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PubMed1993

Dynamin, a GTPase involved in the initial stages of endocytosis.

Dynamin is a high molecular mass (100 kDa) GTPase which binds to and co-purifies with microtubules. Molecular cloning of rat brain dynamin has revealed the three well-established consensus sequence elements for GTP binding within the N-terminal third of the protein, as well as sequence similarity within this region to the interferon-inducible antiviral Mx proteins, the product of the yeast membrane sorting gene VPS1, and the product of the yeast mitochondrial replication gene MGM1. More extensive sequence similarity between rat dynamin and the product of the Drosophila gene shibire, which is involved in endocytosis, has also been found. In in vitro assays microtubules strongly stimulate the dynamin GTPase. This effect can be reversed by removal of the dynamin C-terminus using papain, which abolishes microtubule binding. Overexpression of mutant forms of dynamin in vivo using Cos-7 cells inhibits transferrin uptake and alters the distribution of clathrin and of alpha-adaptin, but not gamma-adaptin. Deletion of the C-terminus of mutant forms of dynamin abolishes these effects. Together these results suggest a critical role for dynamin in the early stages of endocytosis. It is uncertain whether microtubules interact with dynamin in vivo or whether the in vitro effects of microtubules mimic the effects of other regulatory elements in vivo.

Animals↗
PubMed1994

A complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin receptor substrate-1 after insulin treatment.

Insulin drives the formation of a complex between tyrosine-phosphorylated IRS-1 and SH2-containing proteins. The SH2-containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild-type insulin receptor (IR) (CHO-IR cells) in a cell-free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co-immunoprecipitated with dynamin from unstimulated CHO-IR cells. After insulin treatment of CHO-IR cells, anti-dynamin antibodies co-immunoprecipitated the IR beta-subunit and IRS-1, as tyrosine-phosphorylated proteins and PI 3-kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS-1 or the p85 subunit of PI 3-kinase in vitro. Together, these results suggest that in CHO-IR cells, insulin stimulates the binding of dynamin to tyrosine-phosphorylated IRS-1 via Grb2 and that IRS-1 also associates with PI 3-kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus-expressed IRS-1, GST-Grb2 fusion proteins and dynamin peptides containing proline-rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS-1-derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin-Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptor Proteins, Signal Transducing↗
PubMed2004

Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin.

Dynamin, a large GTPase, is located at the necks of clathrin-coated pits where it facilitates the release of coated vesicles from the plasma membrane upon GTP binding, and hydrolysis. Previously, we have shown by negative stain electron microscopy that wild-type dynamin and a dynamin mutant lacking the C-terminal proline-rich domain, DeltaPRD, form protein-lipid tubes that constrict and vesiculate upon addition of GTP. Here, we show by time-resolved cryo-electron microscopy (cryo-EM) that DeltaPRD dynamin in the presence of GTP rapidly constricts the underlying lipid bilayer, and then gradually disassembles from the lipid. In agreement with the negative stain results, the dynamin tubes constrict from 50 to 40 nm, and their helical pitch decreases from approximately 13 to 9.4 nm. However, in contrast to the previous results, examination by cryo-EM shows that the lipid bilayer remains intact and small vesicles or fragments do not form upon GTP binding and hydrolysis. Therefore, the vesicle formation seen by negative stain may be due to the lack of mobility of the dynamin tubes on the grid during the GTP-induced conformational changes. Our results confirm that dynamin is a mechanochemical enzyme and suggest that during endocytosis dynamin is directly responsible for membrane constriction. In the cell, other proteins may enhance the activity of dynamin or the constraints induced by the surrounding coated pit and plasma membrane during constriction may cause the final membrane fission event.

Animals↗
PubMed1999

Dynamin: possible mechanism of "Pinchase" action.

