An Assembly-incompetent Mutant Establishes a Requirement for Dynamin Self-assembly in Clathrin-mediated Endocytosis In Vivo

Abstract

Dynamin GTPase activity is required for its biological function in clathrin-mediated endocytosis; however, the role of self-assembly has not been unambiguously established. Indeed, overexpression of a dynamin mutant, Dyn1-K694A, with impaired ability to self-assemble has been shown to stimulate endocytosis in HeLa cells (Sever et al., Nature 1999, 398, 481). To identify new, assembly-incompetent mutants of dynamin 1, we made point mutations in the GTPase effector/assembly domain (GED) and tested for their effects on self-assembly and clathrin-mediated endocytosis. Mutation of three residues, I690, K694, and I697, suggests that interactions with an amphipathic helix in GED are required for self-assembly. In particular, Dyn1-I690K failed to exhibit detectable assembly-stimulated GTPase activity under all assay conditions. Overexpression of this assembly-incompetent mutant inhibited transferrin endocytosis as potently as the GTPase-defective dominant-negative mutant, Dyn1-K44A. However, worm-like endocytic intermediates accumulated in cells expressing Dyn1-I690K that were structurally distinct from long tubules that accumulated in cells expressing Dyn1-K44A. Together these results provide new structural insight into the role of GED in self-assembly and assembly-stimulated GTPase activity and establish that dynamin self-assembly is essential for clathrin-mediated endocytosis.

SNX9 Regulates Dynamin Assembly and Is Required for Efficient Clathrin-mediated Endocytosis

“…SH3(SNX9) cDNA was amplified by PCR and subcloned into the BamHI/ EcoRI sites of pGEX.KG. C-terminally-tagged dynamin-1 mRFP (designated pmRFP-N1-dyn 1) was generated by replacing EGFP with mRFP in pEGFP-N1-dyn 1 (Song et al, 2004) using AgeI and NotI.…”

“…Wild-type and mutant dynamins were expressed and purified as previously described (Song et al, 2004). The human amphiphysin-I GST fusion construct (GST-amph1) was obtained from Pietro DeCamilli (Yale University) and purified using GST-Sepharose by standard protocols.…”

“…Dynamin self-assembles into a collar-like structure around the necks of deeply invaginated clathrin-coated pits (CCPs) where it is believed to directly mediate membrane fission and vesicle release. Dynamin is also associated with newly formed coated pits (Damke et al, 1994;Evergren et al, 2004) where it may function to regulate early stages of coated pit maturation (Sever et al, 2000a;Song and Schmid, 2003;Song et al, 2004).Although only one isoform of dynamin exists in Drosophila and in Caenorhabditis elegans, mammals express at least three isoforms, which are ϳ70% identical to each other. Dynamin-1, the first identified protein in the dynamin family, is expressed exclusively in neuronal cells where it functions in synaptic vesicle recycling (Shpetner and Vallee, 1989;Nakata et al, 1991;Sontag et al, 1994).…”

“…SH3(SNX9) cDNA was amplified by PCR and subcloned into the BamHI/ EcoRI sites of pGEX.KG. C-terminally-tagged dynamin-1 mRFP (designated pmRFP-N1-dyn 1) was generated by replacing EGFP with mRFP in pEGFP-N1-dyn 1 (Song et al, 2004) using AgeI and NotI.For generation of recombinant adenoviruses, wild-type HA-SNX9 cDNA was cloned into pADT3T7tet vector. HEK293-Cre cells were transfected with pADTetT3T7 vectors and adenoviruses were produced as described (Hardy et al, 1997).…”

Chapter 13 Visualization of Dynamins

“…1). Each domain is required for self-assembly of dynamins into functional, oligomeric structures (Ingerman et al, 2005;Ramachandran et al, 2006;Smirnova et al, 1999;Song et al, 2004). In addition to these conserved motifs, dynamins contain other functional domains specific to the cellular mechanism associated with each protein (Fig.…”