sorting it all out
Coated pits and coated vesicles .Tomas Kirchhausen
Harvard
Medical
School
and Center
for Blood Research,
Boston,
USA.
Clathrin-coated
structures are involved
in the first steps of membrane
vesiculation
which lead to receptor
sorting and directed
traffic. This
review focuses on our current kderstanding
of clathrin and its associated
proteins, the major components
of the coat. Recent experiments
provide
new insights into the interactions
between these proteins.
Current
Opinion
in Structural
Introduction
Clathrin-coated pits and coated vesicles represent a major
pathway of directed membrane tralfic, and are important both in endocytosls and in regulated secretion via
the trans-Golgi network (for recent reviews, see [ 1,2]).
This is a fascinating and apparently basic cellular mechanism which is found in all nucleated eukaryotic cells. The
original electron microscopic observations of invaginated
areas of cell membrane surrounded by a dense ‘coat’
led Roth and Porter [3] to suggest, with remarkable
insight, that these ‘coated pits’ pinch off a section of
membrane, to form ‘coated vesicles’ which transport
specilic proteins. The broad outline of this hypothesis
is still accepted, but today, almost 30 years later, we
are only beginning to learn the details of how this cellular organelle functions. There has been considerable
progress in elucidating the biochemistry and molecular
biology of the clathrln coat, including the cloning of
clathrin itself and the major proteins associated with
isolated coated vesicles. It has proved dillicult to obtain interpretable data on the behavior of the associated
proteins, because these proteins tend to aggregate in physiological solutions. Yet the recent development of new
in vitro systems of clathrln assembly offers hope that new
insights may soon be forthcoming.
Structure
Biology
1993, 3:182-188
main’ [4]. Recent experiments with yeast have shown that
deletion of a poorly conserved 57 amino acid sequence
at the extreme carboxyl-terminal end of the heavy chain
does not prevent trimerlzation [5].
Light chains contain a. central domain of 10 heptads
repeats, characteristic of a helical coiled coil, which is
necessary for the interaction with heavy chain [6-8], and
were originally predicted to have an extended conformation [8] ; polyclonal antibodies to light chain bind along
most of the length of the proximal leg of heavy chain
(16 run) [9]. It has recently been proposed, on the basis
of antibody competition experiments, that the light chain
is instead bent through 180” in the middle of the central
domain, and should therefore extend along only 5-6 nm
of the proximal leg [lo]. We have reexamined this issue
using electron microscopy of epitope-tagged light chains,
and have found that, as predicted, the central domain
of the light chain extends along most of the heavychain proximal leg (12.2 f 4.4~1) [ll]. The carboxyl
terminus is located near the triskelion vertex whereas
the amino terminus is positioned 13.1 f 5.2 nm from the
vertex. The beginning and the end of the amino-terminal
domain are found in essentially the same place, indicating that it either folds back on itself or has a globular
conformation.
Structure
of clathrin
The main protein components found in the coat of
coated vesicles are clathrin and two associated protein
complexes (APs), AR-1 and AR-2. Clathrln comprises
three heavy chains and three light chains, arranged as
shown in Fig. 1. The three legs are extended and relatively stitf, and are held together by interactions towards
the carboxyl terminus of the heavy chain, at the vertex of
the triskelion-shaped molecule. The legs can be divided
into proximal and distal segments, the distal segment
ending with a globular structure called the ‘terminal do-
of the associated
@ Current
Biology
complexes
M-1 and AR-2 are related tetramers found in association with coated structures localized on the trans-Golgi
network and at the plasma membrane (Fig. 2; for a recent review, see [ 121). Each complex contains two large
chains (a and p for AR-2, p’ and y for AR-l), one medium
chain and one small chain. In vitro under physiological
conditions, where clathrin remains trlmeric, AR-1 or M-2
interact with clathrin and assemble with it to form small
coats. The stoichiomeny of the polypeptide chains in Aps
has been controversial, but evidence that each complex
conu%ins one medium and one small chain has recently
Abbreviation
AP-associated protein complex.
182
protein
Ud ISSN 0959-440X
Coated
and
Clathrin
Clathrin
coated
vesicles
Kirchhausen
coat
Arrii;~;art;inal
Central
domain
23-25
pits
/
Membrane
kUa
/
c
Distal
leg
4
Terminal
domain
coated
pit
Fig. 1. Representation
of the heavy and light chains
of a clathrin
trimer
free in solution
and within
the clathrin
lattice.
