An Intramolecular Signaling Element that Modulates Dynamin Function In Vitro and In Vivo

Dynamin exhibits a high basal rate of GTP hydrolysis that is enhanced by self-assembly on a lipid template. Dynamin''s GTPase effector domain (GED) is required for this stimulation, though its mechanism of action is poorly understood. Recent structural work has suggested that GED may physically dock with the GTPase domain to exert its stimulatory effects. To examine how these interactions activate dynamin, we engineered a minimal GTPase-GED fusion protein (GG) that reconstitutes dynamin''s basal GTPase activity and utilized it to define the structural framework that mediates GED''s association with the GTPase domain. Chemical cross-linking of GG and mutagenesis of full-length dynamin establishes that the GTPase-GED interface is comprised of the N- and C-terminal helices of the GTPase domain and the C-terminus of GED. We further show that this interface is essential for structural stability in full-length dynamin. Finally, we identify mutations in this interface that disrupt assembly-stimulated GTP hydrolysis and dynamin-catalyzed membrane fission in vitro and impair the late stages of clathrin-mediated endocytosis in vivo. These data suggest that the components of the GTPase-GED interface act as an intramolecular signaling module, which we term the bundle signaling element, that can modulate dynamin function in vitro and in vivo.     

Domain structure and function of dynamin probed by limited proteolysis

Dynamin is a 100-kDa GTPase with multiple domains. Some of these have known functions, namely, the N-terminal GTPase domain, the PH domain that binds phosphatidylinositol lipids, and the C-terminal proline-arginine-rich domain (PRD) that binds to several SH3 domain-containing dynamin partners. Others, for example, the "middle" located between the GTPase domain and the PH domain and a predicted alpha-helical domain located between the PH domain and PRD, have unknown functions. Dynamin exists as a homotetramer in solution and self-assembles into higher-order structures resembling rings and helical stacks of rings. Dynamin self-assembly stimulates its GTPase activity. We used limited proteolysis to dissect dynamin's domain structure and to gain insight into intradomain interactions that regulate dynamin self-assembly and stimulate GTPase activity. We found that the PH domain functions as a negative regulator of dynamin self-assembly and stimulates GTPase activity and that the alpha-helical domain, termed GED for GTPase effector domain, is required for stimulated GTPase activity.     

Dynamin GTPase regulation is altered by PH domain mutations found in centronuclear myopathy patients

The large GTPase dynamin has an important membrane scission function in receptor‐mediated endocytosis and other cellular processes. Self‐assembly on phosphoinositide‐containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin‐homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C‐terminal α‐helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either ‘sensitize’ dynamin to lipid stimulation or elevate basal GTPase rates by promoting self‐assembly and thus rendering dynamin no longer lipid responsive. We also describe a low‐resolution structure of dimeric dynamin from small‐angle X‐ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self‐assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.     

An assembly-incompetent mutant establishes a requirement for dynamin self-assembly in clathrin-mediated endocytosis in vivo

Dynamin GTPase activity is required for its biological function in clathrin-mediated endocytosis; however, the role of self-assembly has not been unambiguously established. Indeed, overexpression of a dynamin mutant, Dyn1-K694A, with impaired ability to self-assemble has been shown to stimulate endocytosis in HeLa cells (Sever et al., Nature 1999, 398, 481). To identify new, assembly-incompetent mutants of dynamin 1, we made point mutations in the GTPase effector/assembly domain (GED) and tested for their effects on self-assembly and clathrin-mediated endocytosis. Mutation of three residues, I690, K694, and I697, suggests that interactions with an amphipathic helix in GED are required for self-assembly. In particular, Dyn1-I690K failed to exhibit detectable assembly-stimulated GTPase activity under all assay conditions. Overexpression of this assembly-incompetent mutant inhibited transferrin endocytosis as potently as the GTPase-defective dominant-negative mutant, Dyn1-K44A. However, worm-like endocytic intermediates accumulated in cells expressing Dyn1-I690K that were structurally distinct from long tubules that accumulated in cells expressing Dyn1-K44A. Together these results provide new structural insight into the role of GED in self-assembly and assembly-stimulated GTPase activity and establish that dynamin self-assembly is essential for clathrin-mediated endocytosis.     

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.     

Dynamin GTPase domain mutants that differentially affect GTP binding, GTP hydrolysis, and clathrin-mediated endocytosis

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.     

