Practice question

Final separation stage clathrin-coated pit formation yielding free vesicle requires enzymatic fission beyond coat polymerization. Clathrin assembly with adaptor AP2 and BAR proteins epsin, amphiphysin, endophilin generates deeply invaginated bud connected plasma membrane narrow tubule neck diameter around 20 nm bilayer. Separation demands mechanical constriction cutting neck. Specialist protein dynamin large 96 kDa GTPase family founding member N-terminal GTPase domain, middle stalk, PH domain binding PI(4,5)P2 enriched site, GED and PRD binding SH3, polymerizes helical collar around neck. Recruitment mediated curvature-sensing BAR proteins generating curvature. Stoichiometry about 13 dimers per turn aligns G domains efficient hydrolysis. GTP binding tightens helix reducing lumen radius; cooperative hydrolysis drives constriction twisting shear breaking membrane hemi-fission intermediate releasing vesicle. Clathrin provides structural cage not enzymatic fission; Rab GTPases regulate downstream tethering early endosome via EEA1, SNAREs mediate subsequent fusion after uncoating. Temperature-sensitive shibire mutant drosophila dynamin blocks synaptic vesicle recycling restrictive temperature trapping pits long collars demonstrating indispensable scission function receptor-mediated endocytosis essential cellular uptake.