Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages

Abundant evidence has shown that the GTPase dynamin is required for receptor-mediated endocytosis, but its exact role in endocytic clathrin-coated vesicle formation remains to be established. Whereas dynamin GTPase domain mutants that are defective in GTP binding and hydrolysis are potent dominant-negative inhibitors of receptor-mediated endocytosis, overexpression of dynamin GTPase effector domain (GED) mutants that are selectively defective in assembly-stimulated GTPase-activating protein activity can stimulate the formation of constricted coated pits and receptor-mediated endocytosis. These apparently conflicting results suggest that a complex relationship exists between dynamin's GTPase cycle of binding and hydrolysis and its role in endocytic coated vesicle formation. We sought to explore this complex relationship by generating dynamin GTPase mutants predicted to be defective at distinct stages of its GTPase cycle and examining the structural intermediates that accumulate in cells overexpressing these mutants. We report that the effects of nucleotide-binding domain mutants on dynamin's GTPase cycle in vitro are not as predicted by comparison to other GTPase superfamily members. Specifically, GTP and GDP association was destabilized for each of the GTPase domain mutants we analyzed. Nonetheless, we find that overexpression of dynamin mutants with subtle differences in their GTPase properties can lead to the accumulation of distinct intermediates in endocytic coated vesicle formation.     

An InCytes from MBC Selection: Membrane Insertion of the Pleckstrin Homology Domain Variable Loop 1 Is Critical for Dynamin-catalyzed Vesicle Scission

The GTPase dynamin catalyzes the scission of deeply invaginated clathrin-coated pits at the plasma membrane, but the mechanisms governing dynamin-mediated membrane fission remain poorly understood. Through mutagenesis, we have altered the hydrophobic nature of the membrane-inserting variable loop 1 (VL1) of the pleckstrin homology (PH) domain of dynamin-1 and demonstrate that its stable insertion into the lipid bilayer is critical for high membrane curvature generation and subsequent membrane fission. Dynamin PH domain mutants defective in curvature generation regain function when assayed on precurved membrane templates in vitro, but they remain defective in the scission of clathrin-coated pits in vivo. These results demonstrate that, in concert with dynamin self-assembly, PH domain membrane insertion is essential for fission and vesicle release in vitro and 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.     

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.     

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: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.     

The Src homology 3 domain of the beta-subunit of voltage-gated calcium channels promotes endocytosis via dynamin interaction

High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the alpha-interaction-domain in the pore-forming alpha1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the alpha-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the alpha1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing alpha1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the alpha-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltage-gated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the alpha1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.     

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.     

Roles of cortactin, an actin polymerization mediator, in cell endocytosis

Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS 1 oncogene. Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of cortactin expression as well as in vivo introduction of cortactin immunoreagents. Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a cortactin Src homology domain 3 （SH3）-dependent manner. In addition, cortactin was found to function in the late stage of clathrin coated vesicle formation. Taken together, cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.     

The major yolk protein of sea urchins is endocytosed by a dynamin-dependent mechanism

Sea urchin oocytes grow to 10 times their original size during oogenesis by both synthesizing and importing a specific repertoire of proteins to drive fertilization and early embryogenesis. During the vitellogenic growth period, the major yolk protein (MYP), a transferrin-like protein, is synthesized in the gut, transported into the ovary, and actively endocytosed by the oocytes. Here, we begin to dissect this mechanism by first testing the hypothesis that MYP endocytosis is dynamin-dependent. We have identified a sea urchin dynamin cDNA that is highly similar in amino acid sequence, structure, and size to mammalian dynamin I: it contains an N-terminal GTPase domain, a pleckstrin-homology domain, and a C-terminal proline-rich domain. Sea urchin dynamin is enriched at the cortex of oocytes and colocalizes to MYP endocytic vesicles at the oocyte periphery. To test for a functional relationship between MYP endocytosis and dynamin, we used a dominant-negative human dynamin I mutant protein containing an alteration within the GTPase domain (hDyn(K44A)) to specifically compete for dynamin function. Using a fluorescent MYP construct to follow its endocytosis solely, as well as a general endocytosis marker, we demonstrate that the disruption of dynamin function significantly reduces MYP uptake but does not affect fluid-phase endocytosis. Using this specific biochemical approach, we are able to separate distinct pathways of endocytosis during oogenesis and learn that dynamin-mediated endocytosis is responsible for MYP endocytosis but not fluid-phase uptake.     

Dynamin II regulates hormone secretion in neuroendocrine cells

The dynamin family of GTP-binding proteins has been implicated as playing an important role in endocytosis. In Drosophila shibire, mutations of the single dynamin gene cause blockade of endocytosis and neurotransmitter release, manifest as temperature-sensitive neuromuscular paralysis. Mammals express three dynamin genes: the neural specific dynamin I, ubiquitous dynamin II, and predominantly testicular dynamin III. Mutations of dynamin I result in a blockade of synaptic vesicle recycling and receptor-mediated endocytosis. Here, we show that dynamin II plays a key role in controlling constitutive and regulated hormone secretion from mouse pituitary corticotrope (AtT20) cells. Dynamin II is preferentially localized to the Golgi apparatus where it interacts with G-protein betagamma subunit and regulates secretory vesicle release. The presence of dynamin II at the Golgi apparatus and its interaction with the betagamma subunit are mediated by the pleckstrin homology domain of the GTPase. Overexpression of the pleckstrin homology domain, or a dynamin II mutant lacking the C-terminal SH3-binding domain, induces translocation of endogenous dynamin II from the Golgi apparatus to the plasma membrane and transformation of dynamin II from activity in the secretory pathway to receptor-mediated endocytosis. Thus, dynamin II regulates secretory vesicle formation from the Golgi apparatus and hormone release from mammalian neuroendocrine cells.     

Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.     

Essential Role of the Dynamin Pleckstrin Homology Domain in Receptor-Mediated Endocytosis

Pleckstrin homology (PH) domains are found in numerous membrane-associated proteins and have been implicated in the mediation of protein-protein and protein-phospholipid interactions. Dynamin, a GTPase required for clathrin-dependent endocytosis, contains a PH domain which binds to phosphoinositides and participates in the interaction between dynamin and the βγ subunits of heterotrimeric G proteins. The PH domain is essential for expression of phosphoinositide-stimulated GTPase activity of dynamin in vitro, but its involvement in the endocytic process is unknown. We expressed a series of dynamin PH domain mutants in cultured cells and determined their effect on transferrin uptake by those cells. Endocytosis is blocked in cells expressing a PH domain deletion mutant and a point mutant that fails to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. In contrast, expression of a point mutant with unimpaired PI(4,5)P₂ interaction has no effect on transferrin uptake. These results demonstrate the significance of the PH domain for dynamin function and suggest that its role may be to mediate interactions between dynamin and phosphoinositides.     

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.     

Direct dynamin-actin interactions regulate the actin cytoskeleton

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.     

Endocytosis: How dynamin sets vesicles PHree!

The dynamin GTPase is required for clathrin-dependent, receptor-mediated endocytosis. Exciting new studies have shown that dynamin's pleckstrin homology domain binds to phosphatidylinositol 4, 5-bisphosphate in vivo, thus localising dynamin directly at the plasma membrane and ultimately enabling vesiculation.     

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.     

Unique biochemical and behavioral alterations in Drosophila shibire(ts1) mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling

Dynamin is a GTPase protein that is essential for clathrin-mediated endocytosis of synaptic vesicle membranes. The Drosophila dynamin mutation shi(ts1) changes a single residue (G273D) at the boundary of the GTPase domain. In cell fractionation of homogenized fly heads without monovalent cations, all dynamin was in pellet fractions and was minimally susceptible to Triton-X extraction. Addition of Na(+) or K(+) can extract dynamin to the cytosolic (supernatant) fraction. The shi(ts1) mutation reduced the sensitivity of dynamin to salt extraction compared with other temperature-sensitive alleles or wild type. Sensitivity to salt extraction in shi(ts1) was enhanced by GTP and nonhydrolyzable GTP-gammaS. The shi(ts1) mutation may therefore induce a conformational change, involving the GTP binding site, that affects dynamin aggregation. Temperature-sensitive shibire mutations are known to arrest endocytosis at restrictive temperatures, with concomitant accumulation of presynaptic collared pits. Consistent with an effect upon dynamin aggregation, intact shi(ts1) flies recovered much more slowly from heat-induced paralysis than did other temperature-sensitive shibire mutants. Moreover, a genetic mutation that lowers GTP abundance (awd(msf15)), which reduces the paralytic temperature threshold of other temperature-sensitive shibire mutations that lie closer to consensus GTPase motifs, did not reduce the paralytic threshold of shi(ts1). Taken together, the results may link the GTPase domain to conformational shifts that influence aggregation in vitro and endocytosis in vivo, and provide an unexpected point of entry to link the biophysical properties of dynamin to physiological processes at synapses.     

Regulation of phospholipase D activity, membrane targeting and intracellular trafficking by phosphoinositides

Generation of PA (phosphatidic acid) by PLD (phospholipase D)-catalysed hydrolysis of phosphatidylcholine plays a pivotal role in cellular signalling pathways that regulate organization of the actin cytoskeleton, vesicular transport and exocytosis and stimulation of cell growth and survival. PLD regulation and function are intimately linked with phosphoinositide metabolism. Phosphatidyl 4-phosphate 5-kinase is stimulated by PA in vitro and this enzyme is the downstream effector of a significant subset of PLD signalling pathways. Yeast and mammalian PLDs are potently and specifically activated by the product of this kinase, PtdIns(4,5)P2, through interactions mediated by a polybasic motif within the catalytic core of the enzyme. Integrity of this motif is critical for agonist activation of mammalian PLD and for PLD function in secretion, sporulation and exocytosis in vivo. Although dispensable for catalysis in vitro, these PLD enzymes also contain N-terminal PH (pleckstrin) and PX (phox) homology domains. Binding studies using recombinantly expressed PLD fragments indicate that the PH and PX domains also interact specifically with distinct phosphoinositide ligands. Both the PX and PH domains are important for PLD function by controlling the dynamic association of the enzyme with the plasma membrane and its intracellular trafficking by the endocytic pathway. These results identify two distinct modes of regulation of PLD by phosphoinositides: stimulation of catalysis mediated by the polybasic domain and dynamic regulation of membrane targeting mediated primarily by the PH and PX domains.     

The role of dynamin and its binding partners in coated pit invagination and scission

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission.To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits.We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.