Domain structure and intramolecular regulation of dynamin GTPase.

Dynamin is a 100 kDa GTPase required for receptor-mediated endocytosis, functioning as the key regulator of the late stages of clathrin-coated vesicle budding. It is specifically targeted to clathrin-coated pits where it self-assembles into 'collars' required for detachment of coated vesicles from the plasma membrane. Self-assembly stimulates dynamin GTPase activity. Thus, dynamin-dynamin interactions are critical in regulating its cellular function. We show by crosslinking and analytical ultracentrifugation that dynamin is a tetramer. Using limited proteolysis, we have defined structural domains of dynamin and evaluated the domain interactions and requirements for self-assembly and GTP binding and hydrolysis. We show that dynamin's C-terminal proline- and arginine-rich domain (PRD) and dynamin's pleckstrin homology (PH) domain are, respectively, positive and negative regulators of self-assembly and GTP hydrolysis. Importantly, we have discovered that the alpha-helical domain interposed between the PH domain and the PRD interacts with the N-terminal GTPase domain to stimulate GTP hydrolysis. We term this region the GTPase effector domain (GED) of dynamin.     

A model for dynamin self-assembly based on binding between three different protein domains.

Dynamin is a 100-kDa GTPase that assembles into multimeric spirals at the necks of budding clathrin-coated vesicles. We describe three different intramolecular binding interactions that may account for the process of dynamin self-assembly. The first binding interaction is the dimerization of a 100-amino acid segment in the C-terminal half of dynamin. We call this segment the assembly domain, because it appears to be critical for multimerization. The second binding interaction occurs between the assembly domain and the N-terminal GTPase domain. The strength of this interaction is controlled by the nucleotide-bound state of the GTPase domain, as shown with mutations in GTP binding motifs and in vitro binding experiments. The third binding interaction occurs between the assembly domain and a segment that we call the middle domain. This is the segment between the N-terminal GTPase domain and the pleckstrin homology domain. The three different binding interactions suggest a model in which dynamin molecules first dimerize. The dimers are then linked into a chain by a second binding reaction. The third binding interaction might connect adjacent rungs of the spiral.     

Dynamin:GTP controls the formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis

The GTPase dynamin is essential for receptor-mediated endocytosis, but its function remains controversial. A domain of dynamin, termed the GTPase effector domain (GED), controls dynamin's high stimulated rates of GTP hydrolysis by functioning as an assembly-dependent GAP. Dyn(K694A) and dyn(R725A) carry point mutations within GED resulting in reduced assembly stimulated GTPase activity. Biotinylated transferrin is more rapidly sequestered from avidin in cells transiently overexpressing either of these two activating mutants (Sever, S., A.B. Muhlberg, and S.L. Schmid. 1999. Nature. 398:481-486), suggesting that early events in receptor-mediated endocytosis are accelerated. Using stage-specific assays and morphological analyses of stably transformed cells, we have identified which events in clathrin-coated vesicle formation are accelerated by the overexpression of dyn(K694A) and dyn(R725A). Both mutants accelerate the formation of constricted coated pits, which we identify as the rate limiting step in endocytosis. Surprisingly, overexpression of dyn(R725A), whose primary defect is in stimulated GTP hydrolysis, but not dyn(K694A), whose primary defect is in self-assembly, inhibited membrane fission leading to coated vesicle release. Together, our data support a model in which dynamin functions like a classical GTPase as a key regulator of clathrin-mediated endocytosis.     

Dynamin architecture--from monomer to polymer

Dynamins form a family of eukaryotic and prokaryotic proteins involved in membrane fission, fusion and restructuring. They have complex mechanisms of self-assembly, which are coupled to the tubulation and destabilization of lipid bilayers. Recent structural data has revolutionized our understanding and is now yielding detailed insights into dynamin structure, from monomer through to polymer. Traditional division of the dynamin subunit into GTPase domain, middle domain and GTPase effector domain based on sequence alignments and biochemistry is not supported by recent structural data. A unified model of dynamin architecture is presented here, based on observation that the basic dynamin fold is conserved across evolutionary kingdoms.     

Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.     