Dynamin is a GTPase playing an essential role in ubiquitous intra cellular processes involving separation of vesicles from plasma membranes and membranes of cellular compartments. Recent experimental progress (. Cell. 93:1021-1029;. Cell. 94:131-141) has made it possible to attempt to understand the action of dynamin in physical terms. Dynamin molecules are shown to bind to a lipid membrane, to self-assemble into a helicoidal structure constricting the membrane into a tubule, and, as a result of GTP hydrolysis, to mediate fission of this tubule (). In a similar way, dynamin is supposed to mediate fission of a neck connecting an endocytic bud and the plasma membrane, i.e., to complete endocytosis. We suggest a mechanism of this "pinchase" action of dynamin. We propose that, as a result of GTP hydrolysis, dynamin undergoes a conformational change manifested in growth of the pitch of the dynamin helix. We show that this gives rise to a dramatic change of shape of the tubular membrane constricted inside the helix, resulting in a local tightening of the tubule, which is supposed to promote its fission. We treat this model in terms of competing elasticities of the dynamin helix and the tubular membrane and discuss the predictions of the model in relation to the previous views on the mechanism of dynamin action.

Cell Membrane↗
PubMed1997

The role of the PH domain and SH3 binding domains in dynamin function.

Dynamin, a 100 kD GTPase, is necessary for the normal development and function of mammalian neural tissue. In neurons, it is necessary for the biogenesis of synaptic vesicles, and in other cell types dynamin has a general and important role in clathrin mediated receptor endocytosis. Different isoforms function as molecular scissors either during the formation of coated vesicles from plasma membrane coated pits, or during the release of intracellular vesicles from donor membranes. The mechanism entails the formation of a horseshoe-shaped dynamin polymer at the neck of the budding vesicle, followed by neck scission through a GTP hydrolysis dependent activity. The primary sequence of dynamin contains several C-terminal SH3 binding proline motifs, a central pleckstrin homology (PH) domain, and an N-terminal GTPase domain. Each of these domains appears to play a distinct role in dynamin function. Dynamin is activated by stimulus coupled PKC phosphorylation in brain, possibly mediated through PKC interactions with the PH domain. Further, SH3 domain interactions with the C-terminal sequences and phophatidylinositol/G beta gamma interactions with the PH domain also increase dynamin GTPase activity. Each of these various regulatory mechanisms is important in dynamin function during vesicle budding, although the means by which these mechanisms integrate in the overall function of dynamin remains to be elucidated.

Amino Acid Sequence↗
PubMed2006

A bacterial dynamin-like protein.

Dynamins form a superfamily of large mechano-chemical GTPases that includes the classical dynamins and dynamin-like proteins (DLPs). They are found throughout the Eukarya, functioning in core cellular processes such as endocytosis and organelle division. Many bacteria are predicted by sequence to possess large GTPases with the same multidomain architecture that is found in DLPs. Mechanistic dissection of dynamin family members has been impeded by a lack of high-resolution structural data currently restricted to the GTPase and pleckstrin homology domains, and the dynamin-related human guanylate-binding protein. Here we present the crystal structure of a cyanobacterial DLP in both nucleotide-free and GDP-associated conformation. The bacterial DLP shows dynamin-like qualities, such as helical self-assembly and tubulation of a lipid bilayer. In vivo, it localizes to the membrane in a manner reminiscent of FZL, a chloroplast-specific dynamin-related protein with which it shares sequence similarity. Our results provide structural and mechanistic insight that may be relevant across the dynamin superfamily. Concurrently, we show compelling similarity between a cyanobacterial and chloroplast DLP that, given the endosymbiotic ancestry of chloroplasts, questions the evolutionary origins of dynamins.

Animals↗
PubMed1996

A binding site for SH3 domains targets dynamin to coated pits.

