573-577] reported that dynamin II is localized in the trans-Golgi network (TGN) and involved in the formation of constitutive transport vesicles and clathrin-coated vesicles from this compartment. However, immunofluorescence analyses and experiments using cells transfected with dominant-negative dynamin II failed to show any evidence for localization of dynamin II in the TGN or for its involvement in vesicle formation from this compartment. Our data thus indicate that dynamin II is involved in endocytosis but not in the formation of transport vesicles from the TGN.     

Critical but distinct roles for the pleckstrin homology and cysteine-rich domains as positive modulators of Vav2 signaling and transformation

Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unexpectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modulators of Vav2 DH domain function in vivo.     

Dynamin self-assembly and the vesicle scission mechanism: how dynamin oligomers cleave the membrane neck of clathrin-coated pits during endocytosis

Recently, Gao et al. and Chappie et al. elucidated the crystal structures of the polytetrameric stalk domain of the dynamin-like virus resistance protein, MxA, and of the G-domain dimer of the large, membrane-deforming GTPase, dynamin, respectively. Combined, they provide a hypothetical oligomeric structure for the complete dynamin protein. Here, it is discussed how the oligomers are expected to form and how they participate in dynamin mediated vesicle fission during the process of endocytosis. The proposed oligomeric structure is compared with the novel mechanochemical model of dynamin function recently proposed by Bashkirov et al. and Pucadyil and Schmid. In conclusion, the new model of the dynamin oligomer has the potential to explain how short self-limiting fissogenic dynamin assemblies are formed and how concerted GTP hydrolysis is achieved. The oligomerisation of two other dynamin superfamily proteins, the guanylate binding proteins (GBPs) and the immunity-related GTPases (IRGs), is addressed briefly.     

Mammalian dynamin-like protein DLP1 tubulates membranes

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) but not GTP. EM of the GTPgammaS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPgammaS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.     

Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.     

Dynamin and cytokinesis

Animal and plant cytokineses appear morphologically distinct. Recent studies, however, have revealed that these cellular processes have many things in common, including the requirement of co-ordinated membrane trafficking and cytoskeletal dynamics. At the intersection of these two processes are the members of the dynamin family of ubiquitous eukaryotic GTPases. In this review, we highlight the conserved contribution of classical dynamin and dynamin-related proteins during cytokinesis in both animal and plant systems.     

Identification of a novel human Rho protein with unusual properties: GTPase deficiency and in vivo farnesylation.

We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.     

Dynamin isoform-specific interaction with the shank/ProSAP scaffolding proteins of the postsynaptic density and actin cytoskeleton

Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover.     

The GTPase effector domain sequence of the Dnm1p GTPase regulates self-assembly and controls a rate-limiting step in mitochondrial fission

Dnm1p belongs to a family of dynamin-related GTPases required to remodel different cellular membranes. In budding yeast, Dnm1p-containing complexes assemble on the cytoplasmic surface of the outer mitochondrial membrane at sites where mitochondrial tubules divide. Our previous genetic studies suggested that Dnm1p's GTPase activity was required for mitochondrial fission and that Dnm1p interacted with itself. In this study, we show that bacterially expressed Dnm1p can bind and hydrolyze GTP in vitro. Coimmunoprecipitation studies and yeast two-hybrid analysis suggest that Dnm1p oligomerizes in vivo. With the use of the yeast two-hybrid system, we show that this Dnm1p oligomerization is mediated, in part, by a C-terminal sequence related to the GTPase effector domain (GED) in dynamin. The Dnm1p interactions characterized here are similar to those reported for dynamin and dynamin-related proteins that form higher order structures in vivo, suggesting that Dnm1p assembles to form rings or collars that surround mitochondrial tubules. Based on previous findings, a K705A mutation in the Dnm1p GED is predicted to interfere with GTP hydrolysis, stabilize active Dnm1p-GTP, and stimulate a rate-limiting step in fission. Here we show that expression of the Dnm1 K705A protein in yeast enhances mitochondrial fission. Our results provide evidence that the GED region of a dynamin-related protein modulates a rate-limiting step in membrane fission.     