Dynamin-Catalyzed Membrane Fission Requires Coordinated GTP Hydrolysis

Dynamin is the most-studied membrane fission machinery and has served as a paradigm for studies of other fission GTPases; however, several critical questions regarding its function remain unresolved. In particular, because most dynamin GTPase domain mutants studied to date equally impair both basal and assembly-stimulated GTPase activities, it has been difficult to distinguish their respective roles in clathrin-mediated endocytosis (CME) or in dynamin catalyzed membrane fission. Here we compared a new dynamin mutant, Q40E, which is selectively impaired in assembly-stimulated GTPase activity with S45N, a GTP-binding mutant equally defective in both basal and assembly-stimulated GTPase activities. Both mutants potently inhibit CME and effectively recruit other endocytic accessory proteins to stalled coated pits. However, the Q40E mutant blocks at a later step than S45N, providing additional evidence that GTP binding and/or basal GTPase activities of dynamin are required throughout clathrin coated pit maturation. Importantly, using in vitro assays for assembly-stimulated GTPase activity and membrane fission, we find that the latter is much more potently inhibited by both dominant-negative mutants than the former. These studies establish that efficient fission from supported bilayers with excess membrane reservoir (SUPER) templates requires coordinated GTP hydrolysis across two rungs of an assembled dynamin collar.     

Importance of the pleckstrin homology domain of dynamin in clathrin-mediated endocytosis

The GTPase dynamin plays an essential role in clathrin-mediated endocytosis [1] [2] [3]. Substantial evidence suggests that dynamin oligomerisation around the necks of endocytosing vesicles and subsequent dynamin-catalysed GTP hydrolysis is responsible for membrane fission [4] [5]. The pleckstrin homology (PH) domain of dynamin has previously been shown to interact with phosphoinositides, but it has not been determined whether this interaction is essential for dynamin's function in endocytosis [6] [7] [8] [9]. In this study, we address the in vivo function of the PH domain of dynamin by assaying the effects of deletions and point mutations in this region on transferrin uptake in COS-7 fibroblasts. Overexpression of a dynamin construct lacking its entire PH domain potently blocked transferrin uptake, as did overexpression of a dynamin construct containing a mutation in the first variable loop of the PH domain. Structural modelling of this latter mutant suggested that the lysine residue at position 535 (Lys535) may be critical in the coordination of phosphoinositides, and indeed, the purified mutant no longer interacted with lipid nanotubes. Interestingly, the inhibitory phenotype of cells expressing this dynamin mutant was partially relieved by a second mutation in the carboxy-terminal proline-rich domain (PRD), one that prevents dynamin from binding to the Src homology 3 (SH3) domain of amphiphysin. These data demonstrate that dynamin's interaction with phosphoinositides through its PH domain is essential for endocytosis. These findings also support our hypothesis that PRD-SH3 domain interactions are important in the recruitment of dynamin to sites of endocytosis.     

Dynamin 2 Mutants Linked to Centronuclear Myopathies Form Abnormally Stable Polymers

Mutations in the dynamin 2 gene have been identified in patients with autosomal dominant forms of centronuclear myopathy (CNM). Dynamin 2 is a ubiquitously expressed ∼100-kDa GTPase that assembles around the necks of vesiculating membranes and promotes their constriction and scission. It has also been implicated in regulation of the actin and microtubule cytoskeletons. At present, the cellular functions of dynamin 2 that are affected by CNM-linked mutations are not well defined, and the effects of these mutations on the physical and enzymatic properties of dynamin have been not examined. Here, we report the expression, purification, and characterization of four CNM-associated dynamin mutants. All four mutants display higher than wild-type GTPase activities, and more importantly, the mutants form high order oligomers that are significantly more resistant than wild-type dynamin 2 to disassembly by guanine nucleotides or high ionic strength. These observations suggest that the corresponding wild-type residues serve to prevent excessive or prolonged dynamin assembly on cellular membranes or inappropriate self-assembly in the cytoplasm. To our knowledge, this report contains the first identification of point mutations that enhance the stability of dynamin polymers without impairing their ability to bind and/or hydrolyze GTP. We envision that the formation of abnormally large and stable complexes of these dynamin mutants in vivo contributes to their role in CNM pathogenesis.     

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.     