The clathrin
molecule,
which
is triskelion-shaped,
is not flat but has ‘pucker’
appropriate
to the vertices
and edges of a truncated
icosahedron,
which
is the most frequent
design of clathrin
lattice found in bovine
brain coated
vesicles
1601. Both classes of light chain (LCa and LCb)
bind to the same site on the heavy chain, extending
along the whole
length of the proximal
leg. The terminal
domains
project
towards
the membrane.
The APs are located
beneath
the clarhrin
edges.
been provided [IS]. All four large chains can be proteolytically cleaved into two domains, the ‘trunk (the
amino-terminal two thirds of each molecule) and the
‘ear’ (the carboxyl terminus), connected by a linker
which is rich in proline and glycine [13,14,15*]. The p
and p’ chains are very closely related; the trunks of these
chains are 93 % identical in sequence, with most of the
variability residing in the linker region [ 141. There are
two CLchains in AP-2, cLBand tic, and the trunks of these
chains are also 92 % homologous, again with the largest
divergence in the linker region [16]. There is no a-p or
P-r homology, but the trunks of a and y are 31% identical
[171.
The medium chains can be separated into three regions
[ 181. The carboxy-terminal and amino-terminal regions
elegam
are well conserved in the known Caenorhbditti
(P Stenberg, J Le, G Jongeward, personal communication) and rat and yeast homologs [IS]. The AP50 of
AJ?2 has a ttypsin-sensitive site between the central region and the carboxyl-terminal region which is cleaved
when AP-2 is present in clathrin coats but not when the
AT-2 complex is isolated [ 131. A conformational change
in the AP upon binding to clathrin might explain this observation.
Trunk
AP-1
Fig. 2. Relationship
between
the major
associated
protein
complexes
AP-1 and
AP-2. AP-7 contains
the large chains
y
and j3’, the medium
chain
AP47 and
the small chain
AP-19,
whereas
AP-2
contains
CL and p, AP50 and AP-17. C,
carboxyl
terminus;
N, amino
terminus.
183
184
Macromolecular
assemblages
Interactions
between klathrin
associated protein complexes
and the
Initial results indicated that the AP core, which comprises
the trunks of both large chains plus the medium and
small chains (Fig. 2), remains associated with dathrin
after proteolysis resulting in the loss of both ears [ 13,191.
But it has recently been shown, in a different buffer systern, that an intact p or p’ chain is required to maintain
association of@-2 or AP-1 with clathrin lattices, and that
intact p chains can themselves bind clathrin [15*,20]. Although aggregated a chains co-precipitate with clathrin
[ 2I**], there is so far no evidence that monomeric a or
y chains interact directly with clan-u-in. The observation
that an intact p or p’ chain is required for association
with clathrin suggests that both the trunk and the ear
may bind clathrin, perhaps at different sites. The light
chains are not required for Ap binding [ 14,22,23]. TWO
sites on the clathrin heavy chain are important for binding APS, one :in the terminal domain and one either in
the proximal leg or in the first portion of the distal leg
[23,24]. In physiological buffers, intact APs are required
for clathrin coat assembly. Core APs will not substitute
,for intact ones [25], implying that the ears participate
in some way in coat assembly. An exciting development
has been the demonstration that reconstituted APs can
also drive assembly [21**]; this may pemlit an analysis of which domains of the AP chains are important
for coat formation.
The possibility that at least two sites on the APs are required for interaction with clathrin is interesting in view
of the evidence that two sites on clathrin are involved in
AP binding. The importance of the distal/proximal leg
site has been overlooked because electron microscopic
observations appeared to imply that AI% and clathrin
meet only at the clathrin terminal domain [ 261. Analysis
of the radialdistribution of the average electron-density
pro&es of reassembled clathrin-AP coats showed three
peaks: the outer peak corresponded to clathrin legs,
the middle peak to the terminal domains of clathrin,
and the inner peak to the APs. As the intensity of the
clathrin peaks did not change significantly when Aps
were bound, it has been assumed that no part of the
APs can reach towards the clathrin legs. Yet, the ratio of clathrin trimers to AP-2 in reconstituted coats is
N 2:l (T Kirchhausen, unpublished data); thus, there are
six clathrin heavy chains for every AP chain. The mass
expected for six clathrin terminal domains is -300 kDa;
the mass of one P-chain ear is 35 kDa. Therefore, if the fichain ear reaches out to contact the clathrin heavy chain,
the contribution to the electron density might be difficult
to detect. It would be even harder to detect a P-chain ear
in the peak corresponding to the clathrin leg (35 kDa and
750 kDa, respectively). Thus, there is little evidence to refute the idea that p/p’ chain ears bind directly to clathrin.
This hypothesis needs to be tested.
