the strain-specific AGA effect was much larger
than any other effect, which demonstrated that
the increase in ribosome pausing during translation in the B6J and B6J-nmf205–/– cerebellum
occurs specifically at AGA codons (Fig. 4G).
Our data demonstrate that loss of function of a
nuclear encoded tRNA induces ribosome stalling
that is normally resolved by GTPBP2 (fig. S17).
Note that Hbs1l, another ubiquitously expressed
Pelota-binding partner, does not rescue neurodegeneration in the absence of GTPBP2, which
is consistent with nonoverlapping functions of
these proteins (fig. S4B) (19). In addition, the
tissue-specific expression of n-Tr20 suggests
that the regulation of individual isodecoder tRNAs
may enable translational regulation in mammals.
Further, our finding of a pathogenic mutation in
one isodecoder of a five-member gene family underlines the possible deleterious consequences of
epistatic mutations in individual members of cytoplasmic tRNA genes that could affect the readout
of other mutations, including synonymous SNPs.
Finally, these data also emphasize the potential for
regulation and disease of mutations in individual
members of multicopy gene families.
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Data were deposited in GenBank (GSE56127). We thank The Jackson
Laboratory sequencing, histology, microinjection, and multimedia
services for their assistance. We also thank K. Brown for mouse
husbandry assistance and W. Frankel for comments on the manuscript.
This work was supported in part by an institutional CORE grant
CA34196 (JAX) and a National Science Foundation Award 0850155 to
J.H.C. as part of the American Recovery and Reinvestment Act. S. L.A. is
an investigator of the Howard Hughes Medical Institute.
Materials and Methods
Figs. S1 to S17
Tables S1 to S4
16 December 2013; accepted 5 June 2014
AP2 controls clathrin polymerization
with a membrane-activated switch
Bernard T. Kelly,1 Stephen C. Graham,2 Nicole Liska,1 Philip N. Dannhauser,3
Stefan Höning,4 Ernst J. Ungewickell,3 David J. Owen1*
Clathrin-mediated endocytosis (CME) is vital for the internalization of most cell-surface
proteins. In CME, plasma membrane–binding clathrin adaptors recruit and polymerize clathrin
to form clathrin-coated pits into which cargo is sorted. Assembly polypeptide 2 (AP2) is the
most abundant adaptor and is pivotal to CME. Here, we determined a structure of AP2 that
includes the clathrin-binding b2 hinge and developed an AP2-dependent budding assay. Our
findings suggest that an autoinhibitory mechanism prevents clathrin recruitment by cytosolic
AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide
phosphatidylinositol 4,5-bisphosphate and cargo relieves this autoinhibition, triggering clathrin
recruitment and hence clathrin-coated bud formation. This molecular switching mechanism can
couple AP2’s membrane recruitment to its key functions of cargo and clathrin binding.
Clathrin adaptors provide an essential phy- sical bridge connecting clathrin, which itself lacks membrane binding activity (1), to the membrane and to embedded trans- membrane protein cargo. A central player
in clathrin-mediated endocytosis (CME) is the
AP2 (assembly polypeptide 2) complex (Fig. 1A
and fig. S1), which both coordinates clathrin-coated pit (CCP) formation and binds the many
cargo proteins that contain acidic dileucine and
Yxxf endocytic motifs (Y denotes Tyr; x, any
amino acid; and f, a bulky hydrophobic residue)
through its membrane-proximal core (2, 3). Cargo
binding is activated by a large-scale conformational change from the “locked” or inactive cytosolic form to an “open” or active form driven by
localization to membranes containing the plasma
membrane phosphoinositide phosphatidylinositol
4,5-bisphosphate [PtdIns(4,5)P2] (4, 5). The C-terminal “appendages” of the a and b2 subunits
bind other clathrin adaptors as well as CCV (
clathrin-coated vesicle) assembly and disassembly accessory factors (3, 6–8). The flexible hinge separating
the b2 appendage from the b2 trunk binds the N-terminal b-propeller of the clathrin heavy chain
by using a canonical clathrin box motif [LLNLD;
L, Leu; N, Asn; D, Asp (Fig. 1, A and B) (9)]. The
b2 appendage domain also binds clathrin, albeit
weakly, but both interactions are necessary for
robust clathrin binding (10).
A version of AP2 comprising full-length b2, m2,
and s2 subunits and the a trunk domain (FLb.
AP2) (Fig. 1B) (11) was expressed in Escherichia
coli, avoiding contamination with other CCV com-
ponents inherent to purification from brain tis-
sue (12, 13). Despite most FLb.AP2 possessing an
intact b2 subunit (Fig. 1, C to E), it bound clathrin
very poorly in pulldowns when immobilized ei-
ther on glutathione sepharose beads (Fig. 1C) or
via its N-terminal His6 tag [similarly positioned
to the b2 PtdIns(4,5)P2 binding site (Fig. 1B) (4, 5)]
to liposomes containing the nickel-attached nitri-
lotriacetic acid–dioleoylgycerosuccinyl (NiNTA-
DGS) (Fig. 1E): In both cases, the FLb.AP2 will
be in its locked cytosolic conformation (4). FLb.
AP2 also failed to stimulate clathrin cage assem-
bly efficiently at physiological pH (Fig. 1D). In
contrast, the isolated b2 hinge-appendage [glu-
tathione S-transferase (GST)-b2-h+app (fig. S1)]
bound clathrin efficiently (Fig. 1C) and stimu-
lated cage assembly (Fig. 1D). We next compared
clathrin recruitment to synthetic liposomes com-
posed of dioleoylphosphatidylcholine and dio-
either with NiNTA-DGS or with a mixture of
PtdIns(4,5)P2 and a lipid-linked YxxF endocytic
motif (5, 11, 14). b2-h+app fused to His6-tagged
epsin N-terminal homology (ENTH) domain (His6-
ENTH-b2-h+app), which can bind NiNTA-DGS or
PtdIns(4,5)P2, recruited clathrin efficiently to
both types of liposomes. In contrast, FLb.AP2 re-
cruited clathrin only when bound to PtdIns(4,5)P2-
and YxxF-containing liposomes (Fig. 1E). Thus,
no additional proteins are required to prevent
clathrin binding to AP2 in solution, consistent
with immunoprecipitation data (15). We con-
clude that the clathrin-binding activity of AP2 is
autoinhibited in the cytosol to restrict inappro-
priate clathrin recruitment and that only upon
encountering its physiological membrane lig-
ands [PtdIns(4,5)P2 and cargo] can AP2 recruit
clathrin efficiently. Previous reports that AP2
purified from brain could bind and polymerize
clathrin (12) were likely due to other contaminating
clathrin adaptors, such as AP180 (13).
We were unable to crystallize FLb.AP2, so we
determined the structure of a form of AP2
(bhingeHis6.AP2) whose b2 (residues 1 to 650)
includes the clathrin box–containing hinge but
not the b2 appendage. The structure closely
1Cambridge Institute for Medical Research (CIMR), Department
of Clinical Biochemistry, University of Cambridge, Hills Road,
Cambridge CB2 0XY, UK. 2Department of Pathology, University
of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
3Department of Cell Biology, Center of Anatomy, Hannover
Medical School, Carl-Neuberg Strasse 1, D-30625 Hannover,
Germany. 4Institute of Biochemistry I and Center for Molecular
Medicine Cologne, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany.
*Corresponding author. E-mail: email@example.com (B. T.K.);