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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 endophilin 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 were measured using the avidin internalization assay in the presence of the indicated concentrations of GST fusion proteins containing GST alone (filled circles) or GST–endoSH3 (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 the GST–endoSH3 fusion proteins 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–endoSH3 beads. (c) Western blot of cytosol after treatment with GST–endoSH3. 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. (c) 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–endoSH3 (filled diamonds). (e and f) Permeabilized cells were preincubated with GST (filled circles) or GST–endoSH3 (filled squares) at the indicated concentrations for 5 min at 30°C. The membranes were then pelleted by centrifugation and assayed either for avidin inaccessibility (e) or MesNa resistance (f) of B-SS-Tfn in the presence of cytosol (2.5 mg/ml) and ATP.
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Figure 5: The SH3 domain of endophilin 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 were measured using the avidin internalization assay in the presence of the indicated concentrations of GST fusion proteins containing GST alone (filled circles) or GST–endoSH3 (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 the GST–endoSH3 fusion proteins 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–endoSH3 beads. (c) Western blot of cytosol after treatment with GST–endoSH3. 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. (c) 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–endoSH3 (filled diamonds). (e and f) Permeabilized cells were preincubated with GST (filled circles) or GST–endoSH3 (filled squares) at the indicated concentrations for 5 min at 30°C. The membranes were then pelleted by centrifugation and assayed either for avidin inaccessibility (e) or MesNa resistance (f) of B-SS-Tfn in the presence of cytosol (2.5 mg/ml) and ATP.

Mentions: The SH3 domain of endophilin (GST–endoSH3) also binds dynamin, although synaptojanin is its major binding partner (Micheva et al. 1997; Ringstad et al. 1997). Given the specificity of the inhibition of GST–amph2 SH3D36R on invagination, we were interested to see if the SH3 domain of endophilin might also reveal some of the molecular and temporal requirements for invagination and scission. Addition of the SH3 domain of endophilin resulted in a dose-dependent inhibition of both ligand sequestration and internalization (Fig. 5 a). As with the SH3 domain of amphiphysin, we were interested to see if the target of the SH3 domain of endophilin was at the membrane or in the cytosol. The SH3 domain of endophilin can effectively deplete both synaptojanin and, to a lesser extent, dynamin, from cytosol (Fig. 5b and Fig. c). Mock-treated and GST–endoSH3-depleted cytosols were equally efficient at supporting invagination and scission as measured by the avidin inaccessibility and MesNa resistance assays (Fig. 5 c) as was untreated cytosol (data not shown). Similar to the effect of the SH3 domain of amphiphysin, preincubation of the membranes with the SH3 domain of endophilin resulted in a complete inhibition of both transferrin sequestration and internalization (Fig. 5d and Fig. e). However, in contrast to the inhibitory effect of the SH3 domain of amphiphysin, it was not possible to rescue the inhibitory effects of the SH3 domain of endophilin with purified dynamin even at high concentrations (Fig. 6 a; compare with Fig. 2b and Fig. c). Purified synaptojanin was also unable to rescue the inhibitory effects of GST–endoSH3 in either the avidin inaccessibility or MesNa resistance assay (Fig. 6b and Fig. c). Western blot analysis of membranes pretreated with GST–endoSH3 showed that subsequent incubation in the presence of cytosol and either dynamin or synaptojanin had no effect on the amount of SH3 domain associated with the membranes compared with the amount associated with cytosol alone (Fig. 6 d).


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 endophilin 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 were measured using the avidin internalization assay in the presence of the indicated concentrations of GST fusion proteins containing GST alone (filled circles) or GST–endoSH3 (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 the GST–endoSH3 fusion proteins 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–endoSH3 beads. (c) Western blot of cytosol after treatment with GST–endoSH3. 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. (c) 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–endoSH3 (filled diamonds). (e and f) Permeabilized cells were preincubated with GST (filled circles) or GST–endoSH3 (filled squares) at the indicated concentrations for 5 min at 30°C. The membranes were then pelleted by centrifugation and assayed either for avidin inaccessibility (e) or MesNa resistance (f) of B-SS-Tfn in the presence of cytosol (2.5 mg/ml) and ATP.
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Related In: Results  -  Collection

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Figure 5: The SH3 domain of endophilin 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 were measured using the avidin internalization assay in the presence of the indicated concentrations of GST fusion proteins containing GST alone (filled circles) or GST–endoSH3 (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 the GST–endoSH3 fusion proteins 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–endoSH3 beads. (c) Western blot of cytosol after treatment with GST–endoSH3. 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. (c) 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–endoSH3 (filled diamonds). (e and f) Permeabilized cells were preincubated with GST (filled circles) or GST–endoSH3 (filled squares) at the indicated concentrations for 5 min at 30°C. The membranes were then pelleted by centrifugation and assayed either for avidin inaccessibility (e) or MesNa resistance (f) of B-SS-Tfn in the presence of cytosol (2.5 mg/ml) and ATP.
Mentions: The SH3 domain of endophilin (GST–endoSH3) also binds dynamin, although synaptojanin is its major binding partner (Micheva et al. 1997; Ringstad et al. 1997). Given the specificity of the inhibition of GST–amph2 SH3D36R on invagination, we were interested to see if the SH3 domain of endophilin might also reveal some of the molecular and temporal requirements for invagination and scission. Addition of the SH3 domain of endophilin resulted in a dose-dependent inhibition of both ligand sequestration and internalization (Fig. 5 a). As with the SH3 domain of amphiphysin, we were interested to see if the target of the SH3 domain of endophilin was at the membrane or in the cytosol. The SH3 domain of endophilin can effectively deplete both synaptojanin and, to a lesser extent, dynamin, from cytosol (Fig. 5b and Fig. c). Mock-treated and GST–endoSH3-depleted cytosols were equally efficient at supporting invagination and scission as measured by the avidin inaccessibility and MesNa resistance assays (Fig. 5 c) as was untreated cytosol (data not shown). Similar to the effect of the SH3 domain of amphiphysin, preincubation of the membranes with the SH3 domain of endophilin resulted in a complete inhibition of both transferrin sequestration and internalization (Fig. 5d and Fig. e). However, in contrast to the inhibitory effect of the SH3 domain of amphiphysin, it was not possible to rescue the inhibitory effects of the SH3 domain of endophilin with purified dynamin even at high concentrations (Fig. 6 a; compare with Fig. 2b and Fig. c). Purified synaptojanin was also unable to rescue the inhibitory effects of GST–endoSH3 in either the avidin inaccessibility or MesNa resistance assay (Fig. 6b and Fig. c). Western blot analysis of membranes pretreated with GST–endoSH3 showed that subsequent incubation in the presence of cytosol and either dynamin or synaptojanin had no effect on the amount of SH3 domain associated with the membranes compared with the amount associated with cytosol alone (Fig. 6 d).

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