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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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GTP binding and hydrolysis are required for the late stages of coated pit invagination. (a) Permeabilized cells were preincubated with GST–amph2 SH3D36R for 5 min at 30°C. The membranes were collected by centrifugation and assayed for the ability to support B-SS-Tfn sequestration using the avidin inaccessibility assay in the presence of cytosol (2.5 mg/ml) and 5 μg of wild-type (WT), S45N, K44A, T65A, or rat brain (RB) dynamin as indicated. Each point represents the mean ± SD of two experiments each performed in duplicate. (b) Internalization of B-SS-Tfn in HEK293 cells transfected with wild-type, K44A, or T65A dynamin was measured using the avidin inaccessibility or MesNa resistance assays as described in Materials and Methods. Results are from a representative experiment where each point is the mean of duplicates where the range is <10%. The insert shows a Western blot probed with antidynamin antibody showing equivalent protein loadings of HEK293 cells which were mock-transfected (lane 1) or transfected with wild-type, dynamin (lane 2), T65A dynamin (lane 3), or K44A dynamin (lane 4).
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Figure 3: GTP binding and hydrolysis are required for the late stages of coated pit invagination. (a) Permeabilized cells were preincubated with GST–amph2 SH3D36R for 5 min at 30°C. The membranes were collected by centrifugation and assayed for the ability to support B-SS-Tfn sequestration using the avidin inaccessibility assay in the presence of cytosol (2.5 mg/ml) and 5 μg of wild-type (WT), S45N, K44A, T65A, or rat brain (RB) dynamin as indicated. Each point represents the mean ± SD of two experiments each performed in duplicate. (b) Internalization of B-SS-Tfn in HEK293 cells transfected with wild-type, K44A, or T65A dynamin was measured using the avidin inaccessibility or MesNa resistance assays as described in Materials and Methods. Results are from a representative experiment where each point is the mean of duplicates where the range is <10%. The insert shows a Western blot probed with antidynamin antibody showing equivalent protein loadings of HEK293 cells which were mock-transfected (lane 1) or transfected with wild-type, dynamin (lane 2), T65A dynamin (lane 3), or K44A dynamin (lane 4).

Mentions: Dynamin was purified from frozen rat brains (obtained from Harlan Sera-Lab) as described (Stowell et al. 1999). Purified dynamin was dialyzed overnight with three further changes of buffer into KSHM buffer (100 mM potassium acetate, 85 mM sucrose, 20 mM Hepes, 1 mM magnesium chloride, pH 7.4). Dynamin was determined to be >95% pure by Coomassie gel analysis (Fig. 3 a). For the preparation of wild-type and mutant dynamin, human embryonic kidney (HEK)293 cells were transfected with pCMV encoding untagged wild-type or mutant bovine dynamin1 (a generous gift from Harvey McMahon), using calcium phosphate (Sambrook et al. 1989). Cells were harvested 48 h after transfection and dynamin purified as described (Stowell et al. 1999). Dynamin T65A was generated using the Stratagene QuikChange site-directed mutagenesis kit according to the manufacturer's instructions. The sequence of the mutated protein was verified using an ABI automated DNA sequencer (PE Biosystems). Dynamin GTPase assays were performed according to the method of Gout et al. 1993 and Warnock et al. 1996. Synaptojanin was prepared by expressing a cDNA encoding synaptojanin in pEF Bos (a generous gift from Harvey McMahon) in HEK293 cells. The protein was purified using GST–amph2 SH3D36R as an affinity ligand as described for dynamin (Stowell et al. 1999).


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)

GTP binding and hydrolysis are required for the late stages of coated pit invagination. (a) Permeabilized cells were preincubated with GST–amph2 SH3D36R for 5 min at 30°C. The membranes were collected by centrifugation and assayed for the ability to support B-SS-Tfn sequestration using the avidin inaccessibility assay in the presence of cytosol (2.5 mg/ml) and 5 μg of wild-type (WT), S45N, K44A, T65A, or rat brain (RB) dynamin as indicated. Each point represents the mean ± SD of two experiments each performed in duplicate. (b) Internalization of B-SS-Tfn in HEK293 cells transfected with wild-type, K44A, or T65A dynamin was measured using the avidin inaccessibility or MesNa resistance assays as described in Materials and Methods. Results are from a representative experiment where each point is the mean of duplicates where the range is <10%. The insert shows a Western blot probed with antidynamin antibody showing equivalent protein loadings of HEK293 cells which were mock-transfected (lane 1) or transfected with wild-type, dynamin (lane 2), T65A dynamin (lane 3), or K44A dynamin (lane 4).
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Related In: Results  -  Collection

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Figure 3: GTP binding and hydrolysis are required for the late stages of coated pit invagination. (a) Permeabilized cells were preincubated with GST–amph2 SH3D36R for 5 min at 30°C. The membranes were collected by centrifugation and assayed for the ability to support B-SS-Tfn sequestration using the avidin inaccessibility assay in the presence of cytosol (2.5 mg/ml) and 5 μg of wild-type (WT), S45N, K44A, T65A, or rat brain (RB) dynamin as indicated. Each point represents the mean ± SD of two experiments each performed in duplicate. (b) Internalization of B-SS-Tfn in HEK293 cells transfected with wild-type, K44A, or T65A dynamin was measured using the avidin inaccessibility or MesNa resistance assays as described in Materials and Methods. Results are from a representative experiment where each point is the mean of duplicates where the range is <10%. The insert shows a Western blot probed with antidynamin antibody showing equivalent protein loadings of HEK293 cells which were mock-transfected (lane 1) or transfected with wild-type, dynamin (lane 2), T65A dynamin (lane 3), or K44A dynamin (lane 4).
Mentions: Dynamin was purified from frozen rat brains (obtained from Harlan Sera-Lab) as described (Stowell et al. 1999). Purified dynamin was dialyzed overnight with three further changes of buffer into KSHM buffer (100 mM potassium acetate, 85 mM sucrose, 20 mM Hepes, 1 mM magnesium chloride, pH 7.4). Dynamin was determined to be >95% pure by Coomassie gel analysis (Fig. 3 a). For the preparation of wild-type and mutant dynamin, human embryonic kidney (HEK)293 cells were transfected with pCMV encoding untagged wild-type or mutant bovine dynamin1 (a generous gift from Harvey McMahon), using calcium phosphate (Sambrook et al. 1989). Cells were harvested 48 h after transfection and dynamin purified as described (Stowell et al. 1999). Dynamin T65A was generated using the Stratagene QuikChange site-directed mutagenesis kit according to the manufacturer's instructions. The sequence of the mutated protein was verified using an ABI automated DNA sequencer (PE Biosystems). Dynamin GTPase assays were performed according to the method of Gout et al. 1993 and Warnock et al. 1996. Synaptojanin was prepared by expressing a cDNA encoding synaptojanin in pEF Bos (a generous gift from Harvey McMahon) in HEK293 cells. The protein was purified using GST–amph2 SH3D36R as an affinity ligand as described for dynamin (Stowell et al. 1999).

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