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The role of dynamin and its binding partners in coated pit invagination and scission.

Hill E, van Der Kaay J, Downes CP, Smythe E - J. Cell Biol. (2001)

Bottom Line: Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits.We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro.This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Cell Biology, Wellcome Trust Biocentre, Dundee DD1 5EH, United Kingdom.

ABSTRACT
Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

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The SH3 domain of amphiphysin inhibits ligand sequestration and internalization in permeabilized A431 cells. (a) The sequestration of B-SS-Tfn into deeply invaginated coated pits and its internalization into coated vesicles was measured using the avidin internalization assay in the presence of the indicated concentrations of GST fusion proteins containing GST alone (filled circles), or the mutant SH3 domain of amphiphysin GST–amph2 SH3D36R (filled squares). Results are from a typical experiment where each assay point is the mean of duplicates which differed by <10%. (b) Bovine brain cytosol (500 μl of 10 mg/ml) was treated with glutathione-agarose alone or coupled to GST–amph2 SH3D36R for 2 h at 4°C. The beads were pelleted and washed three times in PBS before being electrophoresed on a 10% SDS-PAGE and Coomassie stained. Lane 1, GSH beads alone; lane 2, GST–amph2 SH3D36R beads. (c) Western blot of cytosol after treatment with GST–amph2 SH3D36R. Cytosol was treated as described in b and equivalent volumes of supernatant (lanes 1 and 2) or beads (lanes 3 and 4) were probed on Western blots using either antidynamin or antisynaptojanin antibodies. (d) The avidin inaccessibility assay was carried out in the presence of increasing concentrations of bovine brain cytosol which had been treated with GSH beads alone (filled squares) or with GSH beads coupled to GST–amph2 SH3D36R (filled diamonds). (e and f) Permeabilized cells were preincubated with GST (filled circles) or GST–amph2 SH3D36R (filled squares) at the indicated concentrations for 5 min at 30°C. The membranes were then pelleted by centrifugation and assayed either for (e) avidin inaccessibility or (f) MesNa resistance of B-SS-Tfn in the presence of cytosol (2.5 mg/ml) and ATP.
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Figure 1: The SH3 domain of amphiphysin inhibits ligand sequestration and internalization in permeabilized A431 cells. (a) The sequestration of B-SS-Tfn into deeply invaginated coated pits and its internalization into coated vesicles was measured using the avidin internalization assay in the presence of the indicated concentrations of GST fusion proteins containing GST alone (filled circles), or the mutant SH3 domain of amphiphysin GST–amph2 SH3D36R (filled squares). Results are from a typical experiment where each assay point is the mean of duplicates which differed by <10%. (b) Bovine brain cytosol (500 μl of 10 mg/ml) was treated with glutathione-agarose alone or coupled to GST–amph2 SH3D36R for 2 h at 4°C. The beads were pelleted and washed three times in PBS before being electrophoresed on a 10% SDS-PAGE and Coomassie stained. Lane 1, GSH beads alone; lane 2, GST–amph2 SH3D36R beads. (c) Western blot of cytosol after treatment with GST–amph2 SH3D36R. Cytosol was treated as described in b and equivalent volumes of supernatant (lanes 1 and 2) or beads (lanes 3 and 4) were probed on Western blots using either antidynamin or antisynaptojanin antibodies. (d) The avidin inaccessibility assay was carried out in the presence of increasing concentrations of bovine brain cytosol which had been treated with GSH beads alone (filled squares) or with GSH beads coupled to GST–amph2 SH3D36R (filled diamonds). (e and f) Permeabilized cells were preincubated with GST (filled circles) or GST–amph2 SH3D36R (filled squares) at the indicated concentrations for 5 min at 30°C. The membranes were then pelleted by centrifugation and assayed either for (e) avidin inaccessibility or (f) MesNa resistance of B-SS-Tfn in the presence of cytosol (2.5 mg/ml) and ATP.

Mentions: Dynamin binds via its PRD to a variety of different SH3 domain–containing proteins. Amphiphysin is one such SH3 domain–containing protein which is proposed to recruit dynamin to sites of endocytosis where it participates either directly or indirectly in vesicle scission (Sever et al. 1999; Stowell et al. 1999). We have tested the effect of a GST fusion protein of a mutant form of the SH3 domain of amphiphysin, GST–amph2 SH3D36R, on B-SS-Tfn sequestration and internalization in permeabilized A431 cells. GST–amph2 SH3D36R binds with a higher affinity to dynamin and thus more strongly inhibits endocytosis in vivo than the wild-type SH3 domain (Owen et al. 1998). Addition of this fusion protein to our in vitro assays revealed a potent inhibition of both ligand sequestration into deeply invaginated coated pits and internalization into coated vesicles as measured by the avidin inaccessibility assay (Fig. 1 a). Half-maximal inhibition occurred at a concentration of ∼0.4 μM GST–amph2 SH3D36R. In a separate assay which allows us to measure the invagination of newly formed coated pits only, the adaptor-dependent stimulation of sequestration (Smythe et al. 1992; McLauchlan et al. 1998), B-SS-Tfn sequestration into new pits was also inhibited by GST–amph2 SH3D36R and similar results were obtained with a GST fusion protein encoding the wild-type amphiphysin SH3 domain (data not shown). Similarly, GST alone is not inhibitory and the SH3 domain of Grb2 is inhibitory but only at a 10-fold higher concentration (data not shown). Grb2 has also been demonstrated to bind to dynamin (Gout et al. 1993) but overexpression of the SH3 domain does not inhibit endocytosis (Wang and Moran 1996; Wigge et al. 1997).