Dimeric endophilin A2 stimulates assembly and GTPase activity of dynamin 2

Endophilin, which participates in membrane vesiculation during receptor-mediated endocytosis, is a ∼40 kDa SH3 domain-containing protein that binds to the proline/arginine-rich domain of dynamin, a ∼100 kDa GTPase that is essential for endocytic membrane scission. It has been suggested that endophilin is monomeric in the cytoplasm and dimerizes only after it binds to membranes (or perhaps to dimers or tetramers of dynamin). To clarify this issue, we studied the oligomeric state of endophilin both in vitro using analytical ultracentrifugation and fluorescence anisotropy, and in living cells using two-photon fluorescence fluctuation spectroscopy. We analyzed the fluctuation data using the Q-analysis method, which allowed us to determine the intrinsic brightness of the labeled protein complexes and hence its aggregation state in the cytoplasmic regions of the cell. Although a relatively high K_d (∼5–15 μM) was observed in vitro, the cell measurements indicate that endophilin is dimeric in the cytoplasm, even at submicromolar concentrations. We also demonstrate that endophilin significantly enhances the assembly of dynamin, and that this enhancement is proportional to the fraction of dimeric endophilin that is present. Moreover, there is correlation between the concentrations of endophilin that promote dynamin self-assembly and those that stimulate dynamin GTPase activity. These findings support the view that endophilin-dynamin interactions play an important role in endocytosis.     

The proline/arginine-rich domain is a major determinant of dynamin self-activation

Dynamins induce membrane vesiculation during endocytosis and Golgi budding in a process that requires assembly-dependent GTPase activation. Brain-specific dynamin 1 has a weaker propensity to self-assemble and self-activate than ubiquitously expressed dynamin 2. Here we show that dynamin 3, which has important functions in neuronal synapses, shares the self-assembly and GTPase activation characteristics of dynamin 2. Analysis of dynamin hybrids and of dynamin 1-dynamin 2 and dynamin 1-dynamin 3 heteropolymers reveals that concentration-dependent GTPase activation is suppressed by the C-terminal proline/arginine-rich domain of dynamin 1. Dynamin proline/arginine-rich domains also mediate interactions with SH3 domain-containing proteins and thus regulate both self-association and heteroassociation of dynamins.     

Atlastin-1, the dynamin-like GTPase responsible for spastic paraplegia SPG3A, remodels lipid membranes and may form tubules and vesicles in the endoplasmic reticulum

We examined the effects of wild-type and mutant atlastin-1 on vesicle transport in the endoplasmic reticulum (ER)-Golgi interface and vesicle budding from ER-derived microsomes using the temperature-sensitive reporter vesicular stomatitis virus glycoprotein (VSV-G), and the ability of purified atlastin-1 to form tubules or vesicles from protein-free phosphatidylserine liposomes. A GTPase domain mutation (T162P) altered the cellular distribution of the ER, but none of the mutations studied significantly affected transport from the ER to the Golgi apparatus. The mutations also had no significant effect on the incorporation of VSV-G into vesicles formed from ER microsomes. Atlastin-1, however, was also incorporated into microsome-derived vesicles, suggesting that it might be implicated in vesicle formation. Purified atlastin-1 transformed phosphatidylserine liposomes into branched tubules and polygonal networks of tubules and vesicles, an action inhibited by GDP and the synthetic dynamin inhibitor dynasore. The GTPase mutations T162P and R217C decreased but did not totally prevent this action; the C-terminal transmembrane domain mutation R495W was as active as the wild-type enzyme. Similar effects were observed in human embryonic kidney cells over-expressing mutant atlastin-1. We concluded that atlastin-1, like dynamin, might be implicated in membrane tubulation and vesiculation and participated in the formation as well as the function of the ER.     

Src-dependent tyrosine phosphorylation regulates dynamin self-assembly and ligand-induced endocytosis of the epidermal growth factor receptor

Endocytosis of ligand-activated receptors requires dynamin-mediated GTP hydrolysis, which is regulated by dynamin self-assembly. Here, we demonstrate that phosphorylation of dynamin I by c-Src induces its self-assembly and increases its GTPase activity. Electron microscopic analyses reveal that tyrosine-phosphorylated dynamin I spontaneously self-assembles into large stacks of rings. Tyrosine 597 was identified as being phosphorylated both in vitro and in cultured cells following epidermal growth factor receptor stimulation. The replacement of tyrosine 597 with phenylalanine impairs Src kinase-induced dynamin I self-assembly and GTPase activity in vitro. Expression of Y597F dynamin I in cells attenuates agonist-driven epidermal growth factor receptor internalization. Thus, c-Src-mediated tyrosine phosphorylation is required for the function of dynamin in ligand-induced signaling receptor internalization.     

A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736–1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.