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

Schmid, Sandra L.; Smythe, Elizabeth

doi:10.1016/0962-8924(91)90004-s

Cited by 62 publications

(117 citation statements)

References 26 publications

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“…3A, 125 I-transferrin binding at 4°C to untreated J774 macrophages reached saturation at ϳ1200 fmol ⅐ mg cell protein Ϫ1 , corresponding to ϳ75,000 binding sites ⅐ cell Ϫ1 with a single class of receptors exhibiting an apparent K d of ϳ15 nM, based on Scatchard plot analysis. To study endocytosis and recycling with maximal sensitivity, cells were surface-labeled with 50 nM 125 I-transferrin, washed, and reincubated at 37°C for the indicated intervals, after which they were transferred to 4°C and surface-digested with Pronase to distinguish the pool accessible at the plasma membrane (Pronase-sensitive) from the sequestered pool (Pronase-resistant) [46]. Pronase-resistant transferrin increased very rapidly to level off after ϳ4 min (Fig.…”

Section: Azithromycin Severely Down-regulates Surface Transferrin Recmentioning

confidence: 99%

Azithromycin, a Lysosomotropic Antibiotic, Has Distinct Effects on Fluid-Phase and Receptor-Mediated Endocytosis, but Does Not Impair Phagocytosis in J774 Macrophages

Tyteca

Smissen²,

Mettlen

et al. 2002

Experimental Cell Research

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Section: Azithromycin Severely Down-regulates Surface Transferrin Recmentioning

confidence: 99%

Azithromycin, a Lysosomotropic Antibiotic, Has Distinct Effects on Fluid-Phase and Receptor-Mediated Endocytosis, but Does Not Impair Phagocytosis in J774 Macrophages

Tyteca

Smissen²,

Mettlen

et al. 2002

Experimental Cell Research

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“…Gel-filtered cytosol preparations were rapidly frozen in liquid nitrogen and stored in aliquots at -70~ Bovine brain adaptors were prepared exactly as described by Smythe et al (1992b). Internalization and sequestration assays were performed exactly as previously described (Schmid and Smythe, 1991;Smythe et al, 1992a). Duplicate samples were run for each experimental point.…”

Section: Assays For Ligand Sequestration and Internalizationmentioning

confidence: 99%

“…A431 cells were cultured as previously described (Schmid and Smythe, 1991). Nucleotides and analogs were obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN).…”

Section: Cells and Reagentsmentioning

confidence: 99%

“…Therefore, to begin to dissect the molecular mechanisms of CCV-mediated endocytosis, we have developed stage-specific assays which biochemically distinguish three sequential events leading to coated vesicle formation. These sequential stages are coated pit assembly, coated pit invagination, and coated vesicle budding (Schmid and Smythe, 1991;Smythe et al, 1992a,b). Using these assays we have begun biochemical studies on the mechanism of CCV-mediated endocytosis.…”

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confidence: 99%

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

Carter

Redelmeier

Woollenweber

et al. 1993

The Journal of Cell Biology

175

141

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Abstract. 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/SS. Although coated pit invagination is resistant to GTP'yS, 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-yS, A1F4-, and mastoparan. These results demonstrate that multiple GTP-binding proteins, including heterotfimeric G proteins, participate at discrete stages in receptor-mediated endocytosis via clathrin-coated pits. INTRACELLULAR membrane trafficking is mediated by transport vesicles which bud from one organelle and then fuse with an appropriate target organelle. Vesicle formation occurs at specialized regions of the membrane distinguished by an underlying protein coat. There are now two recognized classes of coat structures which function in transport vesicle formation. The best studied of these are clathrincoated pits and coated vesicles (CCV) 1 which are involved in receptor-mediated endoeytosis and transport from the Golgi complex to lysosomes (reviewed by Brodsky, 1988;Pearse and Robinson, 1990). The major coat constituents of CCVs are clathrin triskelions and adaptors (reviewed by Pearse and Crowther, 1987). Clathrin triskelions are composed of three 180-kD heavy chains and three tightly associated •30-kD light chains. Adaptor complexes are heterotetramers composed of two *100-110-kD adaptin molecules and two smaller subunits of 47-50 and 17-19 kD (reviewed by Pearse and Robinson, 1990;Keen, 1990).More recently a second class of transport vesicles, referred to as "nonclathdn-coated vesicles" or "COP-coated vesicles" has been shown to mediate vesicular traffic along the exoeytic pathway (Orci et al., 1986). The coat constituents of nonclathrin-coated vesicles include polypeptides of 160 (or-cop), 110 (t-cop), 98 ('t-cop), and 68 kD (&cop), smaller subunits of 36 and 35 kD (Maholtra et al., 1989), as well as ADP-ribosylation factor (ARF), a 20-kD GTPbinding protein . Sequence analysis has demonstrated that 13-cop is distantly related to 13-adaptin (17 % homology in the NH2-terminal half of the molecule)1. Abbreviations used in this paper: BFA, brefeldin A; CCV, clathrincoated vesicle; COP..CV, nonclathrin-coated vesicle; GDP~S, guanosine-5'-o-(2-thiodiphosphate); GTP3,S, guanosine-5'-o-(3-thiotriphosphate); MESNa, mercaptoethane sulfonic acid. suggesting some functional relationship between these two coat proteins (Duden et al., 1991).Both classes of coated pits assemble from a cytosolic pool of coat proteins. Clathrin and adaptors exist as distinct soluble pools which appear to assemble sequentially to form cl...

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“…Stage-specific assays for endocytosis show that early, but as yet uncharacterized events in clathrin-coat assembly at the cell surface require ATP hydrolysis (19). Polymerization of AP-2 and clathrin into coats on lysed synaptosomes also requires ATP (20).…”

mentioning

confidence: 99%

Coupled Inositide Phosphorylation and Phospholipase D Activation Initiates Clathrin-coat Assembly on Lysosomes

Arneson¹,

Kunz²,

Anderson³

et al. 1999

Journal of Biological Chemistry

103

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Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) synthesis. PtdIns(4,5)P 2

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

Cited by 62 publications

References 26 publications

Azithromycin, a Lysosomotropic Antibiotic, Has Distinct Effects on Fluid-Phase and Receptor-Mediated Endocytosis, but Does Not Impair Phagocytosis in J774 Macrophages

Azithromycin, a Lysosomotropic Antibiotic, Has Distinct Effects on Fluid-Phase and Receptor-Mediated Endocytosis, but Does Not Impair Phagocytosis in J774 Macrophages

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

Coupled Inositide Phosphorylation and Phospholipase D Activation Initiates Clathrin-coat Assembly on Lysosomes

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