Membrane Dynamics

How does the clathrin coated pit form?

Adaptor proteins such as AP-2, AP180 and CALM (Clathrin-assembly lymphoid myeloid leukaemia protein), which accumulate within the lipid bilayer, are responsible for the recruitment of the triskelion shaped Clathrin trimer. This trimer does not interact with the membrane directly but instead forms a reinforcing lattice structure that acts as a mold in which membrane vesicles may develop. Its influence on membrane curvature is via the adapter proteins which are anchored to the lipid bilayer. Importantly, the adaptor proteins also participate directly in membrane bending and vesicle size determination [1].

AP2 and BAR-domain proteins

The formation of clathrin-coated pits (CCPs) requires various actin-binding proteins such as those belonging to the BAR (Bin/Amphiphysin/Rvs) superfamily. These include amphiphysin and endophilin in mammals and Rvs161p and Rvs167p in yeast [2]. The role of these BAR proteins is in membrane deformation, essentially promoting its tubulation. By binding to negatively charged membranes, a positive curvature is obtained that follows the concave topology of the protein’s amphipathic α -helix dimer [3][4]. F-BAR proteins, which belong to a sub-family of the BAR superfamily, possess a larger domain that is also concave in shape, yet shallower in its curvature. These proteins are proposed to generate vesicles with a larger radius compared to those proteins with possessing a BAR domain [5][6]. In both cases the proteins may act as curvature sensors that reform the membrane into a shape to which they can readily bind [7]. In the case of clathrin mediated endocytosis, the F-BAR proteins are believed to arrive at the site of clathrin-coated pit formation, before the BAR proteins, and as such may also be involved in nucleation of the CCP [8].

While the BAR domain proteins facilitate tubulation of the membrane, the adaptor proteins including AP-2 or those that possess the epsin N-terminal homology (ENTH) domain such as epsin [9], or the AP-180 N-terminal homology (ANTH) domains such as AP-180 continue to recruit the clathrin triskelion and other regulatory proteins required for the later stages of clathrin-coated vesicle (CCV) formation. Both the ENTH and ANTH domains are highly homologous and bind inositol phospholipids; especially PIP2 [10]. Although both protein subclasses stimulate the formation of a clathrin triskelia network, only proteins possessing the ENTH domain influence membrane curvature, with the clathrin lattice produced by AP-180 stimulation having been shown to remain flat [36]. This influence from the ENTH domains is believed to result from the formation of an additional α –helix ‘α0’ between the ENTH domain and the PIP molecule [11]. It has been proposed that insertion of this domain between the lipid heads of the membrane bilayer may be sufficient to alter membrane curvature alone; however it may also be a synergistic response with clathrin assembly [10].

References

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11. Ford MGJ, Mills IG, Peter BJ, Vallis Y, Praefcke GJK, Evans PR, and McMahon HT. Curvature of clathrin-coated pits driven by epsin. Nature 2002; 419(6905):361-6. [PMID: 12353027]