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PubMed2002

The intermolecular interaction between the PH domain and the C-terminal domain of Arabidopsis dynamin-like 6 determines lipid binding specificity.

Dynamin and its related proteins are a group of mechanochemical proteins involved in the modulation of lipid membranes in various biological processes. Here we investigate the nature of membrane binding of the Arabidopsis dynamin-like 6 (ADL6) involved in vesicle trafficking from the trans-Golgi network to the central vacuole. Fractionation experiments by continuous sucrose gradients and gel filtration revealed that the majority of ADL6 is associated with membranes in vivo. Amino acid sequence analysis revealed that ADL6 has a putative pleckstrin homology (PH) domain. In vitro lipid binding assays demonstrated that ADL6 showed high affinity binding to phosphatidylinositol 3-phosphate (PtdIns-3-P) and that the PH domain was responsible for this interaction. However, the PH domain alone binds equally well to both PtdIns-3-P and phosphatidylinositol 4-phosphate (PtdIns-4-P). Interestingly, the high affinity binding of the PH domain to PtdIns-3-P was restored by a protein-protein interaction between the PH domain and the C-terminal region. In addition, deletion of the inserted regions within the PH domain results in high affinity binding of the PH domain to PtdIns-3-P. These results suggest that ADL6 binds specifically to PtdIns-3-P and that the lipid binding specificity is determined by the interaction between the PH domain and the C-terminal domain of ADL6.

Amino Acid Sequence↗

PubMed2002

Endocytosed transferrin receptors recycle via distinct dynamin and phosphatidylinositol 3-kinase-dependent pathways.

Recycling of endocytosed membrane proteins involves passage through early endosomes and recycling endosomes. Previously, we demonstrated a role for clathrin-coated vesicles in transferrin receptor recycling. These clathrin-coated vesicles are formed from recycling endosomes in a process that was inhibited in dynamin-1(G273D)-overexpressing cells. Here we show a second transferrin recycling pathway, which requires phosphatidylinositol 3-kinase activity. Two unrelated phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, retained endocytosed transferrin in early endosomes but did not affect transfer through recycling endosomes. The inhibitory effects of LY294002 and dynamin-1(G273D) on transferrin recycling were additive. In combination with brefeldin A, a drug that prevents the formation of clathrin-coated buds at recycling endosomes, LY294002 inhibited transferrin recycling synergistically. Collectively, these data indicate two distinct recycling pathways. One pathway involves transfer from early endosomes to recycling endosomes, from where clathrin/dynamin-coated vesicles provide for further transport, whereas the other route bypasses recycling endosomes and requires phosphatidylinositol 3-kinase activity.

Chromones↗

PubMed2002

Phosphorylation of key serine residues is required for internalization of the complement 5a (C5a) anaphylatoxin receptor via a beta-arrestin, dynamin, and clathrin-dependent pathway.

The human complement 5a (C5a) anaphylatoxin receptor (CD88) is a G protein-coupled receptor involved in innate host defense and inflammation. Upon agonist binding, C5a receptor (C5aR) undergoes rapid phosphorylation on the six serine residues present in the C-terminal region followed by desensitization and internalization. Using confocal immunofluorescence microscopy and green fluorescent protein-tagged beta-arrestins (beta-arr 1- and beta-arr 2-EGFP) we show a persistent complex between C5aR and beta-arrestins to endosomal compartments. Serine residues in the C5aR C terminus were identified that control the intracellular trafficking of the C5aR-arrestin complex in response to C5a. Two phosphorylation mutants C5aR-A(314,317,327,332) and C5aR-A(314,317,332,334), which are phosphorylated only on Ser(334)/Ser(338) and Ser(327)/Ser(338), respectively, recruited beta-arr 1 and were internalized. In contrast, the phosphorylation-deficient receptors C5aR-A(334,338) and C5aR-A(332,334,338) were not internalized even though observations by confocal microscopy indicated that beta-arr 1-EGFP and/or beta-arr 2-EGFP could be recruited to the plasma membrane. Altogether the results indicate that C5aR activation is able to promote a loose association with beta-arrestins, but phosphorylation of either Ser(334)/Ser(338) or Ser(327)/Ser(338) is necessary and sufficient for the formation of a persistent complex. In addition, it was observed that C5aR endocytosis was inhibited by the expression of the dominant negative mutants of dynamin (K44E) and beta-arrestin 1 (beta-arr 1-(319-418)-EGFP). Thus, the results suggest that the C5aR is internalized via a pathway dependent on beta-arrestin, clathrin, and dynamin.

