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

Schmid, Sandra L.; Smythe, Elizabeth

doi:10.1083/jcb.114.5.869

Cited by 147 publications

(107 citation statements)

References 30 publications

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“…Both the recruitment and assembly of the clathrin coat and dynamin can occur in the absence of nucleotides in vitro (29,30). However, ATP hydrolysis was shown to be required for coat assembly, vesicle budding (19,38), and decoating by the heat-shock cognate protein hsc70 ATPase (39,40) in clathrinmediated endocytosis. ATP may be needed, at least in part, for refolding reactions as well as for the cycle of phosphorylation- dephosphorylation of the endocytic proteins (41).…”

Section: Discussionmentioning

confidence: 99%

Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol

Kinuta

Yamada

Abe

et al. 2002

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

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As a step toward the elucidation of mechanisms in vesicle budding, a cell-free assay that measures cytosol-induced vesicle generation from liposomes was established. This assay then was used to explore the role of phosphoinositides in vesicle formation. Liposomes incubated with brain cytosol in the presence of ATP and GTP massively generated small vesicles, as assessed both quantitatively and qualitatively by a dynamic light-scattering assay. Both ATP and GTP were required. Vesicle formation was inhibited greatly by the immunodepletion of dynamin 1 from the cytosol, indicating a major contribution of this GTPase in this reaction and suggesting that it mimics endocytic vesicle fission. I n eukaryotic cells, transport vesicles are implicated in a variety of intracellular traffic events in both the secretory and endocytic pathways. The formation of a carrier vesicle is initiated by the recruitment of cytosolic coat proteins to the cytoplasmic surface of the donor membrane. One of the functions of the coat is to provide a scaffold for the formation of a high-curvature membrane bud. The coated bud then is pinched off as a coated vesicle, which eventually sheds the coat before fusion with its acceptor compartment. Several types of coats including the COPI, COPII, and clathrin coats have been characterized extensively (reviewed in refs. 1-7).Recently, the role of lipid factors is becoming a focus of the investigation in the field of membrane-coat interactions (7-11). Growing evidence suggests a key function of phosphoinositides and in particular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] in the recruitment of endocytic coats. PtdIns(4,5)P 2 binds to a variety of proteins that function in endocytic traffic including the ␣-subunit of clathrin adaptor protein (AP)2 (12), AP180, CALM (13), dynamin (14), and epsin (15). Phosphoinositides affect AP2's self-assembly (12, 16) and stimulate dynamin's GTPase activity (17,18). Furthermore, disruption of these interactions in living cells resulted in inhibition of endocytosis (15,19). Strong evidence for a physiological role of phosphoinositide metabolism in endocytosis came from the identification and functional characterization of a synaptic polyphosphoinositide phosphatase, synaptojanin 1 (20). Genetic deletion of synaptojanin in mice (21) and Caenorhabditis elegans (22) or disruption of the interactions of synaptojanin with its functional partners (23) resulted in a partial impairment of synaptic vesicle recycling and an increase of clathrin-coated vesicles and the coated pits. Conversely, an enzyme that synthesizes PtdIns(4,5)P 2 from PtdIns 4-monophosphate [PtdIns (4)P], PtdIns (4)P 5-kinase I␥, was shown recently to be concentrated at the synapse (24). Thus, the PtdIns(4,5)P 2 level at the synapse is likely to result from a balance of the enzymatic activities of synaptojanin and PtdIns (4)P 5-kinase.In vitro reconstitution of specific steps of vesicular transport reactions represents an increasingly important strategy to elucidate molecular mechanisms involved ...

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Section: Discussionmentioning

confidence: 99%

Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol

Kinuta

Yamada

Abe

et al. 2002

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

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“…However, although endocytosis is not blocked at this temperature, it is likely to be much reduced (32), so sensitivity to 15°C alone is not sufficient evidence to exclude the possibility that these vesicles are primary endocytic vesicles. In strong support of a primary endocytic identity, two distinct vesicle populations with the characteristics of this vesicle and of synaptic vesicles have been observed in coated vesicle preparations from rat brain after the coats have been removed (13,25).…”

