Instability of coated vesicles in concentrated sucrose solutions.

Nandi, P. K.; Irace, Gaetano; Jaarsveld, P. P. Van; Lippoldt, Roland E.; Edelhoch, Harold

doi:10.1073/pnas.79.19.5881

Cited by 108 publications

(62 citation statements)

References 23 publications

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“…1). This difference may be explained by the differing protocols used by these laboratories to prepare and assay their vesicle preparations, since it is known that the coat structure is sensitive to high sucrose concentrations during centrifugation (22) and that the change in specificity of the vesicle kinase described in this report is correlated with an increase in turbidity suggestive of aggregation. Polyethylene glycol (Mr 3700) at 10% (wt/vol) final concentration did not induce CAP2 phosphorylation (data not shown).…”

Section: Methodsmentioning

confidence: 89%

Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase.

Schook¹,

Puszkin²

1985

Proc. Natl. Acad. Sci. U.S.A.

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Section: Methodsmentioning

confidence: 89%

Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase.

Schook¹,

Puszkin²

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…Preparation of Clathrin-coated Vesicles-Coated vesicles were prepared from NIH 3T3 cells expressing either receptor grown to near confluence or primary rat neurons cultured for 72 h using a 2 H 2 O, 8% sucrose gradient (30). Cells were washed twice with PBS and once with MES buffer (100 mM MES, pH 6.5, 1 mM EGTA, 0.5 mM MgCl 2 , 3 mM NaN 3 , Complete TM proteinase inhibitor mixture).…”

Section: Methodsmentioning

confidence: 99%

Differential Functions of ApoER2 and Very Low Density Lipoprotein Receptor in Reelin Signaling Depend on Differential Sorting of the Receptors

Duit

Mayer

Blake

et al. 2010

Journal of Biological Chemistry

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ApoER2 and very low density lipoprotein (VLDL) receptor transmit the Reelin signal into target cells of the central nervous system. To a certain extent, both receptors can compensate for each other, and only the loss of both receptors results in the reeler phenotype, which is characterized by a gross defect in the architecture of laminated brain structures. Nevertheless, both receptors also have specific distinct functions, as corroborated by analyses of the subtle phenotypes displayed in mice lacking either ApoER2 or VLDL receptor. The differences in their function(s), however, have not been defined at the cellular level. Here, using a panel of chimeric receptors, we demonstrate that endocytosis of Reelin and the fate of the individual receptors upon stimulation are linked to their specific sorting to raft versus non-raft domains of the plasma membrane. VLDL receptor residing in the non-raft domain endocytoses and destines Reelin for degradation via the clathrin-coated pit/clathrin-coated vesicle/endosome pathway without being degraded to a significant extent. Binding of Reelin to ApoER2, a resident of rafts, leads to the production of specific receptor fragments with specific functions of their own and to degradation of ApoER2 via lysosomes. These features contribute to a receptor-specific fine tuning of the Reelin signal, leading to a novel model that emphasizes negative feedback loops specifically mediated by ApoER2 and VLDL receptor, respectively.

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“…Isolation of Clathrin-coated Vesicles and Purification of Hsc70 -Clathrin-coated vesicles were prepared from fresh calf brains according to the procedure of P. Nandi et al (32). Isolated clathrin-coated vesicles were stored at 4°C at a clathrin concentration of 15 M. Bovine brain Hsc70 was prepared as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

Hsc70 Chaperones Clathrin and Primes It to Interact with Vesicle Membranes

Jiang¹,

Gao²,

Prasad³

et al. 2000

Journal of Biological Chemistry

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When Hsc70 uncoats clathrin-coated vesicles in an auxilin-and ATP-dependent reaction, a single round of rapid uncoating occurs followed by very slow steadystate uncoating. We now show that this biphasic time course occurs because Hsc70 sequentially forms two types of complex with the dissociated clathrin triskelions. The first round of clathrin uncoating is driven by formation of a pre-steady-state assembly protein (AP)-clathrin-Hsc70-ADP complex. Then, following exchange of ADP with ATP, a steady-state AP-clathrin-Hsc70-ATP complex forms that ties up Hsc70, preventing further uncoating. This steady-state complex forms only during uncoating in the presence of APs; in the absence of APs, Hsc70 rapidly dissociates from the uncoated clathrin and continues to carry out uncoating. Whether it is complexed with ATP or ADP, the steady-state complex has very different properties from the pre-steady-state complex in that it cannot be immunoprecipitated by anticlathrin antibodies and is readily dissociated by fast protein liquid chromatography. Remarkably, when the steady-state complex is incubated with uncoated vesicle membranes in ATP, the pre-steady-state complex reforms, suggesting that the clathrin triskelions in the steady-state complex rebind to the membranes and are again uncoated by Hsc70. We propose that Hsc70 not only uncoats clathrin but also chaperones it to prevent it from inappropriately polymerizing in the cell cytosol and primes it to reform clathrin-coated pits.Receptor-mediated endocytosis is an essential cellular process required for the rapid import of membrane-bound receptors into cells (1-3). During receptor-mediated endocytosis, clathrin triskelions polymerize and form clathrin-coated pits on the plasma membrane; similar clathrin-coated pits form on the trans-Golgi membrane (3-5). In addition to clathrin and receptors, these coated pits contain assembly proteins that both catalyze the polymerization of clathrin triskelions and bind the receptors that are localized in the coated pit. Several clathrin assembly proteins (APs) 1 have been described (6 -10) including AP1, AP2, AP3, and AP4, which are multimeric subunit complexes, and AP180 and auxilin, which are single subunit, neuronal specific assembly proteins (11,12). In addition to assembly proteins, a number of other proteins, including synaptojanin, amphyphisin, epsin, Eps15, and the small GTPase protein, Rab5-GDI, are involved in the formation and invagination of clathrin-coated pits (13-19). Phospholipids also play an important role in clathrin coat assembly and receptor recruitment (20, 21) as does dephosphorylation of the various proteins involved in the formation of clathrin-coated pits (22,23).Following invagination of the clathrin-coated pits, the GTPbinding protein dynamin plays a key role in the pinching off of the clathrin-coated pits to form clathrin-coated vesicles (4, 5). Then, once in the cytosol, the vesicles are uncoated in an ATP-dependent process by Hsc70 and its partner protein, auxilin, which is both a clathrin assembly protein...

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Instability of coated vesicles in concentrated sucrose solutions.

Cited by 108 publications

References 23 publications

Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase.

Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase.

Differential Functions of ApoER2 and Very Low Density Lipoprotein Receptor in Reelin Signaling Depend on Differential Sorting of the Receptors

Hsc70 Chaperones Clathrin and Primes It to Interact with Vesicle Membranes

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