Other
associated
proteins
Three new APs, called auxilin, ~140 and NP-185 @P-I80
or AP-3) have been identified recently in vesicles from
bovine brain but, so far, little is known about their
biochemistry and function [23,27-291, All three proteins
can drive coat assembly in vitro, and all are monomeric.
Auxilin and NP-185 appear to be specific to the brain. It
will be interesting to determine whether these proteins
are specifically involved in synaptic membrane recycling
and whether analogs exist in other tissues.
Receptor
selection
Coated vesicles transport a highly selected subset of the
proteins available in the membrane; specific receptors are
concentrated up to 100-fold in the coated pit. Sequences
in the cytoplasmic tail of the receptor, and especially the
presence of a tyrosine .residue, are crucial for internalization, although the consensus is very loose (see 1301
for review), indicating that recognition is promiscuous.
It seems unlikely that the clathrin heavy and light chains
are responsible for selection, because they are essentially invariant. AP-1 and AP-2 have significant variability
in the large chains [14,16,17], and are situated between
the clathrin coat and the membrane [26,31]. The AP-1
and AP-2 complexes are specific for the trans-Golgi network and plasma membrane, respectively (see [12] for
review), and AP-2 will bind back to plasma membranes
after coats are stripped with solutions of high molarity
Tris buffers [32]. These observations have led to the
suggestion that AP complexes are responsible for receptor selection. It has however so far proved difficult
to show direct interactions between the APs and receptor tails, perhaps because of the promiscuity of recognition. APs have been shown to bind to affinity columns
composed of receptor cytoplasmic tails made with fusion
proteins or synthetic peptides (A Hille-Rehfeld, personal
communication) [ 33,341. These experiments, although
suggestive, are puzzling in that the extent of AP retention on the column implies association constants in the
micromolar range and that millimolar concentrations of
soluble peptide are necessary to compete with this binding. The relatively high apparent affinity of APs for the
tail columns might therefore reflect the multivalent attachment of aggregated APs. Indeed, APs are known
to associate extensively under the conditions required
for these experiments [ 13,351. Further evidence for direct binding between APs and receptor tails comes from
the demonstration that recombinant receptor tails bind
to the trunk of trypsin-digested p chain after SDS-PAGE
and transfer to nitrocellulose sheet [36]. But the recombinant tails also bound to the trypsin used for digestion,
making the specificity of this interaction questionable.
Because AI% can also drive the formation of coats in
vitro, a two-stage model, in which receptor tails pre-
Coated
associate with APS, then cluster, triggering assembly of
the clathrin lattice, has been widely discussed. It is however still unclear whether the coated pit does form in
stages. Neither receptor-AP pre-association nor receptor pre-clustering has been observed in electron micrographs but, in each case, there are reasons, including the lack of appropriate reagents, for believing that
such events would be hard to see. As the formation of a
coated vesicle takes only 1 min, it is difficult to imagine
how receptor concentration and sorting can be achieved
if receptor pre-clustering does not occur.
The hypothesis that receptor tails are sufficient for AP
binding raises the question of how the specific localization of APs is achieved. Given that APs are also found
in the cytosol, why is it that, when recycling receptors
are expressed in the trans-Golgi network, they do not
attract the plasma membrane complex AP-2? One solution is that membrane-bound ‘docking proteins’ mediate
the attachment of the APs to the correct membrane. A
mechanism for the segregation of docking proteins to
the plasma membrane and trans-Golgi network is also
unknown. Circumstantial evidence for the existence of
a docking protein comes from experiments in which
plasma membranes were treated with elastase before
or after removal of the clathrin coat, and the ability of
the membrane to bind APs was measured [32]. When
the membrane is treated with elastase in the absence of
the coat, it loses its ability to bind APs; the presence of
the coat presumably protects the elastase-sensitive factor.
It is possible to imagine that the complex of an AP with
a docking protein might form a nucleus for the accretion of APs, which then bind to specific receptor tails
present in that membrane. Because the binding events
are constrained to take place in two dimensions, a relatively low binding constant for individual AP-receptor
tail interactions could give a much higher apparent binding constant. Such a model might explain the observed
tendency of APS to aggregate in vitro, as the initial accretion step would require AP-AP binding. This model
would also rationalize the high binding constant obtained
when (presumably associated) APs bind to receptor tails
arrayed on a column.
There are intriguing hints that the aggregation of AP-2
is controlled by molecules involved in cellular signaIling.
Inositol 1,4,5triphosphate and inositol hexaphosphate,
which are presumed second messengers in the polyphosphoinositide signaIling pathway, inhibit AP-2 aggregation
in vitro, probably by binding to the trunk of the a
chain [35,37-391. Phosphatidylinositol 4,5-bisphosphate
also binds to the a chain ofAP-2, but not to the y chain of
AP-1, in vitro, which is interesting because, in mammalian
cells, this lipid is only found in the plasma membrane. It
is clear that endocytosis is controlled in circumstances
such as mitosis, where all endocytic activity stops [40],
and in a more artificial, but nonetheless interesting, system of osmotic shock combined with K+ depletion [41].
