scholarly journals Stage-specific assays for coated pit formation and coated vesicle budding in vitro.

1991 ◽  
Vol 114 (5) ◽  
pp. 869-880 ◽  
Author(s):  
S L Schmid ◽  
E Smythe
Keyword(s):  
Transferrin Receptor ◽  
Total Cell ◽  
Differential Sensitivity ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Intact Cells ◽  
Free System ◽  
Vesicle Budding ◽  
Receptor Mediated Endocytosis ◽  
Morphological Studies

Internalization of biotin-S-S-125I-transferrin (125I-BSST) into semiintact A431 cells were assessed by two different criteria which have allowed us to distinguish partial reactions in the complex overall process of receptor-mediated endocytosis. Early events resulting in the sequestration of ligand into deeply invaginated coated pits were measured by inaccessibility of 125I-BSST to exogenously added antibodies. Later events involving coated vesicle budding and membrane fission were measured by resistance of 125I-BSST to reduction by the membrane impermeant-reducing agent, MesNa. Acquisition of Ab inaccessibility occurred very efficiently in this cell-free system (approximately 50% of total cell-associated 125I-BSST became inaccessible) and could be inhibited by anti-clathrin mAbs and by antibodies directed against the cytoplasmic domain of the transferrin-receptor. In contrast, acquisition of MesNa resistance occurred less efficiently (approximately 10-20% of total cell-associated 125I-BSST) and showed differential sensitivity to inhibition by anti-clathrin and anti-transferrin receptor mAbs. Both partial reactions were stimulated by ATP and cytosol; indicating at least two ATP-requiring events in receptor-mediated endocytosis. The temperature dependence of both reactions was similar to that for 125I-BSST internalization in intact cells with no activity being observed below 10 degrees C. Morphological studies using gold-labeled ligands confirmed that internalization of transferrin receptors into semiintact A431 cell occurred via coated pits and coated vesicles and resulted in delivery of ligand to endosomal structures.

scholarly journals Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis.

1993 ◽  
Vol 120 (1) ◽  
pp. 37-45 ◽  
Author(s):  
L L Carter ◽  
T E Redelmeier ◽  
L A Woollenweber ◽  
S L Schmid
Keyword(s):  
Protein Function ◽  
Binding Proteins ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Vesicle Budding ◽  
Gtp Binding Proteins ◽  
Receptor Mediated Endocytosis ◽  
Gtp Binding ◽  
Gamma S

We have examined the effects of various agonists and antagonists of GTP-binding proteins on receptor-mediated endocytosis in vitro. Stage-specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to GTP gamma S, A1F4-, and mastoparan, late events involved in coated vesicle budding are inhibited by these antagonists of G protein function. Earlier events involved in coated pit assembly are also inhibited by GTP gamma S, A1F4-, and mastoparan. These results demonstrate that multiple GTP-binding proteins, including heterotrimeric G proteins, participate at discrete stages in receptor-mediated endocytosis via clathrin-coated pits.

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 Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes.

1983 ◽  
Vol 97 (2) ◽  
pp. 329-339 ◽  
Author(s):  
C Harding ◽  
J Heuser ◽  
P Stahl
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Intracellular Membrane ◽  
Coated Pits ◽  
Surface Receptors ◽  
Receptor Mediated Endocytosis ◽  
Freeze Dried ◽  
Inner Surface ◽  
Multivesicular Endosomes ◽  
Rat Reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin-resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.

scholarly journals Endocytosis occurs independently of annexin VI in human A431 cells

1994 ◽  
Vol 124 (3) ◽  
pp. 301-306 ◽  
Author(s):  
E Smythe ◽  
PD Smith ◽  
SM Jacob ◽  
J Theobald ◽  
SE Moss
Keyword(s):  
Transferrin Receptor ◽  
Squamous Carcinoma ◽  
Cell Types ◽  
Northern Blotting ◽  
Endocytic Pathway ◽  
A431 Cells ◽  
Coated Pits ◽  
Intact Cells ◽  
Calcium Dependent ◽  
Annexin Vi