Amino Acid Sequence↗

PubMed1995

Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin.

A stable HeLa cell line expressing a dynamin mutant, dynts, exhibits a temperature-sensitive defect in endocytic clathrin-coated vesicle formation. Dynts carries a point mutation, G273D, corresponding to the Drosophila shibirets1 allele. The ts-defect in receptor-mediated endocytosis shows a rapid onset (< 5 min) and is readily reversible. At the nonpermissive temperature (38 degrees C) HRP uptake is only partially inhibited. Moreover, when cells are held at the nonpermissive temperature, fluid phase uptake fully recovers to wild-type levels within 30 min, while receptor-mediated endocytosis remains inhibited. The residual HRP uptake early after shift to the nonpermissive temperature and the induced HRP uptake that occurs after recovery are insensitive to cytosol acidification under conditions that potently inhibit receptor-mediated endocytosis of Tfn. Together, these results suggest that a dynamin- and clathrin-independent mechanism contributes to the total constitutive pinocytosis in HeLa cells and that dynts cells rapidly and completely compensate for the loss of clathrin-dependent endocytosis by inducing an alternate endocytic pathway.

Clathrin↗

PubMed2003

The intramitochondrial dynamin-related GTPase, Mgm1p, is a component of a protein complex that mediates mitochondrial fusion.

A balance between fission and fusion events determines the morphology of mitochondria. In yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related GTPase, Dnm1p. Mitochondrial fusion requires two integral outer membrane components, Fzo1p and Ugo1p. Interestingly, mutations in a second mitochondrial-associated dynamin-related GTPase, Mgm1p, produce similar phenotypes to fzo1 and ugo cells. Specifically, mutations in MGM1 cause mitochondrial fragmentation and a loss of mitochondrial DNA that are suppressed by abolishing DNM1-dependent fission. In contrast to fzo1ts mutants, blocking DNM1-dependent fission restores mitochondrial fusion in mgm1ts cells during mating. Here we show that blocking DNM1-dependent fission in Deltamgm1 cells fails to restore mitochondrial fusion during mating. To examine the role of Mgm1p in mitochondrial fusion, we looked for molecular interactions with known fusion components. Immunoprecipitation experiments revealed that Mgm1p is associated with both Ugo1p and Fzo1p in mitochondria, and that Ugo1p and Fzo1p also are associated with each other. In addition, genetic analysis of specific mgm1 alleles indicates that Mgm1p's GTPase and GTPase effector domains are required for its ability to promote mitochondrial fusion and that Mgm1p self-interacts, suggesting that it functions in fusion as a self-assembling GTPase. Mgm1p's localization within mitochondria has been controversial. Using protease protection and immuno-EM, we have shown previously that Mgm1p localizes to the intermembrane space, associated with the inner membrane. To further test our conclusions, we have used a novel method using the tobacco etch virus protease and confirm that Mgm1p is present in the intermembrane space compartment in vivo. Taken together, these data suggest a model where Mgm1p functions in fusion to remodel the inner membrane and to connect the inner membrane to the outer membrane via its interactions with Ugo1p and Fzo1p, thereby helping to coordinate the behavior of the four mitochondrial membranes during fusion.

Dynamins↗

PubMed2005

Dynein light chain 1 regulates dynamin-mediated F-actin assembly during sperm individualization in Drosophila.

