Fig 1: CCV uncoating revealed that the vATPase is blocked by clathrin coat.(A) Electron micrographs of negatively stained CCVs before and after uncoating with 300 mM Tris-buffer pH 9.0. (B) Western blot of CCVs, uncoated vesicles and supernatant after uncoating shows dissociation of clathrin LC and HC, as well as AP180 and AP2, from the uncoated vesicle (proteins were separated at the 10% gel and detected by chemiluminescence). (C–D) Membrane potential (C) and luminal pH (D) of acidified SVs and CCVs before and after treatment with Tris-buffer (pH 9.0). Error bars represent SD of 3–4 experimental replicates done on over 1000 vesicles each. (E) Membrane potential of acidified CCVs after treatment with 300 mM Tris-buffer (pH 9.0), 500 mM Tris buffer (pH 7.0) and ‘enzymatic’ treatment with 1.7 µg auxilin, 4.8 µg Hsc70 and 1.7 µg Hsp110 proteins. Error bars represent SD of 3 experimental replicates done on over 1000 vesicles each. (F–G) Model of vATPase block by clathin coat: solved structures of vATPase, clathrin tripods and AP2 complex were used to check how vATPase fits within the clathrin lattice. The plasma membrane is depicted in light beige, clathrin triskelia in dark beige/brown; vATPase complex in gray (when inactive), light blue (when active) and dark green (V1H-subunit); AP2 complex in purple/blue/light green. As clathrin triskelia are recruited (through AP2), clathrin ring starts building around the vATPase complex. Insertion of the last triskelion of the clathrin ring would collide with the regulatory V1H-subunit of vATPase (G), thus we hypothesize that the displacement of regulatory V1H-subunit inwards results in the block of the vATPase activity. For more details, see Suppl. Data.