The pleckstrin homology domains of dynamin isoforms require oligomerization for high affinity phosphoinositide binding.
The dynamins are 100-kDa GTPases involved in the scission event required for formation of endocytotic vesicles. The two main described mammalian dynamins (dynamin-1 and dynamin-2) both contain a pleckstrin homology (PH) domain, which has been implicated in dynamin binding to (and activation by) acidic phospholipids, most notably phosphoinositides. We demonstrate that the PH domains of both dynamin isoforms require oligomerization for high affinity phosphoinositide binding. Strong phosphoinositide binding was detected only when the PH domains were dimerized by fusion to glutathione S-transferase, or via a single engineered intermolecular disulfide bond. Phosphoinositide binding specificities agreed reasonably with reported effects of different phospholipids on dynamin GTPase activity. Although they differ in their ability to inhibit rapid endocytosis in adrenal chromaffin cells, the dynamin-1 and dynamin-2 PH domains showed identical phosphoinositide binding specificities. Since oligomerization is required for binding of the dynamin PH domain to phosphoinositides, it follows that PH domain-mediated phosphoinositide binding will favor oligomerization of intact dynamin (which has an inherent tendency to self-associate). We propose that the dynamin PH domain thus mediates the observed cooperative binding of dynamin to membranes containing acidic phospholipids and promotes the self-assembly that is critical for both stimulation of its GTPase activity and its ability to achieve membrane scission.
Department of Biochemistry and Biophysics and Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6089, USA.
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Klein
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Lee
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Frank
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Marks
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Lemmon
D000595:Amino Acid Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D034284:Dynamin I; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001798:Blood Proteins; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D005982:Glutathione Transferase; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins