Self-Assembly of Dynamin

2001 ◽  
Vol 7 (S2) ◽  
pp. 1210-1211
Author(s):  
Dganit Danino ◽  
Jenny E. Hinshaw
Keyword(s):  
Plasma Membrane ◽  
Self Assembly ◽  
Wild Type ◽  
Salt Solutions ◽  
Coated Pits ◽  
Vesicle Recycling ◽  
Rich Domain ◽  
Gtp Binding ◽  
Low Salt ◽  
Over Time

Dynamin is a large GTPase essential for various intracellular processes such as synaptic vesicle recycling, caveolae internalization and trafficking into and out of the Golgi. It is also involved in receptor-mediated endocytosis, and is believed to assemble at the necks of clathrin-coated pits and assist in pinching vesicles from the plasma membrane upon GTP binding and hydrolysis.Purified recombinant dynamin self-assembles into rings and spirals in low salt conditions [1]. A dynamin mutant lacking the c-terminal proline rich domain (APRD) also assembles into rings and spirals, however unlike wild type dynamin APRD constricts in the presence of GTP analogous such as GMP-PCP [2] or GTPγS. to explore differences in the behavior of the wild type and mutant dynamin we dialyzed them into different salt solutions containing various types of nucleotides and studied their assembly over time using negative staining and cryo-TEM.

scholarly journals Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.

1994 ◽  
Vol 127 (4) ◽  
pp. 915-934 ◽  
Author(s):  
H Damke ◽  
T Baba ◽  
D E Warnock ◽  
S L Schmid
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Wild Type ◽  
Coated Pits ◽  
Bulk Fluid ◽  
Vesicle Budding ◽  
Gtp Binding ◽  
Hela Cell Lines

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.

Dynamin Mediates Membrane Vesiculation

1998 ◽  
Vol 4 (S2) ◽  
pp. 1022-1023
Author(s):  
Sharon M. Sweitzer ◽  
Jenny E. Hinshaw
Keyword(s):  
Drosophila Melanogaster ◽  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Mechanism Of Action ◽  
Synaptic Vesicles ◽  
Dominant Negative ◽  
Coated Pits ◽  
Vesicle Recycling ◽  
Membrane Vesiculation ◽  
Helical Tubes

Dynamin, a 100 kDa GTPase, is essential for receptor mediated endocytosis and synaptic vesicle recycling; however its mechanism of action is unknown. The requirement for dynamin was first elucidated by the discovery that the shibire gene product in Drosophila melanogaster was homologous to mammalian dynamin-1 (1,2). The shibire flies exhibit a depletion of synaptic vesicles and an accumulation of collared clathrin-coated pits at the plasma membrane of their nerve termini (3). It was later demonstrated that endocytosis was inhibited by the overexpression of dominant negative mutants of dynamin (4,5), and that purified dynamin can self-associate to form spirals which resemble the collars of shibire and structures seen in synaptosomes treated with GTPγS (6,7). These observations led to the speculation that dynamin pinches the clathrin-coated bud from the plasma membrane. In support of this hypothesis, we show that purified recombinant dynamin can bind to a lipid bilayer in a regular and repeating pattern to form helical tubes which vesiculate upon the addition of GTP.

scholarly journals Ubiquitously Expressed Dynamin-II Has a Higher Intrinsic GTPase Activity and a Greater Propensity for Self-assembly Than Neuronal Dynamin-I

1997 ◽  
Vol 8 (12) ◽  
pp. 2553-2562 ◽  
Author(s):  
Dale E. Warnock ◽  
Takeshi Baba ◽  
Sandra L. Schmid
Keyword(s):  
Self Assembly ◽  
Intrinsic Rate ◽  
Biochemical Properties ◽  
Gtpase Activity ◽  
Coated Pits ◽  
Gtp Hydrolysis ◽  
Rich Domain ◽  
Dynamin I ◽  
Intrinsic Gtpase Activity

To begin to understand mechanistic differences in endocytosis in neurons and nonneuronal cells, we have compared the biochemical properties of the ubiquitously expressed dynamin-II isoform with those of neuron-specific dynamin-I. Like dynamin-I, dynamin-II is specifically localized to and highly concentrated in coated pits on the plasma membrane and can assemble in vitro into rings and helical arrays. As expected, the two closely related isoforms share a similar mechanism for GTP hydrolysis: both are stimulated in vitro by self-assembly and by interaction with microtubules or the SH3 domain-containing protein, grb2. Deletion of the C-terminal proline/arginine-rich domain from either isoform abrogates self-assembly and assembly-dependent increases in GTP hydrolysis. However, dynamin-II exhibits a ∼threefold higher rate of intrinsic GTP hydrolysis and higher affinity for GTP than dynamin-I. Strikingly, the stimulated GTPase activity of dynamin-II can be >40-fold higher than dynamin-I, due principally to its greater propensity for self-assembly and the increased resistance of assembled dynamin-II to GTP-triggered disassembly. These results are consistent with the hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.

scholarly journals The small GTP-binding protein rab6 functions in intra-Golgi transport.

