Download Phosphoinositide regulation of clathrin

Cell Architecture: from Structure to Function
Phosphoinositide regulation of clathrin-mediated
endocytosis
V. Haucke1
Institut für Chemie-Membranbiochemie, Freie Universität Berlin, Takustrasse 6, 14195 Berlin, Germany
Abstract
Endocytosis of transmembrane receptors largely occurs via clathrin-coated vesicles that bud from the
plasma membrane and deliver their cargo to the endosomal system for recycling or degradation. PIs
(phosphoinositides) control the timing and localization of endocytic membrane trafficking by recruiting
adaptors and other components of the transport machinery, thereby being part of a coincidence detection
system in adaptor-mediated vesicle transport. Activation of organelle- and substrate-specific PI kinases by
small GTPases such as Arf (ADP-ribosylation factor) and other factors may result in local changes of PI content,
thereby regulating activity-dependent endocytic events including the recycling of synaptic vesicle membranes at nerve terminals. One such example is the PtdIns(4)P 5-kinase-mediated formation of PI(4,5)P2
[PtdIns(4,5)P2 ], which is required for the exo- and endo-cytic cycling of presynaptic vesicles and secretory
granules. Over the last few years, protein X-ray crystallography in combination with biochemical and cell
biological assays has been used to investigate the structure and function of many PI-binding proteins,
including protein components of the endocytic machinery. These studies have provided molecular insights
into the mechanisms by which PI(4,5)P2 recruits and activates adaptor proteins and their binding partners. In
this mini-review, I will discuss the pathways of PI(4,5)P2 formation and its interactions with endocytic
trafficking adaptors.
Introduction
Eukaryotic cells are compartmentalized into membranebounded organelles that can be distinguished based on their
morphology and biochemical properties. Although distinct
protein and lipid compositions define compartment identity,
organelles are in a dynamic equilibrium governed by the exchange of membrane and luminal cargo. Vesicle-mediated
membrane trafficking can be grossly divided into two main
pathways: biosynthetic transport from the endoplasmic
reticulum via the Golgi complex to the cell surface, and the
endocytic pathway by which plasma-membrane receptors,
nutrients and signalling molecules are targeted to the endosomal–lysosomal system for recycling or degradation.
Plasmalemmal vesicles were first visualized in cells and tissues
in the 1960s by Roth and Porter [1].
Clathrin-mediated endocytosis is the major entry route
for extracellular hormones and signalling factors and serves
to regulate the internalization of transmembrane receptors as
well as the recycling of pre- and postsynaptic membrane proteins [2–4]. Although CCVs (clathrin-coated vesicles) are
found ubiquitously in all eukaryotic cells and tissues, their
components are particularly enriched in brain, where clathrin
and its partner proteins are implicated in the biogenesis of
small clear presynaptic vesicles, the major neurosecretory
Key words: adaptor, ADP-ribosylation factor 6 (Arf6), clathrin, endocytosis, phosphoinositide,
trans-Golgi network.
Abbreviations used: AP, adaptor protein complex; Arf, ADP-ribosylation factor; CALM, clathrin
assembly lymphoid myeloid protein; CCP, clathrin-coated pit; CCV, clathrin-coated vesicle;
PI, phosphoinositide; PI(4)P, PtdIns(4)P; PI(4,5)P2 , PtdIns(4,5)P2 ; PIPK, PtdIns(4)P 5-kinase;
SH, Src homology; TGN, trans-Golgi network.
1
email [email protected]
organelles within the nervous system [5,6]. A related form
of CCVs mediates transport between the TGN (trans-Golgi
network) and the endosomal system [7]. One of the major
issues in the field has been the question of how localized
assembly of CCPs (clathrin-coated pits) and vesicles at the
plasma membrane is achieved and how this is coupled with
the selection of transmembrane cargo proteins destined
for internalization. Over the last decade, a central role for
PIs (phosphoinositides) as spatial landmarks for membrane
trafficking has emerged [8,9]. Based on the intracellular
distribution of differentially phosphorylated PIs, a ‘PI-map’
of the exo- and endo-cytic pathways can be drawn (Figure 1)
[9]. Together with their corresponding vesicle adaptors and
transmembrane cargo proteins, PIs may thus be part of a dualkey strategy for directing membrane trafficking pathways
[10]. PI(4,5)P2 [PtdIns(4,5)P2 ] is required for invagination of
CCPs, fusion of secretory granules with the plasmalemma
and for macropinocytosis. Other PIs have been localized to
distinct intracellular membranes and it now seems that many
of the important components involved in vesicle formation,
fusion or fission are important targets of PIs. Since much of
what we know about the role of PIs in membrane traffic has
been learnt from clathrin-mediated endocytosis, I will mainly
focus on this pathway.