Dynamin is a GTPase that plays a critical role in the very early stages of endocytosis, regulating the scission of clathrin-coated and non-clathrin-coated pits from the plasma membrane. While the ligands through which dynamin exerts its in vivo effects are unknown, dynamin exhibits in vitro binding to several proteins containing Src homology 3 (SH3) domains, as well as to microtubules and anionic phospholipids, via a basic, proline-rich C-terminal domain. To begin to identify the in vivo binding partners of dynamin, we have examined by immunofluorescence the association of mutant and wild-type forms of dynamin with plasma membranes prepared by sonication of transiently transfected cells. Wild-type dynamin was found almost exclusively in association with clathrin-containing domains. Binding to these regions was abolished by removal of a nine-amino acid sequence within the C-terminal domain encoding a candidate SH3 domain binding site. Binding did not require clathrin and resisted extraction at both high and low ionic strength, consistent with an interaction with an SH3 domain. Surprisingly, we also find that dynamin contains multiple regions involved in binding to nonclathrin-containing domains, including a 13-amino acid sequence directly upstream of the C-terminal domain. These observations suggest that a protein containing an SH3 domain is involved in recruiting dynamin to coated pits and provide the first evidence for a biological role for SH3 domains in dynamin function.

Amino Acid Sequence↗
PubMed1997

Role of the basic, proline-rich region of dynamin in Src homology 3 domain binding and endocytosis.

The GTPase dynamin has been implicated in the regulation of the scission of coated and noncoated pits during the early stages of endocytosis. Various macromolecules including microtubules, acidic phospholipids, and Src homology 3 (SH3) domains have been shown to interact with the basic, proline-rich region of dynamin and act as effectors of its GTPase activity. The interaction of dynamin with SH3 domain-containing proteins is of particular interest since SH3 domains are known to mediate protein-protein interactions in signal transducing complexes. In this study, we have systematically defined three distinct SH3 binding regions within the dynamin proline-rich C terminus. These binding regions conform to either the Class I or II SH3 binding consensus sequence, and their location coincides with a region previously shown to be important in the colocalization of dynamin with clathrin-coated pits. Two of these SH3 binding regions are well conserved among four dynamin isoforms, and we show that the overall binding pattern for SH3 domains is comparable among the isoforms. We also demonstrate that neither transferrin nor platelet-derived growth factor receptor uptake is restored upon removal of the basic, proline-rich region in a dominant negative dynamin GTP binding mutant. Together with earlier evidence from our laboratory, these findings suggest that SH3 domains may serve to target dynamin to coated pits and are not the direct targets of dominant inhibitory mutants of dynamin.

Amino Acid Sequence↗
PubMed2000

Essential role of dynamin in internalization of M2 muscarinic acetylcholine and angiotensin AT1A receptors.

Most G protein-coupled receptors (GPCRs), including the M(1) muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M(2) mAChR and the angiotensin AT(1A) receptor (AT(1A)R) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M(2) mAChR and AT(1A)R in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4, 5-bisphosphate, strongly inhibits internalization of M(1) and M(2) mAChRs and AT(1A)Rs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M(1) mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M(1) mAChR internalization. In contrast, M(2) mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M(1) mAChR but also for M(2) mAChR and AT(1A)R internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.

Animals↗
PubMed1999

Disruption of a dynamin homologue affects endocytosis, organelle morphology, and cytokinesis in Dictyostelium discoideum.

The identification and functional characterization of Dictyostelium discoideum dynamin A, a protein composed of 853 amino acids that shares up to 44% sequence identity with other dynamin-related proteins, is described. Dynamin A is present during all stages of D. discoideum development and is found predominantly in the cytosolic fraction and in association with endosomal and postlysosomal vacuoles. Overexpression of the protein has no adverse effect on the cells, whereas depletion of dynamin A by gene-targeting techniques leads to multiple and complex phenotypic changes. Cells lacking a functional copy of dymA show alterations of mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake. They also become multinucleated due to a failure to complete normal cytokinesis. These pleiotropic effects of dynamin A depletion can be rescued by complementation with the cloned gene. Morphological studies using cells producing green fluorescent protein-dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles. Double labeling with vacuolin, a marker of a postlysosomal compartment in D. discoideum, showed an almost complete colocalization of vacuolin and dynamin A. Our results suggest that that dynamin A is likely to function in membrane trafficking processes along the endo-lysosomal pathway of D. discoideum but not at the plasma membrane.

Amino Acid Sequence↗
PubMed1998

Contribution of the GTPase domain to the subcellular localization of dynamin in the nematode Caenorhabditis elegans.

