Practice question

Coat assembly regulated spatiotemporally to occur only at correct donor membranes and when cargo present ensuring energy not wasted. Initiating event activation of small GTPases Sar1 and ARF families from GDP cytosolic to GTP membrane state. Sar1 activated by Sec12 GEF integral ER protein exchanges GDP for GTP exposing amphipathic helix embedding ER leaflet inducing curvature recruiting Sec23-Sec24 heterodimer. Similarly ARF1 activated by GBF1 at ERGIC cis-Golgi and BIG1/2 at TGN upon membrane recruitment exposes myristoylated helix plus effector binding switch regions recruiting COPI coatomer heptamer or AP1/GGA clathrin adaptors beginning lattice nucleation. GTPases serve as membrane-anchored nucleation points; GDP form cytosolic inactive unable to bind coats. Direct clathrin triskelia binding membrane without adaptor or GTPase does not occur physiologically. ATP hydrolysis by NSF drives cis-SNARE disassembly post-fusion not coat initiation. Cargo phosphorylation may modulate adaptor affinity via casein kinase but nucleation itself requires active GTPase switch explaining sensitivity to fungal metabolite brefeldin A stabilizing abortive ARF-GDP-GEF complex and to non-hydrolyzable GTPgammaS locking coats on membranes preventing recycling and fusion competence.