Toward the end of spermiogenesis, spermatid nuclei are compacted and the clonally related spermatids individualize to become mature and active sperm. Studies in Drosophila showed that caudal end-directed movement of a microfilament-rich structure, called investment cone, expels the cytoplasmic contents of individual spermatids. F-actin dynamics plays an important role in this process. Here we report that the dynein light chain 1 (DLC1) of Drosophila is involved in two separate cellular processes during sperm individualization. It is enriched around spermatid nuclei during postelongation stages and plays an important role in the dynein-dynactin-dependent rostral retention of the nuclei during this period. In addition, DDLC1 colocalizes with dynamin along investment cones and regulates F-actin assembly at this organelle by retaining dynamin along the cones. Interestingly, we found that this process does not require the other subunits of cytoplasmic dynein-dynactin complex. Altogether, these observations suggest that DLC1 could independently regulate multiple cellular functions and established a novel role of this protein in F-actin assembly in Drosophila.

Actin Cytoskeleton↗

PubMed1998

A dynamin-like protein in Arabidopsis thaliana is involved in biogenesis of thylakoid membranes.

Dynamin, a GTP-binding protein found in rat brain, plays a role in endocytosis. Suborganellar fractionation studies of Arabidopsis leaf tissue revealed that a dynamin-like protein, ADL1, is localized in the thylakoid membranes of chloroplasts. This notion was supported further by in vivo targeting experiments using an ADL1-green fluorescent fusion protein and immunogold labeling with the anti-ADL1 antibody. Transgenic plants harboring various deletion mutant genes of ADL1 had a yellow leaf phenotype where the cells had very few chloroplasts. In addition, the remaining chloroplasts appeared morphologically not fully developed. The detailed structure of the chloroplasts revealed by electron microscopy showed a greatly reduced amount of thylakoid membranes. Also, the level of thylakoid membrane proteins such as the light-harvesting complex II and CP29 was greatly reduced in these transgenic plants. When we examined the expression of the ADL1 deletion mutant genes, these genes were highly expressed at the transcriptional level. However, the mutant ADL1s were not detectable at the protein level by Western blot analysis. Moreover, the endogenous ADL1 protein level was greatly reduced in these transgenic plants, probably due to a post-transcriptional silencing effect of the transgenes. We propose, therefore, that ADLl is involved in the biogenesis of thylakoid membranes.

Arabidopsis↗

PubMed1999

The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15.

Clathrin-mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins. We have cloned two mammalian genes which code for a number of endocytic adaptor proteins. Two of these proteins, termed Ese1 and Ese2, contain two N-terminal EH domains, a central coiled-coil domain and five C-terminal SH3 domains. Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein-protein interaction surfaces. Yeast two-hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively. Overexpression of Ese1 is sufficient to block clathrin-mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1-Eps15 scaffold. The Ese1-Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast.

Yeast produce two classes of secretory vesicles (SVs) that differ in both density and cargo protein content. In late-acting secretory mutants (e.g. snc1(ala43) and sec6-4), both low- (LDSV) and high-density (HDSV) classes of vesicles accumulate at restrictive temperatures. Here, we have found that disruptions in the genes encoding a dynamin-related protein (VPS1) or clathrin heavy chain (CHC1) abolish HDSV production, yielding LDSVs that contain all secreted cargos. Interestingly, disruption of the PEP12 gene, which encodes the t-SNARE that mediates all Golgi to pre-vacuolar compartment (PVC) transport, also abolishes HDSV production. In contrast, deletions in genes that selectively confer vacuolar hydrolase sorting to the PVC or protein transport to the vacuole (i.e. VPS34 and VAM3, respectively) have no effect. Thus, one branch of the secretory pathway in yeast involves an intermediate sorting compartment and has a specific requirement for clathrin and a dynamin-related protein in SV biogenesis.

Carrier Proteins↗

PubMed2006

Purification and characterization of plant dynamin from tobacco BY-2 cells.

We purified an 84 kDa polypeptide from the MAP (microtubule-associated protein) fraction of tobacco BY-2 cultured cells. LC/MS/MS (liquid chromatography-tandem mass spectrometry) analysis revealed that this polypeptide is a tobacco homolog of AtDRP3 (Arabidopsis thaliana dynamin-related protein 3). Electron microscopy revealed that NtDRP3 (Nicotiana tabacum dynamin-related protein 3) assembles to form a filamentous structure. When GDP was added to the NtDRP3 fraction, the filaments disappeared and many particles appeared. Biochemical analysis revealed that NtDRP3 could bind to and bundle both microtubules and actin filaments in vitro.

