scholarly journals Dynamics of Auxilin1 and GAK in clathrin-mediated traffic

2019 ◽  
Author(s):  
Kangmin He ◽  
Eli Song ◽  
Srigokul Upadhyayula ◽  
Song Dang ◽  
Raphael Gaudin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Temporal Variations ◽  
Kinase Domain ◽  
Coated Vesicles ◽  
Head Group ◽  
Head Groups ◽  
Phosphatidylinositol Phosphate ◽  
J Domain ◽  
Cyclin G ◽  
Lipid Head Group

ABSTRACTClathrin coated vesicles formed at the plasma membrane lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 “uncoating ATPase”. The J-domain containing proteins, auxilin1 (Aux1) and auxilin2/cyclin-G dependent kinase (GAK), recruit Hsc70. Aux1 and GAK are closely related homologs, each with a phosphatase- and tensin-like (PTEN-like) domain, a clathrin-binding region, and a C-terminal J-domain; GAK has an additional, N-terminal Ser/Thr kinase domain. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin mediated membrane scission has released the vesicle from the plasma membrane. We show here that these bursts represent recruitment of a very small number of auxilins such that even 4-6 molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated-pit assembly. We have also shown previously that clathrin coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. We now show that differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.

scholarly journals Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic

2020 ◽  
Vol 219 (3) ◽  
Author(s):  
Kangmin He ◽  
Eli Song ◽  
Srigokul Upadhyayula ◽  
Song Dang ◽  
Raphael Gaudin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phosphatase Activity ◽  
Temporal Variations ◽  
Coated Vesicles ◽  
Head Group ◽  
Timing Mechanism ◽  
Head Groups ◽  
Phosphatidylinositol Phosphate ◽  
Membrane Scission ◽  
Lipid Head Group

Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 “uncoating ATPase.” The J- and PTEN-like domain–containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin-mediated membrane scission has released the vesicle from the plasma membrane. These bursts contain a very small number of auxilins, and even four to six molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated pit assembly. We previously showed that clathrin-coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. The differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.

scholarly journals Internalization of Phospholipids from the Plasma Membrane of Human Osteoblasts Depends on the Lipid Head Group

1999 ◽  
Vol 14 (5) ◽  
pp. 690-699 ◽  
Author(s):  
Jeanette Libera ◽  
Thomas Pomorski ◽  
Oliviera Josimović-Alasević ◽  
Karl-Gerd Fritsch ◽  
Andreas Herrmann
Keyword(s):  
Plasma Membrane ◽  
Head Group ◽  
Human Osteoblasts ◽  
Lipid Head Group

Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin

2001 ◽  
Vol 114 (2) ◽  
pp. 353-365 ◽  
Author(s):  
X. Zhao ◽  
T. Greener ◽  
H. Al-Hasani ◽  
S.W. Cushman ◽  
E. Eisenberg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
J Domain ◽  
Almost All ◽  
Golgi Network ◽  
Assembly Proteins

Although uncoating of clathrin-coated vesicles is a key event in clathrin-mediated endocytosis it is unclear what prevents uncoating of clathrin-coated pits before they pinch off to become clathrin-coated vesicles. We have shown that the J-domain proteins auxilin and GAK are required for uncoating by Hsc70 in vitro. In the present study, we expressed auxilin in cultured cells to determine if this would block endocytosis by causing premature uncoating of clathrin-coated pits. We found that expression of auxilin indeed inhibited endocytosis. However, expression of auxilin with its J-domain mutated so that it no longer interacted with Hsc70 also inhibited endocytosis as did expression of the clathrin-assembly protein, AP180, or its clathrin-binding domain. Accompanying this inhibition, we observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which provided us with an opportunity to determine whether the absence of clathrin from clathrin-coated pits affected the distribution of the clathrin assembly proteins AP1 and AP2. Surprisingly we found almost no change in the association of AP2 and AP1 with the plasma membrane and the trans-Golgi network, respectively. This was particularly obvious when auxilin or GAK was expressed with functional J-domains since, in these cases, almost all of the clathrin was sequestered in granules that also contained Hsc70 and auxilin or GAK. We conclude that expression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it is no longer available to bind to nascent pits but that assembly proteins bind to these pits independently of clathrin.

scholarly journals Exchange Speed of Four-Component Nanorotors Correlates with Hammett Substituent Constants

Chemistry ◽  
2021 ◽  
Vol 3 (1) ◽  
pp. 116-125
Author(s):  
Yi-Fan Li ◽  
Amit Ghosh ◽  
Pronay Kumar Biswas ◽  
Suchismita Saha ◽  
Michael Schmittel
Keyword(s):  
Linear Correlation ◽  
Bond Cleavage ◽  
Head Group ◽  
Ligand Interaction ◽  
Exchange Frequency ◽  
Rate Determining Step ◽  
Head Groups ◽  
Substituent Constants ◽  
First Time ◽  
Determining Factor

