Fig. 2. E/ANTH proteins function in CCP and CCV formation coupled to cycles of PtdIns(4,5)P₂ metabolism. (1) PtdIns(4,5)P₂ (black) is generated from PtdIns by the sequential actions of PtdIns 4-kinase type II and PtdIns 4-phosphate 5-kinase type I. ARF6-GTP can bind to and activate PtdIns 4-phosphate 5-kinase type I on the plasma membrane. (2) E/ANTH proteins bind to PtdIns(4,5)P₂ that may form in localized microdomains within the general pool of membrane phospholipids (light gray). The E/ANTH proteins in turn bind additional endocytic proteins. Other coat components including AP-2 can also bind directly to PtdIns(4,5)P₂. Transmembrane receptors, through specific internalization motifs in their cytoplasmic domains, bind to the AP-2 complex. These cooperative interactions lead to the nucleation of clathrin coats on the plasma membrane. (3) Through their C-terminal regions, several E/ANTH proteins stimulate clathrin assembly, leading to the formation of CCPs. -helix 0, formed at the N-terminus of the ENTH domain in response to phospholipid binding, inserts into the plasma membrane, leading to membrane deformation and curvature. E/ANTH proteins may also function to recruit cargo molecules to nascent CCPs through binding to their cytoplasmic tails. (4) CCVs pinch off from the plasma membrane in a dynamin-dependent mechanism. (5) The lipid phosphatase synaptojanin is recruited to CCVs, where it converts PtdIns(4,5)P₂ to PtdIns. This leads to destabilization of clathrin coats with Hsc70 and auxilin (not pictured) driving coat disassembly.