scholarly journals Multi-modal adaptor-clathrin contacts drive coated vesicle assembly

2021 ◽  
Author(s):  
Sarah M Smith ◽  
Gabrielle Larocque ◽  
Katherine M Wood ◽  
Kyle L Morris ◽  
Alan M Roseman ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Binding Sites ◽  
Potential Role ◽  
Fundamental Property ◽  
Structural Knowledge ◽  
Coated Vesicle ◽  
Functional Importance ◽  
Coated Pits ◽  
Interaction Sites ◽  
Cross Links

Clathrin-coated pits are formed by the recognition of membrane and cargo by the heterotetrameric AP2 complex and the subsequent recruitment of clathrin triskelia. A potential role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the b2 subunit of AP2, and several binding sites on b2 and on the clathrin heavy chain have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of b2 hinge and appendage (b2HA) domains. We find that the b2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, b2-appendage cross-links two adjacent terminal domains from different triskelia below the vertex. Functional analysis of b2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the ''sandwich site'' on the appendage, with the appendage ''platform site'' having less importance. From these studies and the work of others, we propose that b2-appendage binding to more than one clathrin triskelion is a key feature of the system and likely explains why clathrin assembly is driven by AP2.

scholarly journals AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

1998 ◽  
Vol 140 (5) ◽  
pp. 1055-1062 ◽  
Author(s):  
Alexandre Benmerah ◽  
Christophe Lamaze ◽  
Bernadette Bègue ◽  
Sandra L. Schmid ◽  
Alice Dautry-Varsat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Coated Vesicle ◽  
Mutant Form ◽  
A431 Cells ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Terminal Domain

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.

scholarly journals Endocytosis from coated pits of Shiga toxin: a glycolipid-binding protein from Shigella dysenteriae 1.

1989 ◽  
Vol 108 (4) ◽  
pp. 1331-1343 ◽  
Author(s):  
K Sandvig ◽  
S Olsnes ◽  
J E Brown ◽  
O W Petersen ◽  
B van Deurs
Keyword(s):  
Shiga Toxin ◽  
Binding Sites ◽  
Vero Cells ◽  
Lipid Binding ◽  
Atp Depletion ◽  
Shigella Dysenteriae ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Normal Medium ◽  
Toxin Binding

Evidence is presented that endocytosis is involved in the transport to the cytosol of the cytotoxin from Shigella dysenteriae 1, Shiga toxin, which acts by removal of a single adenine residue in 28-S ribosomal RNA. Inhibition of endocytosis by ATP depletion of the cells prevented toxin uptake. Exposure of HeLa S3 and Vero cells to toxin at low extracellular pH, where translocation to the cytosol, but not endocytosis is inhibited, allowed the toxin to accumulate in a compartment where it was protected against antibodies to the toxin. Upon transfer of the cells to normal medium endocytosed toxin entered the cytosol. Electron microscopical studies of cells exposed at 0 degrees C to a toxin-horseradish peroxidase (HRP) conjugate, or to unconjugated toxin followed by horse antitoxin antibodies and then protein G-gold, revealed that the Shiga toxin binding sites were randomly distributed on the cell surface, without any preference to, for example, coated pits. In contrast, when cells were exposed to toxin at 37 degrees C, the binding sites were preferentially localized in coated pits. The Shiga-HRP conjugate was also seen in endosomes, lysosomes, and in the Golgi region. Endocytosis by the coated pit/coated vesicle pathway was selectively inhibited by acidification of the cytosol. Under these conditions, both the uptake of toxin-HRP conjugates and intoxication of the cells were inhibited. Evidence from the literature as well as our own results suggest that Shiga toxin binding sites are glycolipids. Thus, Shiga toxin appears to be the first example of a lipid-binding ligand that is endocytosed from coated pits.

Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

2019 ◽  
Author(s):  
Patrick R. A. Zanon ◽  
Lisa Lewald ◽  
Stephan M. Hacker
Keyword(s):  
Binding Sites ◽  
Bacterial Resistance ◽  
Mevalonate Pathway ◽  
Rapid Development ◽  
High Reactivity ◽  
Functional Importance ◽  
Essential Proteins ◽  
Covalent Inhibitors ◽  
Druggable Targets ◽  
Covalent Ligands

Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors. Here, we describe isotopically labeled desthiobiotin azide (isoDTB) tags that are easily synthesized, shorten the chemoproteomic workflow and allow an increased coverage of cysteines in bacterial systems. We quantify 59% of all cysteines in essential proteins in <i>Staphylococcus aureus</i> and discover 88 cysteines with high reactivity, which correlates with functional importance. Furthermore, we identify 268 cysteines that are engaged by covalent ligands. We verify inhibition of HMG-CoA synthase, which will allow addressing the bacterial mevalonate pathway through a new target. Overall, a comprehensive map of the bacterial cysteinome is obtained, which will facilitate the development of antibiotics with novel modes-of-action.

scholarly journals Analysis of functional importance of binding sites in the Drosophila gap gene network model

BMC Genomics ◽  
2015 ◽  
Vol 16 (Suppl 13) ◽  
pp. S7 ◽  
Author(s):  
Konstantin Kozlov ◽  
Vitaly V Gursky ◽  
Ivan V Kulakovskiy ◽  
Arina Dymova ◽  
Maria Samsonova
Keyword(s):  
Network Model ◽  
Binding Sites ◽  
Gene Network ◽  
Functional Importance ◽  
Gap Gene

scholarly journals Structural and Functional Analysis of the Metal-binding Sites ofClostridium thermocellumEndoglucanase CelD

1995 ◽  
Vol 270 (17) ◽  
pp. 9757-9762 ◽  
Author(s):  
Sylvie Chauvaux ◽  
Hélène Souchon ◽  
Pedro M. Alzari ◽  
Patrick Chariot ◽  
Pierre Beguin
Keyword(s):  
Functional Analysis ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Binding Sites

scholarly journals Coated Vesicle-Mediated Transport and Deposition of Vitellogenic Ferritin in the Rapid Growth Phase of Snail Oocytes

1982 ◽  
Vol 53 (1) ◽  
pp. 173-191 ◽  
Author(s):  
W. BOTTKE ◽  
I. SINHA ◽  
I. KEIL
Keyword(s):  
Growth Phase ◽  
Rapid Growth ◽  
Lymnaea Stagnalis ◽  
Follicle Cells ◽  
Coated Vesicle ◽  
Secretory Cells ◽  
Basal Region ◽  
Coated Pits ◽  
Iron Storage ◽  
Basement Lamina

The main yolk component in oocytes of the pulmonate freshwater snails Planorbarius corneus L. and Lymnaea stagnalis L. consists of the iron storage protein ferritin and iron-free apoferritin. Both compounds are deposited in the yolk in the form of large paracrystalloids, tubular structures and randomly dispersed particles. In addition, the plasm contains lysosome-like inclusions with depositions of haemosiderin. Haemosiderin is interpreted as the product of proteolytic degradation of ferritin. During the rapid growth phase of the oocytes vitellogenic ferritin is transported across the basement lamina and taken up by adsorptive endocytosis via coated pits and vesicles. Formation of yolk bodies occurs by fusion of ferritin-containing vacuoles and empty vesicles that are probably derived from the Golgi apparatus. Uptake of ferritin is restricted to the basal region of the oocyte. No involvement of the follicle cells in synthesis and deposition of ferritin could be detected. Secretory cells of the midgut gland are the most likely site of synthesis of vitellogenic ferritin. Under conditions of iron overload large masses of ferritin are encountered in the basement lamina of the oocytes. However, no significant increase in the uptake of ferritin could be observed. With the use of a tannic acid-glutaraldehyde fixation procedure a hitherto unobserved filamentous or rod-like material was detected inside the lamina and in coated pits. This material is probably also taken up by the oocytes and integrated into yolk platelets. Though ferritin is a rather unusual vitellogenic protein, the mode of its uptake and deposition in the oocyte plasm is highly reminiscent of that of typical hormone-induced vitellogenins in other animal groups.

scholarly journals In vivo functional analysis reveals specific roles for the integrin-binding sites of talin

2011 ◽  
Vol 124 (11) ◽  
pp. 1844-1856 ◽  
Author(s):  
S. J. Ellis ◽  
M. Pines ◽  
M. J. Fairchild ◽  
G. Tanentzapf
Keyword(s):  
Functional Analysis ◽  
Binding Sites
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