PtdIns(4,5)P2-activated and cargo-stabilized AP2
is sufficient to drive bud formation on liposomes,
these data suggest that in vivo the dominant
mechanism for endocytic CCP initiation will be
the recruitment of AP2 to PtdIns(4,5)P2-enriched
sites in its locked form with its clathrin binding
autoinhibited, followed by transition to the open
form if sufficient PtdIns(4,5)P2 is present (5, 19).
AP2’s conformational change will expel the b2
hinge, allowing clathrin triskelia to bind. The
presence of cargo will stabilize AP2’s open form
and increase its dwell time on the membrane
(5, 14, 19, 22), thus increasing the chances of it
binding clathrin and forming a sufficiently stable
nucleating structure. Once a small nucleus of AP2
and clathrin has formed, further AP2, clathrin, and
other clathrin adaptors that bind the a appendage
can then be recruited in random order to produce
a CCP, which can ultimately be severed from the
membrane (3, 23, 24).
REFERENCES AND NOTES
1. P. N. Dannhauser, E. J. Ungewickell, Nat. Cell Biol. 14, 634–639 (2012).
2. E. Cocucci, F. Aguet, S. Boulant, T. Kirchhausen, Cell 150,
3. L. M. Traub, PLOS Biol. 9, e1001037 (2011).
4. B. M. Collins, A. J. McCoy, H. M. Kent, P. R. Evans, D. J. Owen,
Cell 109, 523–535 (2002).
462 25 JULY 2014 • VOL 345 ISSUE 6195
Fig. 4. AP2 and clathrin are sufficient to generate clathrin-coated buds
on membranes. (A) Clathrin recruitment to liposomes. Synthetic liposomes
supplemented with PtdIns(4,5)P2, or with PtdIns(4,5)P2 and TGN38 peptide
(as indicated), were incubated with adaptor and clathrin as shown (both at
0.4 mM); supernatants (s) and pellets (p) were then separated and analyzed by
gel electrophoresis. (B) Liposomes supplemented with PtdIns(4,5)P2 and lipid-
linked TGN38 internalization were incubated sequentially with FLb.AP2 and
clathrin and analyzed by negative stain EM, showing clathrin-coated membrane
buds (arrowheads). (C) Mean number of buds formed per mm2 of membrane,
estimated for various combinations of lipid and adaptor. Note that no buds
were found on PtdIns(4,5)P2 liposomes with or without TGN38 incubated
sequentially with the AP2 core and clathrin or with clathrin only (fig. S9). Error
bars indicate SEM, three experiments. (D and E) Liposomes supplemented with
PtdIns(4,5)P2 and lipid-linked TGN38 internalization incubated sequentially with
FLb.AP2 and clathrin and analyzed by ultrathin sectioning. Arrowheads indicate
examples of clathrin-coated structures; arrows indicate coated buds where the
connection to the liposome is visible. (E) shows an enlarged image of a bud
showing the “neck.” (F) Examples of liposomes supplemented with the lipids
indicated [PtdIns(4,5)P2 or NiNTA-DGS] incubated sequentially with adaptors
(as indicated) and clathrin, examined by negative stain EM. Some buds produced
by FLb.AP2 on PtdIns(4,5)P2-only liposomes seemed less invaginated than those
shown here; this can be seen more clearly in ultrathin sections (fig. S10).