Annexin VI is one of a family of calcium-dependent phospholipid-binding proteins. Although the function of this protein is not known, various physiological roles have been proposed, including a role in the budding of clathrin-coated pits (Lin et al., 1992. Cell. 70:283-291.). In this study we have investigated a possible endocytotic role for annexin VI in intact cells, using the human squamous carcinoma cell line A431, and report that these cells do not express endogenous annexin VI, as judged by Western and Northern blotting and PCR/Southern blotting. To examine whether endocytosis might in some way be either facilitated or inhibited by the presence of annexin VI, a series of A431 clones were isolated in which annexin VI expression was achieved by stable transfection. These cells expressed annexin VI at similar levels to other human cell types. Using assays for endocytosis and recycling of the transferrin receptor, we report that each of these cellular processes occurs with identical kinetics in both transfected and wild-type A431 cells. In addition, purified annexin VI failed to support the scission of coated pits in permeabilized A431 cells. We conclude that annexin VI is not an essential component of the endocytic pathway, and that in A431 cells, annexin VI fails to exert any influence on internalization and recycling of the transferrin receptor.

scholarly journals Clathrin-mediated endocytosis in AP-2–depleted cells

2003 ◽  
Vol 162 (5) ◽  
pp. 909-918 ◽  
Author(s):  
Alison Motley ◽  
Nicholas A. Bright ◽  
Matthew N.J. Seaman ◽  
Margaret S. Robinson
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Ldl Receptor ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Golgi Membrane ◽  
Coated Pits ◽  
Ldl Receptors ◽  
Cargo Proteins ◽  
Membrane Compartments

We have used RNA interference to knock down the AP-2 μ2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2–depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2–depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2–depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

scholarly journals Dynamin:Gtp Controls the Formation of Constricted Coated Pits, the Rate Limiting Step in Clathrin-Mediated Endocytosis

2000 ◽  
Vol 150 (5) ◽  
pp. 1137-1148 ◽  
Author(s):  
Sanja Sever ◽  
Hanna Damke ◽  
Sandra L. Schmid
Keyword(s):  
Self Assembly ◽  
Point Mutations ◽  
Coated Vesicle ◽  
Primary Defect ◽  
Coated Pits ◽  
Gtp Hydrolysis ◽  
Receptor Mediated Endocytosis ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

The GTPase dynamin is essential for receptor-mediated endocytosis, but its function remains controversial. A domain of dynamin, termed the GTPase effector domain (GED), controls dynamin's high stimulated rates of GTP hydrolysis by functioning as an assembly-dependent GAP. Dyn(K694A) and dyn(R725A) carry point mutations within GED resulting in reduced assembly stimulated GTPase activity. Biotinylated transferrin is more rapidly sequestered from avidin in cells transiently overexpressing either of these two activating mutants (Sever, S., A.B. Muhlberg, and S.L. Schmid. 1999. Nature. 398:481–486), suggesting that early events in receptor-mediated endocytosis are accelerated. Using stage-specific assays and morphological analyses of stably transformed cells, we have identified which events in clathrin-coated vesicle formation are accelerated by the overexpression of dyn(K694A) and dyn(R725A). Both mutants accelerate the formation of constricted coated pits, which we identify as the rate limiting step in endocytosis. Surprisingly, overexpression of dyn(R725A), whose primary defect is in stimulated GTP hydrolysis, but not dyn(K694A), whose primary defect is in self-assembly, inhibited membrane fission leading to coated vesicle release. Together, our data support a model in which dynamin functions like a classical GTPase as a key regulator of clathrin-mediated endocytosis.

scholarly journals Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors.