The role of dynamin and its binding partners in coated pit invagination and scission.

Hill E, van Der Kaay J, Downes CP, Smythe E - J. Cell Biol. (2001)

The SH3 domain of amphiphysin inhibits ligand sequestration and internalization in permeabilized A431 cells. (a) The sequestration of B-SS-Tfn into deeply invaginated coated pits and its internalization into coated vesicles was measured using the avidin internalization assay in the presence of the indicated concentrations of GST fusion proteins containing GST alone (filled circles), or the mutant SH3 domain of amphiphysin GST–amph2 SH3D36R (filled squares). Results are from a typical experiment where each assay point is the mean of duplicates which differed by <10%. (b) Bovine brain cytosol (500 μl of 10 mg/ml) was treated with glutathione-agarose alone or coupled to GST–amph2 SH3D36R for 2 h at 4°C. The beads were pelleted and washed three times in PBS before being electrophoresed on a 10% SDS-PAGE and Coomassie stained. Lane 1, GSH beads alone; lane 2, GST–amph2 SH3D36R beads. (c) Western blot of cytosol after treatment with GST–amph2 SH3D36R. Cytosol was treated as described in b and equivalent volumes of supernatant (lanes 1 and 2) or beads (lanes 3 and 4) were probed on Western blots using either antidynamin or antisynaptojanin antibodies. (d) The avidin inaccessibility assay was carried out in the presence of increasing concentrations of bovine brain cytosol which had been treated with GSH beads alone (filled squares) or with GSH beads coupled to GST–amph2 SH3D36R (filled diamonds). (e and f) Permeabilized cells were preincubated with GST (filled circles) or GST–amph2 SH3D36R (filled squares) at the indicated concentrations for 5 min at 30°C. The membranes were then pelleted by centrifugation and assayed either for (e) avidin inaccessibility or (f) MesNa resistance of B-SS-Tfn in the presence of cytosol (2.5 mg/ml) and ATP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199618&req=5

Figure 1: The SH3 domain of amphiphysin inhibits ligand sequestration and internalization in permeabilized A431 cells. (a) The sequestration of B-SS-Tfn into deeply invaginated coated pits and its internalization into coated vesicles was measured using the avidin internalization assay in the presence of the indicated concentrations of GST fusion proteins containing GST alone (filled circles), or the mutant SH3 domain of amphiphysin GST–amph2 SH3D36R (filled squares). Results are from a typical experiment where each assay point is the mean of duplicates which differed by <10%. (b) Bovine brain cytosol (500 μl of 10 mg/ml) was treated with glutathione-agarose alone or coupled to GST–amph2 SH3D36R for 2 h at 4°C. The beads were pelleted and washed three times in PBS before being electrophoresed on a 10% SDS-PAGE and Coomassie stained. Lane 1, GSH beads alone; lane 2, GST–amph2 SH3D36R beads. (c) Western blot of cytosol after treatment with GST–amph2 SH3D36R. Cytosol was treated as described in b and equivalent volumes of supernatant (lanes 1 and 2) or beads (lanes 3 and 4) were probed on Western blots using either antidynamin or antisynaptojanin antibodies. (d) The avidin inaccessibility assay was carried out in the presence of increasing concentrations of bovine brain cytosol which had been treated with GSH beads alone (filled squares) or with GSH beads coupled to GST–amph2 SH3D36R (filled diamonds). (e and f) Permeabilized cells were preincubated with GST (filled circles) or GST–amph2 SH3D36R (filled squares) at the indicated concentrations for 5 min at 30°C. The membranes were then pelleted by centrifugation and assayed either for (e) avidin inaccessibility or (f) MesNa resistance of B-SS-Tfn in the presence of cytosol (2.5 mg/ml) and ATP.
Mentions: Dynamin binds via its PRD to a variety of different SH3 domain–containing proteins. Amphiphysin is one such SH3 domain–containing protein which is proposed to recruit dynamin to sites of endocytosis where it participates either directly or indirectly in vesicle scission (Sever et al. 1999; Stowell et al. 1999). We have tested the effect of a GST fusion protein of a mutant form of the SH3 domain of amphiphysin, GST–amph2 SH3D36R, on B-SS-Tfn sequestration and internalization in permeabilized A431 cells. GST–amph2 SH3D36R binds with a higher affinity to dynamin and thus more strongly inhibits endocytosis in vivo than the wild-type SH3 domain (Owen et al. 1998). Addition of this fusion protein to our in vitro assays revealed a potent inhibition of both ligand sequestration into deeply invaginated coated pits and internalization into coated vesicles as measured by the avidin inaccessibility assay (Fig. 1 a). Half-maximal inhibition occurred at a concentration of ∼0.4 μM GST–amph2 SH3D36R. In a separate assay which allows us to measure the invagination of newly formed coated pits only, the adaptor-dependent stimulation of sequestration (Smythe et al. 1992; McLauchlan et al. 1998), B-SS-Tfn sequestration into new pits was also inhibited by GST–amph2 SH3D36R and similar results were obtained with a GST fusion protein encoding the wild-type amphiphysin SH3 domain (data not shown). Similarly, GST alone is not inhibitory and the SH3 domain of Grb2 is inhibitory but only at a 10-fold higher concentration (data not shown). Grb2 has also been demonstrated to bind to dynamin (Gout et al. 1993) but overexpression of the SH3 domain does not inhibit endocytosis (Wang and Moran 1996; Wigge et al. 1997).

Bottom Line: Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits.We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro.This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Cell Biology, Wellcome Trust Biocentre, Dundee DD1 5EH, United Kingdom.

ABSTRACT
Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

Show MeSH
Related in: MedlinePlus