These observations may allow investigation of the cellular
control of coated pit and coated vesicle formation.
Pit formation
nits
and
and vesicle
coated
vesicles
Kirchhausen
budding
Although the formation of coats in vitro in physiological
buffers requires only clathrin and AI%, there is increasing
evidence that other factors are required for the formation
and budding of coated pits. Three new in vitro systems
involving broken cells have allowed the dissection of
some of the events involved. In the first of these, the top
membrane of a cell is allowed to adhere to a glass coverslip, then torn away by removing the coverslip. Clathnn
and APs can then be removed from the membrane by
washing with two high molar&y Tris buffers. Although
purified APs bind to these stripped membranes, purified
clathrin does not, and no coated pits can form [42]. Interestingly, in the absence of stripping and in the presence
of added cytosol, this system allows the budding of preformed coated pits [43]. Thus, it appears that a factor(s)
in the cytosol or in the membrane is required for the
formation of pits, at least in this system. It is also possible that the clathrin present in the cytosol is modified
in some unknown way. Similarly, in a second broken-cell
system featuring mechanicaUy disrupted cell membranes
which can support one round of endocytosis, clathrin puriiied from cytosol, but not clathrin purified from coated
vesicles, facilitates the budding of coated vesicles. In this
system, purified APs can support budding [44]. This is
exciting, as it may be possible to use this broken ceU
system to dissect which elements of the APs are involved
in the different stages of budding. The third new in vitro
system uses cells broken by freeze-thawing [45] or by
mechanical disruption [46], and allows one to infuse
coat components and follow their localization in the
cell. Localization of AP-1 to the trans-Golgi network can
be seen in this system. It is enhanced by the addition of
GTP-yS, and blocked by Brefeldin A. The effect of the
latter is reversed by A+ The influence of all these compounds parallels their effects on trimeric G proteins. The
participation of such proteins in AP-1 localization therefore seems likely.
Recently, two proteins have been implicated in coated
vesicle formation. Using the glass-adhered-membrane
model described above, it has been shown that depletion of cytosol using antibodies to annexin VI, a
phospholipid-binding protein, blocks coated vesicle formation at a point just before budding occurs [47]. It
has therefore been proposed tha? annexin VI may be
involved in the membrane fusion event that is required
for the bud to pinch off from the membrane. The genetic
defect in Drosophila sbibire mutants which cause a failure
to complete the closure of coated pits has also recently
been identified [48]. The gene encodes dynamin, which
was originally isolated from rat brain as a microtubulebinding protein with GTPase activity [49].
A further clue that unidentiIied factors may be involved
in vesicle formation comes from the observation that the
size and topology of the clan-u-inlattices produced in vivo
is different from the smaU clathrin-AP coats produced in
vitro, and vanes according to the cells from which the
vesicles are purified. The average size of vesicles from
tissues other than brain is - 100-150 nm, whereas that
of brain vesicles is - 70-90 nm and that of coats assem-
185
186
Macromolecular
assemblaees
bled in vitro is - 70 MI. Thus, tissue- and site-specific
proteins might regulate the size of the vesicles formed
in vivo. It is also possible that the kinetics of vesicle formation, or the need to distort the membrane to form a
vesicle, influence the size of vesicle produced.
In the glass-adhered membrane system, clathrin is often
observed as sheets of hexagons underlying the plasma
membrane, which disappear as coated pits are formed
[43,50]. It is frequently assumed that these are an in$ial
stage of coated pit formation. But the introduction of the
proper number of pentagons for curvature requires major molecular rearrangement (see Fig. 3) and seems unlikely to be favored energetically. That clathrin assembly
is the driving force in vesicle budding [ 511 would be eas
ier to accept if the curved vesicle were assembled from
soluble clathrin (see Fig. 1) and not rearranged from an
assembled sheet. It is also hard to see how the correct
number of pentagons could *be inserted at the correct
places. It is worth noting that no structures intennediate
between soluble clathrin and complete coats have been
observed in r&-o. It is possible that, in the glass-adhered
membrane system, the flat sheets depolymerize, providing the material for coated pit formation. The possibility
that soluble clathrin is a necessar)’ intermediate in this
process could be tested.
In vivo experiments
The biological functions of clathrin and its associated
proteins have also been probed using genetic techniques.