Arabidopsis Proteins↗

PubMed2005

Golgi vesiculation induced by cholesterol occurs by a dynamin- and cPLA2-dependent mechanism.

It was recently demonstrated that an increase in the cellular cholesterol level leads to vesiculation of the Golgi apparatus. This vesiculation affects the entire Golgi apparatus and is a reversible process. We have now started to elucidate the mechanism behind this cholesterol-induced vesiculation of the Golgi apparatus. Transient transfection of cells with dominant negative mutant constructs of dynamin 1 and 2 inhibited the vesiculation; expression of dynK44A in HeLa cells stably transfected with this construct had the same effect. However, the vesiculation seems to be independent of clathrin, as cholesterol-induced vesiculation still occurred following knock down of clathrin heavy chain in HeLa cells using RNA interference as well as in BHK cells where expression of antisense to clathrin heavy chain had been induced. Importantly, the cPLA2 inhibitor MAFP and the chelator BAPTA-AM that binds cytosolic Ca2+ inhibited the cholesterol-induced vesiculation, suggesting involvement of a cPLA2 that requires cytosolic Ca2+ for translocation to membranes. Furthermore, in response to an increased cellular cholesterol level, an EGFP-cPLA2 fusion protein translocated to the Golgi apparatus. Thus, our results demonstrate that the cholesterol-induced vesiculation of the Golgi apparatus is mediated by a cPLA2- and dynamin-dependent mechanism.

Animals↗

PubMed2006

The high-affinity immunoglobulin-E receptor (FcepsilonRI) is endocytosed by an AP-2/clathrin-independent, dynamin-dependent mechanism.

Aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcepsilonRI), expressed on mast cells and basophils, initiates the immediate hypersensitivity reaction. Aggregated FcepsilonRI has been reported to rapidly migrate to lipid rafts in RBL-2H3 cells. We confirmed that aggregated FcepsilonRI is found in the lipid raft fractions of cellular lysates. Furthermore, we show that the cross-linked FcepsilonRI remains associated with detergent-resistant structures upon internalization. Previous morphological studies have reported that aggregated FepsiloncRI is endocytosed via clathrin-coated pits, which in general are not lipid raft associated. To address this apparent discrepancy, we employed siRNA to suppress expression of components of the clathrin-mediated internalization machinery, namely, clathrin heavy chain, and the AP-2 (alpha-adaptin or mu2-subunit). Transferrin receptor (TfR) is endocytosed by a clathrin-mediated process and, as expected, each transfected siRNA caused a two to threefold elevation of TfR surface expression and almost completely inhibited its endocytosis. In contrast, there was no effect on surface expression levels of FcepsilonRI nor on the endocytosis of the dinitrophenyl-human serum albumin (DNP-HSA)/IgE/FcepsilonRI complex. On the contrary, internalization of DNP-HSA/IgE/FcepsilonRI was inhibited by overexpression of a dominant-negative dynamin mutant. We conclude that internalization of cross-linked FcRI does not require the AP-2/clathrin complex but is dynamin-dependent and may be lipid raft mediated.

Animals↗

PubMed2005

Dynamin-related proteins and Pex11 proteins in peroxisome division and proliferation.

The abundance and size of cellular organelles vary depending on the cell type and metabolic needs. Peroxisomes constitute a class of cellular organelles renowned for their ability to adapt to cellular and environmental conditions. Together with transcriptional regulators, two groups of peroxisomal proteins have a pronounced influence on peroxisome size and abundance. Pex11-type peroxisome proliferators are involved in the proliferation of peroxisomes, defined here as an increase in size and/or number of peroxisomes. Dynamin-related proteins have recently been suggested to be required for the scission of peroxisomal membranes. This review surveys the function of Pex11-type peroxisome proliferators and dynamin-related proteins in peroxisomal proliferation and division.

Animals↗

PubMed2006

Structural characterization of the large soluble oligomers of the GTPase effector domain of dynamin.