1992 ◽  
Vol 119 (5) ◽  
pp. 1163-1171 ◽  
Author(s):  
E Smythe ◽  
L L Carter ◽  
S L Schmid
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicle ◽  
A431 Cells ◽  
Coated Pits ◽  
Vesicle Budding ◽  
Membrane Fission ◽  
Cytosolic Factors ◽  
Insight Into ◽  
Stimulation Of

Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-dependent stimulation of Tfn sequestration requires cytosolic clathrin, but is unaffected by clathrin purified from coated vesicles suggesting that soluble and assembled clathrin pools are functionally distinct. In addition to adaptors and cytosolic clathrin other, as yet unidentified, cytosolic factors are also required for efficient coated pit invagination. These results provide new insight into the mechanisms and regulation of coated pit assembly and invagination.

scholarly journals Reconstitution of clathrin-coated pit budding from plasma membranes.

1991 ◽  
Vol 114 (5) ◽  
pp. 881-891 ◽  
Author(s):  
H C Lin ◽  
M S Moore ◽  
D A Sanan ◽  
R G Anderson
Keyword(s):  
Plasma Membranes ◽  
Ldl Receptor ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Gold Label ◽  
Cytosolic Factors

Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.

scholarly journals Mutations in human dynamin block an intermediate stage in coated vesicle formation

1993 ◽  
Vol 122 (3) ◽  
pp. 553-563 ◽  
Author(s):  
AM van der Bliek ◽  
TE Redelmeier ◽  
H Damke ◽  
EJ Tisdale ◽  
EM Meyerowitz ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Mammalian Cells ◽  
Intermediate Stage ◽  
Binding Domain ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Gtp Binding

The role of human dynamin in receptor-mediated endocytosis was investigated by transient expression of GTP-binding domain mutants in mammalian cells. Using assays which detect intermediates in coated vesicle formation, the dynamin mutants were found to block endocytosis at a stage after the initiation of coat assembly and preceding the sequestration of ligands into deeply invaginated coated pits. Membrane transport from the ER to the Golgi complex was unaffected indicating that dynamin mutants specifically block early events in endocytosis. These results demonstrate that mutations in the GTP-binding domain of dynamin block Tfn-endocytosis in mammalian cells and suggest that a functional dynamin GTPase is required for receptor-mediated endocytosis via clathrin-coated pits.

scholarly journals Dynamin GTPase Domain Mutants Block Endocytic Vesicle Formation at Morphologically Distinct Stages

2001 ◽  
Vol 12 (9) ◽  
pp. 2578-2589 ◽  
Author(s):  
Hanna Damke ◽  
Derk D. Binns ◽  
Hideho Ueda ◽  
Sandra L. Schmid ◽  
Takeshi Baba
Keyword(s):  
Dominant Negative ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Complex Relationship ◽  
Protein Activity ◽  
Coated Pits ◽  
Gtpase Domain ◽  
Receptor Mediated Endocytosis ◽  
Gtpase Cycle

Abundant evidence has shown that the GTPase dynamin is required for receptor-mediated endocytosis, but its exact role in endocytic clathrin-coated vesicle formation remains to be established. Whereas dynamin GTPase domain mutants that are defective in GTP binding and hydrolysis are potent dominant-negative inhibitors of receptor-mediated endocytosis, overexpression of dynamin GTPase effector domain (GED) mutants that are selectively defective in assembly-stimulated GTPase-activating protein activity can stimulate the formation of constricted coated pits and receptor-mediated endocytosis. These apparently conflicting results suggest that a complex relationship exists between dynamin's GTPase cycle of binding and hydrolysis and its role in endocytic coated vesicle formation. We sought to explore this complex relationship by generating dynamin GTPase mutants predicted to be defective at distinct stages of its GTPase cycle and examining the structural intermediates that accumulate in cells overexpressing these mutants. We report that the effects of nucleotide-binding domain mutants on dynamin's GTPase cycle in vitro are not as predicted by comparison to other GTPase superfamily members. Specifically, GTP and GDP association was destabilized for each of the GTPase domain mutants we analyzed. Nonetheless, we find that overexpression of dynamin mutants with subtle differences in their GTPase properties can lead to the accumulation of distinct intermediates in endocytic coated vesicle formation.

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