Three distinct four-component supramolecular nanorotors were prepared, using, for the first time, bipyridine instead of phenanthroline stations in the stator. Following our established self-sorting protocol to multicomponent nanodevices, the nanorotors were self-assembled by mixing the stator, rotators with various pyridine head groups, copper(I) ions and 1,4-diazabicyclo[2.2.2]octane (DABCO). Whereas the exchange of a phenanthroline vs. a bipyridine station did not entail significant changes in the rotational exchange frequency, the para-substituents at the pyridine head group of the rotator had drastic consequences on the speed: 4-OMe (k298 = 35 kHz), 4-H (k298 = 77 kHz) and 4-NO2 (k298 = 843 kHz). The exchange frequency (log k) showed an excellent linear correlation with both the Hammett substituent constants and log K of the copper(I)–ligand interaction, proving that rotator–copper(I) bond cleavage is the key determining factor in the rate-determining step.

scholarly journals Very Cool Clathrin

2005 ◽  
Vol 13 (6) ◽  
pp. 3-7
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicles ◽  
Membrane Bilayer ◽  
Membrane Bound ◽  
Membranous Component

Clathrin-coated vesicles are the shuttle containers within cells. The vesicles carry lipids and proteins between membrane-bound compartments. Clathrin forms a cage-like structure around the membrane-bound vesicle that is pinched off from the plasma membrane (in endocytosis) or a membranous component of the cytoplasm. Clathrin recruits cargo that is within a vesicle through intermediary proteins known as adaptors that help select membrane-anchored protein and form an interface between the clathrin cage and the membrane bilayer.

EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain

1999 ◽  
Vol 112 (12) ◽  
pp. 1957-1965 ◽  
Author(s):  
K. Venkateswarlu ◽  
F. Gunn-Moore ◽  
J.M. Tavare ◽  
P.J. Cullen
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Laser Scanning ◽  
Time Course ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Head Group ◽  
Nucleotide Exchange ◽  
Ph Domain

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.

Orientation of the v-erb-B gene product in the plasma membrane

1986 ◽  
Vol 6 (4) ◽  
pp. 1329-1333
Author(s):  
R C Schatzman ◽  
G I Evan ◽  
M L Privalsky ◽  
J M Bishop
Keyword(s):  
Endoplasmic Reticulum ◽  
Epidermal Growth Factor Receptor ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Amino Terminus ◽  
Protein Kinase Domain ◽  
Epidermal Growth

The retroviral oncogene v-erb-B encodes a truncated version of the receptor for epidermal growth factor. To define the disposition of the v-erb-B protein within cells and across the plasma membrane, we raised antibodies against defined epitopes in the protein and used these in immunofluorescence to analyze cells transformed by v-erb-B. A small fraction of the v-erb-B protein was found on the plasma membrane in a clustered configuration. The bulk of the protein was located in the endoplasmic reticulum and Golgi apparatus. Epitopes near the amino terminus of the v-erb-B protein were displayed on the surface of the cell, whereas epitopes in the protein kinase domain were located exclusively within cells. We conclude that the v-erb-B protein spans the plasma membrane in a manner similar or identical to that of the epidermal growth factor receptor, even though the viral transforming protein does not possess the signal peptide that is thought to direct insertion of the receptor into the membrane.

scholarly journals Correction: Effect of lipid head group interactions on membrane properties and membrane-induced cationic β-hairpin folding

2016 ◽  
Vol 18 (38) ◽  
pp. 26998-26998
Author(s):  
Sai J. Ganesan ◽  
Hongcheng Xu ◽  
Silvina Matysiak
Keyword(s):  
Chem Phys ◽  
Membrane Properties ◽  
Head Group ◽  
Group Interactions ◽  
Correction Effect ◽  
Lipid Head Group ◽  
Phys Chem Chem Phys

Correction for ‘Effect of lipid head group interactions on membrane properties and membrane-induced cationic β-hairpin folding’ by Sai J. Ganesan et al., Phys. Chem. Chem. Phys., 2016, 18, 17836–17850.

scholarly journals ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

1998 ◽  
Vol 9 (8) ◽  
pp. 2217-2229 ◽  
Author(s):  
Lisa A. Hannan ◽  
Sherri L. Newmyer ◽  
Sandra L. Schmid
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Dual Role ◽  
Vesicular Trafficking ◽  
Coated Vesicles ◽  
Golgi Network ◽  
Cytosolic Factor

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.

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