1994 ◽  
Vol 127 (6) ◽  
pp. 1575-1588 ◽  
Author(s):  
O Martinez ◽  
A Schmidt ◽  
J Salaméro ◽  
B Hoflack ◽  
M Roa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Binding Protein ◽  
Retrograde Transport ◽  
Wild Type ◽  
Gtp Binding Protein ◽  
Anterograde Transport ◽  
Golgi Transport ◽  
Gtp Binding ◽  
Hemagglutinin Protein

Rab6 is a ubiquitous ras-like GTP-binding protein associated with the membranes of the Golgi complex (Goud, B., A. Zahraoui, A. Tavitian, and J. Saraste. 1990. Nature (Lond.). 345:553-556; Antony, C., C. Cibert, G. Géraud, A. Santa Maria, B. Maro, V. Mayau, and B. Goud. 1992. J. Cell Sci. 103: 785-796). We have transiently overexpressed in mouse L cells and human HeLa cells wild-type rab6, GTP (rab6 Q72L), and GDP (rab6 T27N) -bound mutants of rab6 and analyzed the intracellular transport of a soluble secreted form of alkaline phosphatase (SEAP) and of a plasma membrane protein, the hemagglutinin protein (HA) of influenza virus. Over-expression of wild-type rab6 and rab6 Q72L greatly reduced transport of both markers between cis/medial (alpha-mannosidase II positive) and late (sialyl-transferase positive) Golgi compartments, without affecting transport from the endoplasmic reticulum (ER) to cis/medial-Golgi or from the trans-Golgi network (TGN) to the plasma membrane. Whereas overexpression of rab6 T27N did not affect the individual steps of transport between ER and the plasma membrane, it caused an apparent delay in secretion, most likely due to the accumulation of the transport markers in late Golgi compartments. Overexpression of both rab6 Q72L and rab6 T27N altered the morphology of the Golgi apparatus as well as that of the TGN, as assessed at the immunofluorescence level with several markers. We interpret these results as indicating that rab6 controls intra-Golgi transport, either acting as an inhibitor in anterograde transport or as a positive regulator of retrograde transport.

scholarly journals KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Timothy H. Tran ◽  
Albert H. Chan ◽  
Lucy C. Young ◽  
Lakshman Bindu ◽  
Chris Neale ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Crystal Structures ◽  
Regulatory Region ◽  
Binding Domain ◽  
Published Data ◽  
Wild Type ◽  
Kras Mutations ◽  
Rich Domain ◽  
Structural Entity ◽  
Cysteine Rich Domain

AbstractThe first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

scholarly journals Dynamin is primed at endocytic sites for ultrafast endocytosis

2021 ◽  
Author(s):  
Yuuta Imoto ◽  
Sumana Raychaudhuri ◽  
Pascal Fenske ◽  
Eduardo Sandoval ◽  
Kie Itoh ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Splice Variant ◽  
Synaptic Vesicle Recycling ◽  
Vesicle Recycling ◽  
Rich Domain ◽  
Interacting Domain

SummaryDynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane when the proline-rich domain of this variant is dephosphorylated. When this domain is mutated to include phosphomimetic residues or Syndapin 1’s dynamin-interacting domain is mutated, Dynamin 1xA becomes diffuse, and consequently, ultrafast endocytosis slows down by ∼100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

scholarly journals Endocytosis of the ASGP receptor H1 is reduced by mutation of tyrosine-5 but still occurs via coated pits.

1991 ◽  
Vol 114 (3) ◽  
pp. 423-431 ◽  
Author(s):  
C Fuhrer ◽  
I Geffen ◽  
M Spiess
Keyword(s):  
Plasma Membrane ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Membrane Receptors ◽  
Immunogold Localization ◽  
Wild Type ◽  
Coated Pits ◽  
Residual Rate ◽  
Tyrosine Residues ◽  
Plasma Membrane Receptors

The clustering of plasma membrane receptors in clathrin-coated pits depends on determinants within their cytoplasmic domains. In several cases, individual tyrosine residues were shown to be necessary for rapid internalization. We have mutated the single tyrosine at position 5 in the cytoplasmic domain of the major subunit H1 of the asialoglycoprotein receptor to alanine. Expressed in fibroblasts cells, the mutant protein was accumulated in the plasma membrane, and its rate of internalization was reduced by a factor of four. The residual rate of endocytosis, however, was still significantly higher than that of resident plasma membrane proteins. Upon acidification of the cytoplasm, which specifically inhibits the formation of clathrin-coated vesicles but not uptake of the fluid phase marker Lucifer yellow, residual endocytosis was blocked. By immunoelectron microscopy mutant H1 could be directly demonstrated in coated pits. The fraction of wild-type and mutant H1 present in coated pits as determined by immunogold localization correlated well with the respective rates of internalization. Thus, mutation of tyrosine-5 only partially inactivates recognition of H1 for incorporation into coated pits.

scholarly journals Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments.

1995 ◽  
Vol 128 (6) ◽  
pp. 1003-1017 ◽  
Author(s):  
P J Peters ◽  
V W Hsu ◽  
C E Ooi ◽  
D Finazzi ◽  
S B Teal ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Membrane System ◽  
Endocytic Pathway ◽  
Coat Proteins ◽  
Wild Type ◽  
Gtp Binding ◽  
Defective Mutant

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.

scholarly journals KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

2020 ◽  
Author(s):  
Timothy H. Tran ◽  
Albert H. Chan ◽  
Lucy C. Young ◽  
Lakshman Bindu ◽  
Chris Neale ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Crystal Structures ◽  
Kras Mutation ◽  
Regulatory Region ◽  
Binding Domain ◽  
Published Data ◽  
Wild Type ◽  
Rich Domain ◽  
Structural Entity ◽  
Cysteine Rich Domain

ABSTRACTA vital first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we solved crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures revealed that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutation at the KRAS-CRD interface resulted in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

Sign in / Sign up

Export Citation Format

Share Document