In yeast, the lack of clathrin heavy or light chain is
not lethal, though the cells grow slowly [ 52-541; the
major phenotype is mislocalization of some proteins normally
found in the trans-Golgi network to the cell surface
[ 55,561. Similarly, in Diciyosfelitrm, heavy-chain depletion
by expression of antisense mRNA is not lethal and leads
to an absence of coated structures and decreased endocytosis [ 571. In contrast, in Drosophila, lack of clathrin
heavy chain is lethal, although embryos develop to a
relatively late stage, presumably because of the presence
of maternally derived clathrin (C Bazinet, personal cornmunication). It seems, therefore, that clathrin-mediated
vesicular tral%c is crucial for the complex development
and differentiation of tissues and is more important for
multicellular than unicellular organisms, perhaps because
cellLcell communication is tiected.
The yeast homologs of APs p and p’, AP-47, AP-17 and
M-19, most of which were identified by database search
[l&58,59], have been disrupted in individual mutants but
no phenotype has been detected (G Payne, S Lemmon,
T Kirchhausen, unpublished data). It is hard to reconcile this lack of phenotype with the hypothesis that the
Fig. 3. Rearrangements
of clathrin
mole-
cules
in a flat hexagonal
sheet
that
would
be required
to transform
a single hexagon
into a pentagon.
If hexagonal
sheets
are indeed
intermediates
in the budding
of coated
pits, major
rearrangements
of this kind
would
be
necessary.
Thick lines show the clathrin
molecules
that will need to be re-folded
into the new
lattice:
dark
grey
lines
indicate
clathrin
molecules
originating
on the left side of the removed
section;
black
lines those
originating
on
the right. The reader
is invited
to copy
the diagram
and to fold it along
the
lines indicated
to see the new shape of
the sheet. To make a spherical
clathrin
lattice,
12 such
rearrangements
would
need to be performed
at precise
locations within
the sheet.
As clathrin
has
Fold
here
to
join
A and
B
K
B
natural
‘pucker’,
with leg angles that are
close to those required
for the final cage
1601, it is easier to explain
correct
assembly of the clathrin
coat if soluble clathrin
’
forms
the curved
coated
pit directly
by
assembly
on sequential
vertices,
instead
of via a flat hexagonal
intermediate.
Coated
APs either select receptors for inclusion into the coated
vesicle or trigger the formation of clathrin coats, as disruption of either function would be expected to result
in a mislocalization of trans-Golgi network proteins similar to the one observed in yeast for the clathrin heavy
and light chain knockout. Yet, in C elegurzs, disruption
of AP-47 suppresses the effect of the let-23 gene, a mutation of the membrane receptor responsible for vulva
development, implying that AP-47 is involved in the receptor signalling pathway (P Stenberg, J Le, G Jongeward,
personal communication). The expression of light-chain
mutants and AP mutants in mammalian cells has so far
failed to yield dramatic phenotypes, although it is possible that more subtle effects of these mutations might
be detected upon further analysis.
5.
LEMMONSK, PEUICENA P~t.uz A, CONLEV K, FREUNDCL Sequence of the Clathrin Heavy Chain from Succharomyces
cerevisiue and Requirement of the COOH Terminus for
Clathrin Function. J cell SibI 1991, 112:65-80.
6.
S~ARMAT~
7.
BRODSKVFM, G-WAY CJ, BIANK GS, JACKSONAP, SEOW HF,
DIUCKAMERK, Pm
P: Localization of Clathrin Light-Chain
Sequences Mediating Heavy-Chain Binding and Coated Vesicle Diversity. Nature 1987, 326:203205.
8.
KIRCHHAUSEN
T, SCARMATOP, HARRISONSC, MONROEJJ, CHOW
EP, MATTALL~NORJ, RAMACHANDRAN
KL, St&RT JE, AHN AH,
BROSIUSJ: Clathrin Light Chains LCA and LCB Are Similar,
Polymorphic, and Share Repeated Heptad Motifs. Science
1987, 236:320-324.
9.
UNGEWICKEU.E: Biochemical and Immunological Studies on
Clathrin tight Chains and Their Binding Sites on Clathrin
Triskelions. EMBO J 1983, 8:1401-1408.
10.
NATHKE IS, HEUSERJ, LUPASA, STOCKJ, TURCKCW, BRODSKY
FM: Folding and Ttimerixation of Clathrin Subunits at the
Triskelion Hub. Cell 1992, 68:899-910.
11.
TOYODA T, KIRCHHAUSENT: Immuno-Electron
Microscopic
Evidence for the Extended Conformation of Light Chains
in Clathrin Trimetx. I Biol &em 1333, in press.
12.
ROBINSONMS: Adaptins. Trenak Ceil Biol 1992, 2:293-297.