CCV assembly at the plasma-membrane
PI(4,5)P2
CCVs are formed by the co-ordinated assembly of clathrin
triskelia formed from three tightly linked heavy and associated light chains on to the plasma membrane. The recruitment
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Figure 1 PI regulation of the exocytic and endocytic pathways
PI(4,5)P2 (blue) is required for the assembly of CCPs as well as for macropinocytosis and for fusion of secretory vesicles and
granules (SVs). PI(3)P [PtdIns(3)P] (green) is present in early endosomes and internal vesicles of the multivesicular body
(MVB). At the boundary membrane of the MVB, PI(3)P is converted into PI(3,5)P2 [PtdIns(3,5)P2 ]; (purple). PI(4)P (red)
localizes to the Golgi complex and in particular to the TGN. EV, endocytic vesicle.
and polymerization of the outer clathrin layer is assisted by
mono- and hetero-tetrameric adaptor proteins, which simultaneously bind to clathrin, membrane lipids and in many cases
to transmembrane cargo proteins [7,11]. CCV formation at
the plasmalemma is co-ordinated by the heterotetrameric
adaptor complex AP-2 (adaptor protein complex 2) comprising two large subunits (α and β2), a medium subunit (µ2)
and a small subunit (σ 2). The two large subunits together
with σ 2 and the N-terminal domain of µ2 (N-µ2) form the
trunk or core domain of AP-2 [12], which are joined by
extended, flexible linkers or ‘hinges’ to the appendage or ear
domains of α- and β2-adaptins. By associating with clathrin,
accessory endocytic proteins, membrane lipids and cargo receptors, AP-2 may serve as a central protein recruitment hub
during CCV assembly [13]. Many accessory proteins such
as epsins, AP180/CALM (clathrin assembly lymphoid
myeloid protein) and amphiphysin also serve an adaptor
function. These monomeric adaptors possess a folded lipidbinding domain [14] linked to a more flexible portion of
the protein harbouring short clathrin- and AP-2-binding
motifs, which aid in stabilizing nascent coated pits during
the assembly process. During CCP assembly, transmembrane
cargo proteins are recognized by adaptor proteins, most notably the AP-2 complex, which bind to endocytic sorting
motifs within their cytoplasmic tails. These motifs include
tyrosine-based YXX (where is a bulky hydrophobic residue) and dileucine motifs, which bind directly to distinct
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sites within the AP-2 core domain [15]. YXX motifs have
been co-crystallized with the distal portion of the AP-2
µ-subunit (C-µ2) to which they bind in an extended conformation [16]. C-µ2 also harbours a structurally unresolved
binding site for basic internalization motifs found in a
variety of multimeric membrane proteins including postsynaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and GABAA (γ -aminobutyric acid type A)
receptors (J.T. Kittler, personal communication), the α 1B adrenergic receptor [17] and members of the synaptotagmin
family of calcium sensor proteins [18–20]. Most, if not all, of
these proteins undergo ligand- or stimulus-dependent endocytosis, suggesting that perhaps basic AP-2-binding signals
may be preferentially used for regulated internalization.
Addition of mono- or multi-ubiquitin moieties to signalling receptors has been shown to serve as a signal for endocytosis [21] and sorting for lysosomal degradation [22,23].
Multiple lines of evidence suggest that the assembly of plasmalemmal CCPs is dependent on the presence of PI(4,5)P2
within the membrane. PI(4,5)P2 is concentrated on and serves
as a marker for the plasma membrane [9]. Elevated levels of
PI(4,5)P2 due to genetic inactivation of the inositol phosphatase synaptojanin 1 lead to perinatal lethality in mice and
to the accumulation of CCVs within nerve endings due to
decreased rates of presynaptic vesicle cycling [24]. Conversely, depletion of PI(4,5)P2 by overexpression of the membrane-targeted 5-phosphatase domain of synaptojanin 1 [25]
Cell Architecture: from Structure to Function
Table 1 Overview of PI(4,5)P2 -binding proteins
Proteins with a proven or assumed function in endocytosis are shown
in the top half of the Table. CAP23, cytoskeleton-associated protein of
23 kDa; E/ANTH, epsin/AP180 N-terminal homology domain; GAP43,
growth-associated protein 43; HIP, Huntingtin-interacting protein;
MARCKS, myristoylated alanine-rich C-kinase substrate; PH, pleckstrin
homology; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; PLD,
phospholipase D; PX, Phox homology; WASP, Wiskott–Aldrich syndrome
protein.
