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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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Dynamin can rescue the inhibitory effects of GST–amph2 SH3D36R in the avidin inaccessibility but not the MesNa resistance assay. (a) Dynamin, purified from rat brain was electrophoresed on a 10% SDS-PAGE gel and visualized by Coomassie staining. Permeabilized cell membranes were preincubated in the presence or absence of GST–amph2 SH3D36R (0.1 mg/ml) for 5 min. They were then collected by centrifugation and assayed in the presence of ATP, cytosol (2.5 mg/ml), and dynamin (5 μg) as indicated for the ability to support avidin inaccessibility (b) or MesNa resistance (c) of B-SS-Tfn. Results are expresssed as the mean ± SEM of three experiments each performed in duplicate. Permeabilized cell membranes were preincubated in the presence or absence of GST–amph2 SH3D36R (0.1 mg/ml) for 5 min. They were then collected by centrifugation and assayed in the presence of ATP, cytosol (2.5 mg/ml), and synaptojanin (5 μg) as indicated for the ability to support avidin inaccessibility (d) or MesNa resistance (e) of B-SS-Tfn. Results are expresssed as the mean ± SD of two experiments each performed in duplicate. (f) Permeabilized membranes which had been preincubated in the presence of GST–amph2 SH3D36R and then subsequently incubated for 30 min in the presence of ATP and cytosol (lanes 1–3), plus dynamin (5 μg; lane 2), plus synaptojanin (5 μg; lane 3) were solubilized and electrophoresed on a 10% SDS gel, transferred to nitrocellulose, and probed for the presence of GST–amph2 SH3D36R using anti-GST antibodies.
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Figure 2: Dynamin can rescue the inhibitory effects of GST–amph2 SH3D36R in the avidin inaccessibility but not the MesNa resistance assay. (a) Dynamin, purified from rat brain was electrophoresed on a 10% SDS-PAGE gel and visualized by Coomassie staining. Permeabilized cell membranes were preincubated in the presence or absence of GST–amph2 SH3D36R (0.1 mg/ml) for 5 min. They were then collected by centrifugation and assayed in the presence of ATP, cytosol (2.5 mg/ml), and dynamin (5 μg) as indicated for the ability to support avidin inaccessibility (b) or MesNa resistance (c) of B-SS-Tfn. Results are expresssed as the mean ± SEM of three experiments each performed in duplicate. Permeabilized cell membranes were preincubated in the presence or absence of GST–amph2 SH3D36R (0.1 mg/ml) for 5 min. They were then collected by centrifugation and assayed in the presence of ATP, cytosol (2.5 mg/ml), and synaptojanin (5 μg) as indicated for the ability to support avidin inaccessibility (d) or MesNa resistance (e) of B-SS-Tfn. Results are expresssed as the mean ± SD of two experiments each performed in duplicate. (f) Permeabilized membranes which had been preincubated in the presence of GST–amph2 SH3D36R and then subsequently incubated for 30 min in the presence of ATP and cytosol (lanes 1–3), plus dynamin (5 μg; lane 2), plus synaptojanin (5 μg; lane 3) were solubilized and electrophoresed on a 10% SDS gel, transferred to nitrocellulose, and probed for the presence of GST–amph2 SH3D36R using anti-GST antibodies.

Mentions: The major binding partners for the SH3 domain of amphiphysin are dynamin and the inositol 5-phosphatase, synaptojanin (David et al. 1996; Micheva et al. 1997; Wigge et al. 1997) (see Fig. 1b and Fig. c). We wanted to investigate whether the inhibitory effects of the SH3 domain were specific either for dynamin or synaptojanin. Therefore, we investigated whether purified dynamin and synaptojanin could rescue the membranes which had been preincubated with GST–amph2 SH3D36R. Dynamin was purified from rat brain and synaptojanin was purified after overexpression in HEK293 cells using the wild-type GST–amph2 SH3 fusion protein as an affinity ligand (Stowell et al. 1999). Purified dynamin (Fig. 2 a) could restore the ability of the preincubated membranes to support ligand sequestration into deeply invaginated coated pits (Fig. 2 b). Strikingly, however, dynamin was unable to rescue the inhibitory effects of GST–amph2 SH3D36R in the MesNa resistance assay (Fig. 2 c). By contrast, purified synaptojanin was unable to rescue the inhibitory effects of GST–amph2 SH3D36R in either the avidin inaccessibility or MesNa resistance assays (Fig. 2d and Fig. e). To assess whether dynamin effected rescue by competing the SH3 domain of amphiphysin from the membrane, membranes which had been treated with GST–amph2 SH3D36R were incubated in the presence of cytosol alone or cytosol plus dynamin or synaptojanin. They were then Western blotted and probed with an antibody raised against GST. In the presence of dynamin or synaptojanin there was no reduction in the amount of GST–amph2 SH3D36R associated with the membrane (Fig. 2 f). This shows that when dynamin rescues pretreated membranes, it is not acting simply to displace GST–amph2 SH3D36R from the membranes. The inability of dynamin to restore MesNa resistance may be due to depletion of another protein by the SH3 domain of amphiphysin, but it could equally well be due to disruption of the sequential order and timing of events during endocytic vesicle formation.