Section: Fig 5 Distributions Of Vesicle Populations Containing Endomentioning

confidence: 99%

Evidence for a Primary Endocytic Vesicle Involved in Synaptic Vesicle Biogenesis

Provoda¹,

Waring

Buckley

2000

Journal of Biological Chemistry

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The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitterfilled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.The biogenesis of synaptic vesicles is a complex orchestration of events culminating in a vesicle population responsible for the uptake, storage, and regulated secretion of neurotransmitters. Current models of synaptic vesicle biogenesis suggest that at least two pathways exist for the formation of new synaptic vesicles after exocytosis: directly from the plasma membrane and indirectly via an intermediate endosomal compartment (1, 2). In neurons, both a direct pathway, which is at the active zone, and a more distal indirect pathway have been demonstrated at synapses in the Drosophila shibire ts1 mutant (3). Studies of membrane recycling in hippocampal synapses using the fluorescent dye FM1-43 also suggest that synaptic vesicles formed by endocytosis can fuse with the plasma membrane directly, without passing through an endosomal compartment (4). In the PC12 neuroendocrine cell line, synaptic vesicles are also derived from two different pathways as follows: directly from the plasma membrane (5, 6) as well as from an endosomal intermediate (7)(8)(9)(10). Together these data form an emerging model of multiple pathways to recycle both synaptic vesicle proteins and synaptic vesicle membrane.Although presumably all synaptic vesicle proteins undergo endocytic trafficking, relatively little is known about the intermediate steps in the pathway to mature synaptic vesicles. In order to understand fully the mechanisms underlying synaptic vesicle biogenesis, it is critical that the synaptic vesicle protein trafficking pathways are fully elucidated. In neurons, synaptic vesicle proteins are presen...

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“…In these studies, wildtype or K44A dynamin were co-expressed with Kir2.3 in Xenopus oocytes, and Kir2.3 surface expression was evaluated before and after BFA treatment with whole cell potassium current measurements and cell surface biotinylation. Biotinylation was preferentially used for labeling endosomal pools over antibody binding experiments, because the K44A dynamin mutant arrests endocytosis at a point of vesicle fission where endocytic cargo may be inaccessible to surface antibody binding (52,53). Following the injection of the cognate cRNAs, EGFP-tagged wild-type and K44A dynamin 2, proteins of the appropriate molecular mass (ϳ120 kDa) were readily detected in oocytes with anti-GFP antibodies by immunoblot analysis (Fig.…”

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confidence: 99%

AP-2-dependent Internalization of Potassium Channel Kir2.3 Is Driven by a Novel Di-hydrophobic Signal

Mason¹,

Jacobs

Welling

2008

Journal of Biological Chemistry

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The localization and density of Kir2.3 channels are influenced by the balance between PDZ protein interaction at the cell surface and routing into the endocytic pathway. Here, we explore mechanisms by which the Kir2.3 channel is directed into the endocytic pathway. We found that Kir2.3 channels are constitutively internalized from the cell surface in a dynamin-dependent manner, indicative of vesicle-mediated endocytosis. The rate of Kir2.3 endocytosis was dramatically attenuated following RNA interference-mediated knockdown of either ␣ adaptin (AP-2 clathrin adaptor) or clathrin heavy chain, revealing that Kir2.3 is internalized by an AP-2 clathrin-dependent mechanism. Structure-rationalized mutagenesis studies of a number of different potential AP-2 interaction motifs indicate that internalization of Kir2.3 is largely dependent on a non-canonical di-isoleucine motif (II413) embedded within the C terminus. Internalization assays using CD4-Kir2.3 chimeras demonstrate that the di-isoleucine signal acts in an autonomous and transplantable manner. Kir2.3 co-immunoprecipitates with ␣ adaptin, and disruption of the di-isoleucine motif decreased interaction of the channel with AP-2. Replacement of the di-isoleucine motif with a canonical di-leucine internalization signal actually blocked Kir2.3 endocytosis. Moreover, in yeast three-hybrid studies, the Kir2.3 di-isoleucine motif does not bind the AP-2 ␣C-2 hemicomplex in the way that has been recently observed for canonical di-leucine signals. Altogether, the results indicate that Kir2.3 channels are marked for clathrin-dependent internalization from the plasma membrane by a novel AP-2-dependent signal.

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

Cited by 147 publications

References 30 publications

Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol

Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol

Evidence for a Primary Endocytic Vesicle Involved in Synaptic Vesicle Biogenesis

AP-2-dependent Internalization of Potassium Channel Kir2.3 Is Driven by a Novel Di-hydrophobic Signal

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