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 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 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

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.

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.

Identification of intracellular sites of superoxide production in stimulated neutrophils

1998 ◽  
Vol 111 (1) ◽  
pp. 81-91 ◽  
Author(s):  
T. Kobayashi ◽  
J.M. Robinson ◽  
H. Seguchi
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Time Course ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Morphological Evidence ◽  
Marker Enzyme ◽  
Intracellular Compartments

In this study, we show that superoxide production is carried out within intracellular compartments of human neutrophils and not at the plasma membrane following stimulation with phorbol myristate acetate. Oxidant production was not observed in unstimulated cells. Stimulated cells exhibited superoxide production in two distinct types of intracellular organelles. Initially, activity was detected in slender rod-shaped granules and in spherical or elliptical granules. The oxidant-producing granules fused directly with the plasma membrane or fused to form larger intracellular vesicles which then became associated with the plasma membrane. Longer periods of stimulation with PMA resulted in a decrease in the number of vesicles containing oxidant reaction product only, and an increase in structures containing both the oxidant-reaction product and ferritin particles; the latter was used herein as a marker for endocytosis. Thus a complex pattern of intracellular vesicular trafficking occurs in stimulated neutrophils. Alkaline phosphatase activity, a marker enzyme for a type of intracellular neutrophil granule was co-localized in the oxidant reaction-positive intracellular compartments. The time course of up-regulation of alkaline phosphatase activity to the cell surface parallelled the release of superoxide from stimulated cells. Results from this study demonstrate for the first time cytochemical and morphological evidence that superoxide is released from stimulated neutrophils through exocytosis of an oxidant-producing intracellular granule.

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