13.
MATSUIW, K~RCHHAUSEN
T: Stabilization of Clatbrin Coats by
the Core of the Clathrin-Associated Protein Complex AP-2.
Biochem&tiy 1990, 29:10791-10798.
14.
K~RCHHAUSEN
T, NATHANSONKI, MATSUIW, iri- AL: Stntcttual
and Functional Division into ‘lwo Domains of the Large
(loo- to 115-kDa) Chains of the Clathrii-Associated Protein
Complex AP-2. Proc Nat1 Acud Sci USA 1989, 86:2612-2616.
SCHRODERS, UNGEWICKEUE: Subunit Interaction and Function of Clathrin-Coated Vesicle Adaptors from the Golgi
and the Plasma Membrane. J Biol @em 1991, 266:7910-7918.
The study indicates that an intact B or p’ chain is necessary to maintain
association of AP complexes with clathrin lattices.
16.
ROBINSONMS: Cloning of cDNAs Encoding ‘Iwo Related
lOO-kD Coated Vesicle Proteins (Alpha-Adaptins). J Cell Etiol
1990, 108:833-842.
17.
ROBINSONMS: Cloning and Expression of Gamma-Adapt@
a Component of’ Clathrii-Coated Vesicles Associated with
the Golgi Apparatus. 1 Cell Eiol 1990, 111:2319-2326.
18.
NAKAYAMA Y, GOEBL M, O’BRINE GRECO B, LEMMON S,
PINGCHANGCHOW E, K~RCHHAUSEN
T: The Medium Chains
of the Mammalian Clathrin-Associated Proteins Have a
Homolog in Yeast. Eur J Biocbem 1991, 202:56$574.
KEEN JH, BECK KA: Identification of the Clatbti+3iiding
Domain of Assembly Protein AP-2. Biocbena Biopbys Res
Cbmmun 1989, 158:17-23.
reading
Papers of panicular interest, published within the annual period of
review, have been highlighted as:
.
of special interest
..
of outstanding interest
1.
RODMAN JS, MERCER RW, STAHL PD: Endocytosis and
Tran~cytosis. Curr Opin Cell Biol 1990, 2664-672.
2.
SW
E, WARREN G: The Mechanism of Receptor-Mediated
Endocytosis. Eur J Biofbem 1331, 202:&?~99.
3.
Row TF, PORTERKR: Yolk Protein Uptake in the Oocyte of
the Mosquito Aedes aegy@ J Cell Biol 1964, 20:313-331.
P, KI~~~HHA~~ENT: Analysis of Clathfin Light
Chain-Heavy Chain Interactions Using Truncated .Mutams of Rat Liver Light Chain LCB3. Biopby &em 1990,
265:3661-3668.
15.
.
I would like to thank S Lemmon, M Roth, S Schmid and B Ward
for their insightful comments on this review.
and recommended
Kirchhausen
KIRCHHAUSEN
T, NATHAN~ONKL, MATsul W, VA&BERGA, CHOW
EP, BURNEC, KEEN JH, DAVIS AE: Structural and Functional
Division into Two Domains of the Large (IOO- to 115
kDa) Chains of the Clathcin-Associated Protein Complex
AF-2. h-cc Nat1 Acud Sci (I S A 1989, 86:2612-2616.
Acknowledgements
References
vesicles
4.
Conclusion
Although there is still limited understanding of the
biological importance of clathrin-coated structures or
the molecular mechanism by which directed tratfic is
achieved, several important tools have recently been developed. The demonstration that renatured APs can drive
the formation of clathrin coats in physiological buffers
should allow the dissection of which parts of the APs are
crucial in this process. Mutants produced by genetic engineering might provide valuable reagents for further study
in reconstitution experiments. In particular, the ability to
study the budding of coated vesicles and the targeting of
APs in in vitro membrane systems offers hope for a better understanding of the factors involved. In vivo knockout and replacement experiments will provide the crucial
test of such insights gained from in vitro experiments.
Finally, the expression of clathrin and its associated proteins by recombinant methods offers the possibility of
using direct high-resolution structural methods to probe
their structure, which may eventually shed fight onto their
function.
pits and coated
19.
20.
21.
..
ABLE S, UNGEwtCKELLE: Identification of a Clathrin Binding
Subunit in the HA2 Adaptor Protein Complex. J Biol C%em
1989, 264:20089-20093.
PRAS~DK, KEEN JH: Interaction of Assembly Froteio AF-2
. and Its isolated Subunits with Clathrin. Biochmishy 1991,
30:559t35597.
The first paper to demonstrate that APs, refolded after complete denatutation in vitro, recover their ability to associate with clatbtin and to
promote the assembly of coats.
