Movement of proteins through the Golgi stack: a molecular dissection of vesicular transport
            <sup>1</sup>

Rothman, James E.; Orci, Lelio

doi:10.1096/fasebj.4.5.2407590

Cited by 150 publications

(74 citation statements)

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“…Bar, 10 um. inhibited in the presence of GTPyS (28,31,36) and, in turn, the effect of BFA on ß-COP is blocked by GTPyS or aluminium fluoride (10) . Preliminary studies have shown that the dissociation of p200 from the Golgi in the presence of BFA is also blocked by GTPyS and aluminium fluoride, suggesting a role for G proteins in its membrane association (J .…”

Section: Discussionmentioning

confidence: 99%

Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes

Narula

McMorrow

Plopper

et al. 1992

The Journal of Cell Biology

114

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Section: Discussionmentioning

confidence: 99%

Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes

Narula

McMorrow

Plopper

et al. 1992

The Journal of Cell Biology

114

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“…This commonality has led to the isolation and cloning of NEM-sensitive factor (Rothman and Orci, 1990), a cytosolic factor that is required for membrane-membrane fusion between vesicles in these membrane transportpathways. SinceNEM treatment ofcytosol did not affect coated pit budding in vitro, apparently this factor is not required.…”

Section: Discussion Purified Plasma Membranes Support Coated Pit Buddingmentioning

confidence: 99%

Reconstitution of clathrin-coated pit budding from plasma membranes.

Lin

Moore

Sanan

et al. 1991

The Journal of Cell Biology

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Abstract. 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°C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4°C and then gently sonicating them to remove everything except the adherent membrane . Initially the gold label was clustered over flat, clathrincoated pits. After these membranes were warmed to 37°C for 5-10 min in the presence of buffer that con-1THIN a cell, coated vesicles form from coated pits (Anderson et al ., 1977;van Deurs et al., 1989) . Besides transforming into endocytic vesicles, coated pits also control the clustering of receptors that ligands use to enter cells by receptor-mediated endocytosis (Anderson, 1991). Selective mutagenesis ofthese receptors has revealed that their cytoplasmic domains contain an amino acid sequence that is necessary for efficient internalization (Chen et al ., 1990;Collawn et al., 1990;Ktistakis et al., 1990). However, the identification ofthe molecular elements(s) within coated pits that recognize these cytoplasmic domains remains to be made.Isolated coated vesicles have been useful for analyzing the structure, protein composition, and in vitro assembly of the clathrin lattice (Keen, 1990) . The lattice is composed of two subunits: (a) the clathrin triskelion, which is constructed from three 180-kD and three 30-35 kD proteins and forms the polygonal mesh work and (b) the AP (Assembly Protein [Keen, 1990] or Adaptor Protein [Pearse, 1988])' subunit, which is created from two sets of threeproteins (100-110, 50, and 16-18 kD) and appears to link the lattice to the plasma membrane (Vigers et al ., 1986). There are two different forms ofthe AP subunit: AP-1 and AP-2. These two isoforms differ in protein composition and location within the cell . tained cytosol extract, Cal+, 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°C; (b) the ATP, 150 /AM Cal+, and cytosolic factor(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.

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“…If removal of ARFs uncouples fusion from budding and leads to direct fusion of donor and acceptor membranes, then the lag in glycosylation-coupled transport thought to reflect the time required to form vesicles on the donor Golgi membranes (Balch et al, 1984b;Rothman and Orci, 1990) should disappear. Assays containing saturating concentrations of cytosol were incubated for various intervals at 30°C and then transferred to ice to arrest further transport (Balch et al, 1984a Figure 4A), the relative kinetics of transport in Figure 6.…”

Section: Removal Of Arfs From Transport Assays Has No Effect On the Rmentioning

confidence: 99%

Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: evidence for coated vesicle-independent transport.

Taylor

Kanstein

Weidman

et al. 1994

MBoC

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We investigated the role of ADP-ribosylation factors (ARFs) in Golgi function using biochemical and morphological cell-free assays. An ARF-free cytosol produced from soluble Chinese hamster ovary (CHO) extracts supports intra-Golgi transport by a mechanism that is biochemically indistinguishable from control transport reactions: ARF-free transport reactions are NSF-dependent, remain sensitive to the donor Golgi-specific inhibitor ilimaquinone, and exhibit kinetics that are identical to that of control reactions containing ARFs. In contrast, ARF-free cytosol does not support the formation of coated vesicles on Golgi cistemae. However, vesicle formation is reconstituted upon the addition of ARF1. These data suggest that neither soluble ARFs nor coated vesicle formation are essential for transport. We conclude that cell-free intra-Golgi transport proceeds via a coated vesicle-independent mechanism regardless of vesicle formation on Golgi cistemae.

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Movement of proteins through the Golgi stack: a molecular dissection of vesicular transport ¹

Cited by 150 publications

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Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes

Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes

Reconstitution of clathrin-coated pit budding from plasma membranes.

Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: evidence for coated vesicle-independent transport.

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Movement of proteins through the Golgi stack: a molecular dissection of vesicular transport 1

Cited by 150 publications

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Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes

Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes

Reconstitution of clathrin-coated pit budding from plasma membranes.

Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: evidence for coated vesicle-independent transport.

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Movement of proteins through the Golgi stack: a molecular dissection of vesicular transport ¹