Caenorhabditis elegans dynamin is expressed at high levels in neurons and at lower levels in other cell types, consistent with the important role that dynamin plays in the recycling of synaptic vesicles. Indirect immunofluorescence showed that dynamin is concentrated along the dorsal and ventral nerve cords and in the synapse-rich nerve ring. Green fluorescent protein (GFP) fused to the N terminus of dynamin is localized to synapse-rich regions. Furthermore, this chimera was detected along the apical membrane of intestinal cells, in spermathecae, and in coelomocytes. Dynamin localization was not affected by disrupting axonal transport of synaptic vesicles in the unc-104 (kinesin) mutant. To investigate the alternative mechanisms that dynamin might use for translocation to the synapse, we systematically tested the localization of different protein domains by fusion to GFP. Localization of each chimera was measured in one specific neuron, the ALM. The GTPase, a middle domain, and the putative coiled coil each contribute to synaptic localization. Surprisingly, the pleckstrin homology domain and the proline-rich domain, which are known to bind to coated-pit constituents, did not contribute to synaptic localization. The GFP-GTPase chimera was most strongly localized, although the GTPase domain has no known interactions with proteins other than with dynamin itself. Our results suggest that different dynamin domains contribute to axonal transport and the sequestration of a pool of dynamin molecules in synaptic cytosol.

Animals↗
PubMed1997

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.

Dynamins↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed1999

Participation of dynamin in the biogenesis of cytoplasmic vesicles.

Dynamin is a 100-kDa GTPase that has been implicated in endocytosis. To extend our understanding of its cellular functions, we have microinjected specific affinity-purified anti-dynamin antibodies into cultured mammalian epithelial cells. Using this approach, dynamin function can be inhibited specifically and rapidly in single cells. Effects of microinjected inhibitory antibodies on distinct endocytic processes and plasmalemmal morphology were then assayed by fluorescence microscopy (FM) and ultrastructural analysis. Micro-injected antibodies inhibit the clathrin-mediated endocytosis of fluorophore-labeled transferrin and cause a marked invagination of the plasma membrane. Many of these long plasmalemmal invaginations had clathrin-coated pits along their cytoplasmic surface. A number of distinct noncoated pits resembling plasmalemmal caveolae also accumulated in anti-dynamin antibody-injected cells. Further, the cellular uptake of cholera toxin B, which normally occurs by the internalization of caveolae, was inhibited in these cells. In support of these observations, immunoisolation techniques, double-label immuno-FM, and immunoelectron microscopy (immuno-EM) provided biochemical and morphological evidence that dynamin associates with plasmalemmal caveolae. Together, these observations indicate that dynamin mediates scission from the plasma membrane of both clathrin-coated pits and caveolae during distinct endocytic processes. These results demonstrate that dynamin isoforms are involved in an additional endocytic process that is distinct from clathrin-mediated endocytosis and provide significant insights into the molecular mechanisms governing the GTP-mediated internalization of caveolae. Evidence is provided demonstrating that dynamin isoforms have a differential distribution in mammalian cells. Targeting information for these isoforms is provided at least in part by regions of alternative splicing. Thus, the different dynamin isoforms may be localized to distinct cellular compartments but provide a similar scission function during the biogenesis of nascent cytoplasmic vesicles.

Animals↗
PubMed2006

Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network.