187
188
Macromolecdar
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34
35.
36.
37.
38.
assemblages
42.
KEEN JH, BECK KA, KIRCHHAUSEN
T, JARR!XI’T: Clathrin Domains Involved in Recognition by Assembly Protein AP-2.
J Biol C&em 1991, 266:7950-7956.
ZAREMBAS, KEENJH: Limited Proteolytic Digestion of Coated
Vesicle Assembly Polypeptide Abolishes Reassembly A&ivity. J Cell Biocbem 1985, 2847-58.
VIGERS GP, CROWIHER & PEAI&E BM: Location of the
lOOkd-50 kd Accessory Proteins in Clathrin Coats. /MB0
J 1986, 5:2079-2085.
MURPHYJE, PLEASUREIT, PUSZKINS, PKA.%D K, KEEN p-l:
Clathrin Assembly Protein AP-3. The Identity of the 155K
Protein, AP 180, and NP185 and Demonstration of a
Clatbrin Binding Domain. J Biol G~em 1991, 266:44014408.
AHIE S, UNGNVICKEUE: Auxjlin, a Newly Identified ClathrinAssociated Protein in Coated Vesicles ftom Bovine Brain.
J Cc/l Biti 1990, 111:19-29.
LINDNERR, UNGEWICKELLE: Clathrin-Associated Proteins of
Bovine Brain Coated Vesicles: an Analysis of Their Number and Assembly-Promoting Activity. J Eiol Uwm 1992.
26116567-16573.
KTISTAKISNT, THO~W D, ROM MG: Characteristics of the Tyrosine R&ognition Signal for Internalization of Transmembrane Surface Glycoproteins. J Cell Biol1990, ll1:1393-1407.
HEUSERJ, *KIRCHHAUSEN
T: Deep-Etch Views of Clathrin Assemblies. J Ultrasfruci
Rcs 1985. 92:1-27.
MAHAFFF(DT, PEELERJS, BROD~KYFM, ANDERSONRG: ClathrinCoated Pits Contain an Integral Membrane Protein that
Binds the AP-2 Subunit with High Amenity. J Biol Chem
1990, 265:16514-16520.
GUCKMANJN, CONIBEARE, Pm BM: Specificity of Binding of Clathrin Adaptors to Signals on the Mannose-6Phosphate/Insulin-Like Growth Factor 11 Receptor. EMBO
J 1989, 8:1041-1047.
POE BM: Receptors Compete for Adaptors Found in
Plasma Membrane Coated Pits. EMBO J 1988, 7:3331-3336.
BECKKA, KEENJH: Self-Association of the Plasma MembraneAssociated Clathrin Assembly Protein AP-2. J Biol aem
1991, 2664437-4441.
44.
SMYIHE E, CARTERLL, SCHMIDSL: Cytosol- and Clathrin-Dependent Stimulation of Endocytosis in vitro by Purified
Adaptors. J Ce// Eiol 1992, 119:11631171.
45.
ROBINSONMS, KREISTE: Recruitment of Coat Proteins onto
Golgi Membranes in Intact and Permeabiied
Cells: Effects of Brefeldin A and G Protein Activators. Ce// 1992,
69:129-138.
46.
WONC DH, BRODKSYFM: 100.kD Proteins of Golgi- and
Trans-Golgi Network-Associated Coated Vesicles Have Related but Distinct Membrane Binding Properties. J Ce// Biol
1992, 117:1171-1179.
47.
IJN HC, Suo~ior; TC, ANDERSONKG: Annexin VI Is Required
for Budding of Clathrin-Coated Pits. Cell 1992, 70:283291.
VAN DERBUEK AM, MEYEROWIX EM: Dynamin-Like Protein
Encoded by the Drosophila
shibire
Gene Associated with
Vesicular Traffic. Nature 1991, 351:411-414.
OBAR R4, COLUNS Cq ~IMARBACK JA, SHPEINER HS,
VALLEE RB: Molecular Cloning of the Microtubule-Associated Mechanochemical Enzyme Dynamin Reveals Homology with a New Family of GTP-Binding Proteins. Nafure
19’90, 30256261.
SANAN DA, ANDERSONRGW: Simultaneous Visualization of
LDL Receptor Distribution and Clathrin Lattices on Membranes Tom from the Upper Surface of Cultured Cells. J
Hisltiem
CJlfochem 1991, 39~1017-1024.
BELIZERJP, SP~ESSM: In u/t*0 Binding of the Asialoglycoprotein Receptor to the p Adaptin of Plasma Membrane
Coated Vesicles. EMBO J 1991, 10:3735-3742.
