Inner layer : membrane containing cargo

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Clathrin-Coated Vesicles
J. Heuser
Inner layer membrane containing cargo Middle
layer adaptors and accessory proteins Outer
layer mechanical scaffold
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CCV formation at the plasma membrane
John Heuser
Mark Bretscher
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AP Complexes
Family of 300kDa heterotetrameric protein
complexes (AP1, AP2, AP3, AP4, bgdz COP) play
central role in vesicle trafficking events
AP2 is a major component of endocytic
CCVs Large subunits (110-120kDa) a and
b2 -subdivided into trunk, linker and
appendage Medium subunit (50kDa) m2 Small subunit
(17kDa) s2
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AP2 function
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AP2 appendages
b2 appendage
a appendage
C-terminal 30kDa of large a and b2 subunits
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AP2 appendages bind DFF motifs in a single
hydrophobic pocket.
b2-appendage
a-appendage
DFF motifs are found in proteins with many
different accessory and regulatory roles in CCV
formation including amphiphysin, CALM, epsin,
eps15, GAK/AAK and synaptojanin
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m2
The
The 30kDa C-terminal signal- binding domain of m2
binds YxxF signals The motif forms an extra
strand on a pre-existing b-sheet The Y and F
side chains fit into Chemically compatible pockets
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Y and F residues fit in compatible pockets
W421
R423
D176
Y
F
Y2
W421
Y2
Y site
F site
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A model for AP2
The structures and the molecular basis of of the
functions of various domains have been
determined..but... How do the subunits interact
with each other in the whole complex? How does
the complex interact with cargo that is embedded
in a phospholipid membrane? What is the
molecular mechanism by which the complexs many
functions are regulated?
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Structure of AP2 core
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a b2 m2 s2
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Assembly of the AP2 core
a b2 m2 s2
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The AP2 Complex is composed of hetero-dimers
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Membrane phospholipid binding
PI(3,4,5)P3
The various membranes in the cell have specific
phosphatidylinositol polyphosphate
composition. AP2 binds PIP2 and PIP3 on the
N-terminus of the a-trunk domain. PIP2/PIP3
binding by the a-trunk domain is a major
factor in AP2 localisation/recruitment
PIP3
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Binding of IP6 by m2
IP6 binds to a patch of positive electrostatic
potential Site demonstrated in vivo and in vitro
to bind PIP2 (V. Haucke)
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YxxF motif binding
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The YxxF motif binding site on m2 is blocked by
b2
b2Y405
m2
YxxF peptide binding site
b2 V365
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YxxF motif binding site
R423
R423
D176
D176
W421
W421
Peptide binding does not change the conformation
of m2 The Y site is pre-formed and empty but the
F site is blocked
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Model of AP2 function
Binding site for YxxF motifs is blocked and in
the wrong orientation with respect to the
membrane. There are two PIP2/PIP3 binding sites
located on perpendicular faces of the
complex. Phosphorylation of Thr156 in linker
joining the two domains of m2 increases binding
to signals 25 fold.
.a conformational change must occur
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m2T156 phosphorylation By AAK/GAK
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Conformational change in AP2
A conformational change should allow simultaneous
binding to YxxF containing cargo and both
PIP2/PIP3 binding sites.
The conformational change would be triggered by
AAK1 phosphorylation of Thr156 in the m2
linker and the presence of the charged, planar
plasma membrane containing high local
concentrations of PIP2/PIP3
During vesicle disassembly the process must be
reversed by dephosphorylation and breakdown of
PIP2/PIP3
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A model for whole AP2?
Central core and appendages are compact folded
domains. Appendages grab hold of
regulatory/accessory proteins. Linkers joining
the appendages are approximately 100 amino acids
long, proteolytically sensitive low
complexity regions. Linkers have little
secondary structure as determined by circular
dichroism (lt10 310 helix). Linkers could
therefore stretch 300-400Å from the core.
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or
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Fig. 4. Loosely connected domains. (a) A
representation of full-length epsin, showing the
folded ENTH domain at the bottom and an unfolded
C-terminal region containing multiple binding
sites for ubiquitin, adaptors (AP2), clathrin and
Eps15. (b) The entire heterotetrameric AP2
complex, compiled from crystal structures of the
core (trunk) 7 and the two appendage' domains
24 and 26. The loose linkers (hinges) attaching
the appendages are schematic. The superhelical
trunk domains of - and 2-adaptin wrap round the
small domains of 2-adaptin and the N-terminal
domain of 2-adaptin. The signal-binding site on
the C-terminal domain of 2-adaptin is blocked by
2-adaptin. The two InsP6 sites correspond to the
single site between molecules in the crystal.
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