The role of dynamin and so-called accessory proteins in endocytosis is well established. However, molecular details of the function(s) of dynamin II at the Golgi are largely unclear. We demonstrate that the ubiquitously expressed syndapin II isoform interacts with the proline-rich domain (PRD) of dynamin II through its Src-homology 3 (SH3) domain. Co-immunoprecipitation of endogenous syndapin II and dynamin II, and successful reconstitutions of such complexes at membranes in COS-7 cells, show the in vivo relevance of the interaction. Syndapin II can associate with Golgi membranes and this association increases upon Golgi exit block. Brefeldin A treatment clearly shows that the observed perinuclear localization of syndapin II co-localizing with syntaxin 6 reflects the Golgi complex and that it requires functional integrity of the Golgi. Syndapins are crucial for Golgi vesicle formation because anti-syndapin antibodies, used either in in vitro reconstitutions or in living cells, inhibited this process. Both types of assays additionally revealed the essential role of syndapin II SH3 interactions with the dynamin II PRD in vesicle formation. An excess of the syndapin SH3 domain strongly inhibited budding from Golgi membranes in vitro. Likewise, overexpression of the syndapin SH3 domain or of a dynamin II variant incapable of associating with syndapin II (dynamin IIDeltaPRD) impaired trafficking of vesicular stomatitis virus glycoprotein (VSVG)-GFP in vivo. By contrast, full-length syndapin II-l had no negative effect, and instead promoted VSVG-GFP export from the Golgi. Importantly, a cytosolic fraction containing endogenous syndapin-dynamin complexes was sufficient to promote vesicle formation from Golgi membranes in a syndapin-dependent manner. Thus, syndapin-dynamin complexes are crucial and sufficient to promote vesicle formation from the trans-Golgi network.

Animals↗
PubMed1994

Association of Ash/Grb-2 with dynamin through the Src homology 3 domain.

Ash/Grb-2 is an adaptor protein composed only of Src homology (SH) 2 and SH3 domains that is considered to be essential for Ras activation. To clarify the downstream of Ash signaling, we investigated Ash-bound proteins. Ash-glutathione S-transferase (GST) fusion proteins were used to affinity-purify proteins bound to Ash. We found 180-, 150-, 100-, and 70-kDa proteins bound to GST-Ash, among which the 100 kDa protein was found to be dynamin by amino acid sequencing and Western blot with anti-dynamin antibody. Next, the in vitro and in vivo associations between Ash and dynamin were examined using PC12 cells. Dynamin in PC12 cell lysates bound to GST-Ash independent of NGF treatment. Also, Ash and dynamin co-precipitated when cell lysates of PC12 were immunoprecipitated with anti-Ash antibody or anti-dynamin antibody. Using various GST-Ash constructs, we studied the importance of the individual domains in binding and found that the SH3 domain is necessary for binding. This binding was inhibited by a synthetic peptide (GPPQVPSRPNRC, amino acids 827-838 in dynamin). These data show that Ash SH3 domains bind to the proline-rich region of dynamin. Considering the function of dynamin in membrane trafficking, Ash may regulate endocytosis in addition to Ras activation.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids.

Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 +/- 51 nM. Optimal binding occurred with mixtures of phosphatidylserine and phosphatidylcholine of 1:3 with little binding to phosphatidylcholine or phosphatidylserine alone. Phospholipid binding was abolished after dynamin I phosphorylation by PKC and was restored after dephosphorylation by calcineurin. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry revealed the phosphorylation site in PKCalpha-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus, phosphorylation of dynamin I on Ser-795 prevents its association with phospholipid, providing a basis for the cytosolic localization of the minor pool of phospho-dynamin I that mediates synaptic vesicle retrieval in nerve terminals.

Adenine Nucleotides↗
PubMed2002

Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles.

Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the trans-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that is served by endosome-derived clathrin-coated vesicles, we used cells that overexpressed a temperature-sensitive mutant of dynamin-1 (dynamin-1(G273D)) or, as a control, dynamin-1 wild type. In dynamin-1(G273D)-expressing cells, 29-36% of endocytosed transferrin failed to recycle at the nonpermissive temperature and remained associated with tubular recycling endosomes. Sorting of endocytosed transferrin from fluid-phase endocytosed markers in early endosome antigen 1-labeled sorting endosomes was not inhibited. Dynamin-1(G273D) associated with accumulated clathrin-coated buds on extended tubular recycling endosomes. Brefeldin A interfered with the assembly of clathrin coats on endosomes and reduced the extent of transferrin recycling in control cells but did not further affect recycling by dynamin-1(G273D)-expressing cells. Together, these data indicate that the pathway from recycling endosomes to the plasma membrane is mediated, at least in part, by endosome-derived clathrin-coated vesicles in a dynamin-dependent manner.

Blotting, Western↗