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)

Dynamin can rescue the inhibitory effects of GST–amph2 SH3D36R in the avidin inaccessibility but not the MesNa resistance assay. (a) Dynamin, purified from rat brain was electrophoresed on a 10% SDS-PAGE gel and visualized by Coomassie staining. Permeabilized cell membranes were preincubated in the presence or absence of GST–amph2 SH3D36R (0.1 mg/ml) for 5 min. They were then collected by centrifugation and assayed in the presence of ATP, cytosol (2.5 mg/ml), and dynamin (5 μg) as indicated for the ability to support avidin inaccessibility (b) or MesNa resistance (c) of B-SS-Tfn. Results are expresssed as the mean ± SEM of three experiments each performed in duplicate. Permeabilized cell membranes were preincubated in the presence or absence of GST–amph2 SH3D36R (0.1 mg/ml) for 5 min. They were then collected by centrifugation and assayed in the presence of ATP, cytosol (2.5 mg/ml), and synaptojanin (5 μg) as indicated for the ability to support avidin inaccessibility (d) or MesNa resistance (e) of B-SS-Tfn. Results are expresssed as the mean ± SD of two experiments each performed in duplicate. (f) Permeabilized membranes which had been preincubated in the presence of GST–amph2 SH3D36R and then subsequently incubated for 30 min in the presence of ATP and cytosol (lanes 1–3), plus dynamin (5 μg; lane 2), plus synaptojanin (5 μg; lane 3) were solubilized and electrophoresed on a 10% SDS gel, transferred to nitrocellulose, and probed for the presence of GST–amph2 SH3D36R using anti-GST antibodies.
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Related In: Results  -  Collection

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Figure 2: Dynamin can rescue the inhibitory effects of GST–amph2 SH3D36R in the avidin inaccessibility but not the MesNa resistance assay. (a) Dynamin, purified from rat brain was electrophoresed on a 10% SDS-PAGE gel and visualized by Coomassie staining. Permeabilized cell membranes were preincubated in the presence or absence of GST–amph2 SH3D36R (0.1 mg/ml) for 5 min. They were then collected by centrifugation and assayed in the presence of ATP, cytosol (2.5 mg/ml), and dynamin (5 μg) as indicated for the ability to support avidin inaccessibility (b) or MesNa resistance (c) of B-SS-Tfn. Results are expresssed as the mean ± SEM of three experiments each performed in duplicate. Permeabilized cell membranes were preincubated in the presence or absence of GST–amph2 SH3D36R (0.1 mg/ml) for 5 min. They were then collected by centrifugation and assayed in the presence of ATP, cytosol (2.5 mg/ml), and synaptojanin (5 μg) as indicated for the ability to support avidin inaccessibility (d) or MesNa resistance (e) of B-SS-Tfn. Results are expresssed as the mean ± SD of two experiments each performed in duplicate. (f) Permeabilized membranes which had been preincubated in the presence of GST–amph2 SH3D36R and then subsequently incubated for 30 min in the presence of ATP and cytosol (lanes 1–3), plus dynamin (5 μg; lane 2), plus synaptojanin (5 μg; lane 3) were solubilized and electrophoresed on a 10% SDS gel, transferred to nitrocellulose, and probed for the presence of GST–amph2 SH3D36R using anti-GST antibodies.
Mentions: The major binding partners for the SH3 domain of amphiphysin are dynamin and the inositol 5-phosphatase, synaptojanin (David et al. 1996; Micheva et al. 1997; Wigge et al. 1997) (see Fig. 1b and Fig. c). We wanted to investigate whether the inhibitory effects of the SH3 domain were specific either for dynamin or synaptojanin. Therefore, we investigated whether purified dynamin and synaptojanin could rescue the membranes which had been preincubated with GST–amph2 SH3D36R. Dynamin was purified from rat brain and synaptojanin was purified after overexpression in HEK293 cells using the wild-type GST–amph2 SH3 fusion protein as an affinity ligand (Stowell et al. 1999). Purified dynamin (Fig. 2 a) could restore the ability of the preincubated membranes to support ligand sequestration into deeply invaginated coated pits (Fig. 2 b). Strikingly, however, dynamin was unable to rescue the inhibitory effects of GST–amph2 SH3D36R in the MesNa resistance assay (Fig. 2 c). By contrast, purified synaptojanin was unable to rescue the inhibitory effects of GST–amph2 SH3D36R in either the avidin inaccessibility or MesNa resistance assays (Fig. 2d and Fig. e). To assess whether dynamin effected rescue by competing the SH3 domain of amphiphysin from the membrane, membranes which had been treated with GST–amph2 SH3D36R were incubated in the presence of cytosol alone or cytosol plus dynamin or synaptojanin. They were then Western blotted and probed with an antibody raised against GST. In the presence of dynamin or synaptojanin there was no reduction in the amount of GST–amph2 SH3D36R associated with the membrane (Fig. 2 f). This shows that when dynamin rescues pretreated membranes, it is not acting simply to displace GST–amph2 SH3D36R from the membranes. The inability of dynamin to restore MesNa resistance may be due to depletion of another protein by the SH3 domain of amphiphysin, but it could equally well be due to disruption of the sequential order and timing of events during endocytic vesicle formation.

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