VOG~MA~ERSM. KEEN JH, MURPHYJE, FERRISCD, PRESNV~CH
GD, SNYDERSH, THEBERT AB: Inositol Hexakisphosphate
Receptor Identified as the Clathrin Assembly Protein Ap-2.
Biochem Biq@s
Res Commun
1992. 187:158-163.
Tvmttd~ AP. MAYRIE~ER MM, LLIKAS TJ, CHADWICKCc,
SAITO A, WATI-E~ON DM, SCHINDIERH, F~ZXHER S: Inosito1 PolyphoSphate Receptor and Clathrin Assembly Protein
AP.2 Are Related Proteins that Form Potassium-Selective
Ion Channels in Planar Lipid Bilayers. Proc Nat/ Acud Sci /J
43.
.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
S A 1952, 89:8976+980.
39.
40.
41.
MAHAFFEVDT, MOOUEMS, BRODSKYFM, ANDERSONRG: Coat
Proteins Isolated from Clathrin Coated Vesicles Can Assemble into Coated Pita. J Cell B/o/ 1989, lO8:1615-1624.
LIN HC, MOOREMS, SANANDA, ANDERSONGW: Reconstruction
of Clathrin-Coated Pit Budding f?om Plasma Membranes. J
Ceil Biol 1991, 1148814391.
.
R, UNGEWXKEUE: Light-Chain-Independent
Binding
of Adaptors, AP180, and Auxilin to Clathrin. Biocbemisl~~
1991, 30:9097~101.
MURPHYJE, KEENJH: Recognition Sites for Clathrin-&sociated Proteins Ap-2 and AP-3 on Clathrin Triskelia. / Biol
Uxm 1992, 267:1085(t10855.
LINDNER
BECKKA, KEENJH: Interaction of Phosphoinositide Cycle Intermediates with the Plasma Membrane-Associated Clathrin
Assembly Protein AP-2. J Biol Cbem 1991, 266:4442-4447.
P~%RT M, MUNDY D, SOIJTERE, IABBE J-C, WARRENG: Mitotic Cytosol Inhibits Invagination of Coated Pits in Broken
.Mitotic Cells. J cell Biol 1991, 114:1159-1166.
m
JM, DONZEUWC, ANDERSONRG: Potassium-Dependent
Assembly of Coated Pits: New Coated Pits Form as Planer
Clathrin Lattices. J Cell Biol 1986, 103:261%2627.
59
60
HARRISONSC, KIRCHHAUSEN
T: Clathrin. Cages, and Coated
Vesicles. CeN 1983, 33:650-652.
PAYNE GS, SCHEK~VIANR: A Test of Clathrin Function
in Protein Secretion and Cell Growth. Science 1985,
230:100~1014.
SILVEIRAL4, WONG DH, MASIARZFR. SCHEK~~ANR Yeast
Clathrin Has a Distinctive Light Chain that Is Important
for Cell Growth. J Cell Biol 1990, 111:1437-1449.
IJZMMONSK, JONES EW: Clathrin Requirement for Normal
Growth of Yeast. Science 1987, 238:504-509.
SEEGERM, PARVEGS: A Role for Clathrin in the Sorting of
Vacuolar Proteins in the Golgi Complex of Yeast. WcfgO J
1992, 1 I:281 1-2818.
SEEGERM, PAYNE GS: Selective and Immediate Effects of
Clathrin Heavy Chain Mutations on Golgi Membrane Protein Retention in Saccharomyces
cerehsiae.
J Cell Bio/
1992, 118:531-540.
O’HARRLORANTJ, ANDERSONRG: Characterization of the
Clathrin Heavy Chain from Dictyostelium
discofdeum
DNA
Cell Biol 1992, 11321-330.
KIRCI~HAUSEN
T. DAVIS AC, FRUCHTS, GRECO BO, PAYNEGS,
TUBB B: AP17 and AP19, the Mammalian Small Chains of
the Clathrin-Associated Protein Comolexes Show Homolow
to Yapl7p, Their Putative Homolog in Yeast. J Biol G.&I
1991, 266:1115311157.
KIRCHHAUSEN
T: Identification of a Putative Yeast Homolog
of the Mammalian Beta Chains of the Clathrin-Associated
Protein Complexes. Mol Ceil Biof 1990, 10:6089-6090.
KIRCHHAUSEN
T, HARRISONSC, HEUSERJ: Configuration of
Clathrin Trimers: Evidence from Electron Microscopy. J
Uhstruct
MO/ Struct
Res 1986, 94:199-208.
T Kirchhausen, Haward Medical School and Center for Blood Research,
200 Longwood Avenue, Boston, Massachusetts02115, USA