Regulation of dynamin family proteins by post-translational modifications

Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agents involved in several biological processes such as endocytosis, maintenance of organelle morphology and viral resistance. These large GTPases couple GTP hydrolysis with membrane alterations such as fission, fusion or tubulation by undergoing repeated cycles of self-assembly/disassembly. The functions of these proteins are regulated by various post-translational modifications that affect their GTPase activity, multimerization or membrane association. Recently, several reports have demonstrated variety of such modifications providing a better understanding of the mechanisms by which dynamin proteins influence cellular responses to physiological and environmental cues. In this review, we discuss major post-translational modifications along with their roles in the mechanism of dynamin functions and implications in various cellular processes.     

Study of the mechanism of Ras-dva small GTPase intracellular localization

An analysis of amino acid sequences of small GTPases of the Ras-dva family allowed us to determine the C-terminal prenylation motif, which could be responsible for the membrane localization of these proteins. We demonstrated using in vivo EGFP tracing that the Ras-dva small GTPases from Xenopus laevis embryo cells and NIH-3T3 fibroblasts are localized on both plasma membranes and endomembranes (the endoplasmic reticulum, the Golgi apparatus, and vesicles). At the same time, the replacement of the Cys residue, the SH group of which must be theoretically farnesylated, in the C-terminal prenylation motif of the Ras-dva small GTPase by the Ser residue prevented the membrane localization of the protein. These results indicate that the C-terminal prenylation site is critical for the membrane localization of small Ras-dva GTPases.     

RHO GTPase Signaling for Axon Extension: Is Prenylation Important?

Many lines of evidence indicate the importance of the Rho family guanine nucleotide triphosphatases (GTPases) in directing axon extension and guidance. The signaling networks that involve these proteins regulate actin cytoskeletal dynamics in navigating neuronal growth cones. However, the intricate patterns that regulate Rho GTPase activation and signaling are not yet fully defined. Activity and subcellular localization of the Rho GTPases are regulated by post-translational modification. The addition of a geranylgeranyl group to the carboxy (C-) terminus targets Rho GTPases to the plasma membrane and promotes their activation by facilitating interaction with guanine nucleotide exchange factors and allowing sequestering by association with guanine dissociation inhibitors. However, it is unclear how these modifications affect neurite extension or how subcellular localization alters signaling from the classical Rho GTPases (RhoA, Rac1, and Cdc42). Here, we review recent data addressing this issue and propose that Rho GTPase geranylgeranylation regulates outgrowth.     

Dominant-negative inhibition of receptor-mediated endocytosis by a dynamin-1 mutant with a defective pleckstrin homology domain

The dynamins are 100 kDa GTPases involved in the scission of endocytic vesicles from the plasma membrane [1]. Dynamin-1 is present in solution as a tetramer [2], and undergoes further self-assembly following its recruitment to coated pits to form higher-order oligomers that resemble 'collars' around the necks of nascent coated buds [1] [3]. GTP hydrolysis by dynamin in these collars is thought to accompany the 'pinching off' of endocytic vesicles [1] [4]. Dynamin contains a pleckstrin homology (PH) domain that binds phosphoinositides [5] [6], which in turn enhance both the GTPase activity [5] [7] [8] and self-assembly [9] [10] of dynamin. We recently showed that the dynamin PH domain binds phosphoinositides only when it is oligomeric [6]. Here, we demonstrate that interactions between the dynamin PH domain and phosphoinositides are important for dynamin function in vivo. Full-length dynamin-1 containing mutations that abolish phosphoinositide binding by its PH domain was a dominant-negative inhibitor of receptor-mediated endocytosis. Mutated dynamin-1 with both a defective PH domain and impaired GTP binding and hydrolysis also inhibited receptor-mediated endocytosis. These findings suggest that the role of the PH domain in dynamin function differs from that seen for other PH domains. We propose that high-avidity binding to phosphoinositide-rich regions of the membrane by the multiple PH domains in a dynamin oligomer is critical for dynamin's ability to complete vesicle budding.