Dynamin, a protein playing crucial roles in endocytosis, oligomerizes to form spirals around the necks of incipient vesicles and helps their scission from membranes. This oligomerization is known to be mediated by the GTPase effector domain (GED). Here we have characterized the structural features of recombinant GED using a variety of biophysical methods. Gel filtration and dynamic light scattering experiments indicate that in solution, the GED has an intrinsic tendency to oligomerize. It forms large soluble oligomers (molecular mass > 600 kDa). Interestingly, they exist in equilibrium with the monomer, the equilibrium being largely in favour of the oligomers. This equilibrium, observed for the first time for GED, may have regulatory implications for dynamin function. From the circular dichroism measurements the multimers are seen to have a high helical content. From multidimensional NMR analysis we have determined that about 30 residues in the monomeric units constituting the oligomers are flexible, and these include a 17 residue stretch near the N-terminal. This contains two short segments with helical propensities in an otherwise dynamic structure. Negatively charged SDS micelles cause dissociation of the oligomers into monomers, and interestingly, the helical characteristics of the oligomer are completely retained in the individual monomers. The segments along the chain that are likely to form helices have been predicted from five different algorithms, all of which identify two long stretches. Surface electrostatic potential calculation for these helices reveals that there is a distribution of neutral, positive and negative potentials, suggesting that both electrostatic and hydrophobic interactions could be playing important roles in the oligomer core formation. A single point mutation, I697A, in one of the helices inhibited oligomerization quite substantially, indicating firstly, a special role of this residue, and secondly, a decisive, though localized, contribution of hydrophobic interaction in the association process.

Amino Acid Sequence↗

PubMed2000

Overexpression of dynamin is induced by chronic stimulation of mu- but not delta-opioid receptors: relationships with mu-related morphine dependence.

Several studies using selective opioid agonists or mice with a deletion of the mu-opioid receptor, have shown that morphine dependence is essentially due to chronic stimulation of mu- but not delta-opioid receptors. Because dependence is assumed to be related to persistent intracellular modifications, we have investigated modifications putatively induced by chronic activation of mu receptors with morphine or selective agonists in vitro in SH-SY5Y cells and in vivo in different strains of mice, including mice lacking the mu-opioid receptor gene. The results show a similar down-regulation and desensitization of mu and delta binding sites, whereas an overexpression of dynamin occurred only with mu agonists, strongly suggesting the relevance of this up-regulation with the opiate dependence. Moreover, translocation of overexpressed dynamin from intracellular pools to plasma membranes was observed in chronic morphine-treated rats. This recruitment could be critically involved in long-lasting changes such as alterations of axonal transport observed in opioid dependence.

Animals↗

PubMed2005

Vesicle endocytosis requires dynamin-dependent GTP hydrolysis at a fast CNS synapse.

Molecular dependence of vesicular endocytosis was investigated with capacitance measurements at the calyx of Held terminal in brainstem slices. Intraterminal loading of botulinum toxin E revealed that the rapid capacitance transient implicated as "kiss-and-run" was unrelated to transmitter release. The release-related capacitance change decayed with an endocytotic time constant of 10 to 25 seconds, depending on the magnitude of exocytosis. Presynaptic loading of the nonhydrolyzable guanosine 5'-triphosphate (GTP) analog GTPgS or dynamin-1 proline-rich domain peptide abolished endocytosis. These compounds had no immediate effect on exocytosis, but caused a use-dependent rundown of exocytosis. Thus, the guanosine triphosphatase dynamin-1 is indispensable for vesicle endocytosis at this fast central nervous system (CNS) synapse.

Animals↗

PubMed1998

Adenovirus internalization and infection require dynamin.

The cell receptors that facilitate adenovirus internalization into cells have been identified; however, the infectious pathway of virus entry has not been established. Adenovirus entry and infection were examined in HeLa cells lacking or overexpressing mutant dynamin, a protein that specifically regulates clathrin-mediated endocytosis. Expression of mutant dynamin significantly reduced adenovirus internalization and gene delivery, indicating a functional requirement for this molecule. These findings are consistent with virus entry via the clathrin-coated pit pathway.

Adenoviruses, Human↗

PubMed2003

The mitochondrial protein hFis1 regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein DLP1.

The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p. In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined. In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells. Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission. Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria. Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology. Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1. These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol. We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency.

Amino Acid Sequence↗