E/ANTH
PH
PX
Others
Epsin 1
Dynamin 1
Type II PI3K
AP-2α
Epsin 2
AP180
CALM
Dynamin 2
C2α
AP-2µ
Dab2
PLD
HIP1
HIP1R
Arf
βIII-spectrin
WASP
Profilin
PLCε
GAP43
MARCKS
CAP23
Ezrin
Moesin
or knockdown of PIPKIγ [PtdIns(4)P 5-kinase type Iγ ]
impairs receptor-mediated uptake of transferrin [26] or
epidermal growth factor due to mislocalization of AP-2 and
clathrin [25]. Similarly, mice lacking the major brain-enriched
PIPKIγ display defects in both the exo- and endo-cytic limbs
of the synaptic vesicle cycle [27].
A number of endocytic proteins including the α [28] and
µ2 [29] subunits of AP-2, AP180/CALM [30], HIP1/1R,
Dab2 [31], epsin [32] and dynamin [33] specifically bind to
PI(4,5)P2 . Structurally, binding is accomplished by a variety
of domain architectures including a PH (pleckstrin homology) domain in the case of dynamin, ANTH (AP180 N-terminal homology) or ENTH (epsin N-terminal homology)
domains within AP180/CALM and epsin, as well as surfaceexposed basic patches within AP-2α and µ2. Moreover, a
number of proteins related to the function of the actin cytoskeleton, such as WASP (Wiskott–Aldrich syndrome protein) and profilin, which may also affect clathrin-dependent
endocytosis have been shown to associate with PI(4,5)P2
[9] (Table 1). In the case of AP-2, PI(4,5)P2 may play an
active role in stabilizing the open conformation of the complex, thus enabling cargo recognition by its µ2 subunit,
facilitating the stable association of AP-2 with the membrane
[15]. Consistent with this scenario, clathrin/AP-2-coated pits
have been shown to become stabilized in living cells upon
encounter of cargo receptors [11]. Similarly, specialized
monomeric adaptors such as Dab2 or ARH (autosomal recessive hypercholesterolaemia) are capable of simultaneously
associating with NPXY motif-containing cargo receptors and
PI(4,5)P2 [23]. These combined results suggest a dual-key
recognition strategy for endocytic adaptor binding to the
plasmalemma using a combined coincidence detection mode
based on cargo recognition in the context of a PI(4,5)P2 -rich
membrane [10,15]. A similar mechanism may operate at the
TGN where PI(4)P [PtdIns(4)P] [9] regulates budding of
clathrin/AP-1-coated vesicles (Figure 1).
Regulation of PI(4,5)P2 synthesis
PIs generally constitute <10% of the total cellular phospholipids; yet, as outlined above, they are key regulators
of intracellular membrane traffic and cell signalling. PI(4)P,
the immediate substrate for PI(4,5)P2 synthesis, is enriched
within Golgi membranes, at the TGN, and is generated within
secretory vesicles (Figure 1). In mammals, three PIPKIγ isoenzymes exist (α, β and γ ), all of which have been shown
to partition between the cytosol and the plasma membrane, thereby generating the plasmalemmal pool of PI(4,5)P2
[34]. While the division of labour between the α and β isoenzymes appears to slightly differ between different cell
types, PIPKIγ is most highly expressed in neurons, where
it is enriched in presynaptic nerve terminals [35]. The type Iγ
kinase, in addition, is an important component of focal
adhesion sites where it undergoes Src tyrosine kinase-dependent interactions with talin, resulting in massive stimulation
of its enzymatic activity [36,37]. A similar mechanism operates at nerve terminals where PIPKIγ -mediated PI(4,5)P2
synthesis regulates clathrin-mediated cycling of endocytic
vesicles. Another profound stimulator of type I PIPK is the
small GTPase Arf6 (ADP-ribosylation factor 6) [38]. Activated Arf6 localizes to the plasma membrane in a variety of cell
types and is enriched in presynaptic boutons [25] but
can also be detected within neuronal dendrites. Nucleotide
exchange on Arf6 by the Sec7 domain-containing ArfGEF (guanine nucleotide-exchange factor) mSec7 facilitates
exocytic–endocytic cycling of presynaptic vesicles [39]
and Arf6-GTP in turn is capable of directly activating
PIPKIγ (Figure 2), resulting in increased PI(4,5)P2 levels
within presynaptic membranes and to increased recruitment
of clathrin and endocytic adaptors [25]. The direct activation of PIPKIγ isoenzymes by Arf6 may synergize with
other Arf-dependent effector pathways that affect clathrinmediated endocytosis. This includes Arf-mediated stimulation of phospholipase D, another potent activator of
type I PIPK, Rac-induced actin rearrangements and NM23H1-catalysed GTP formation, subsequently made available
to GTPases including dynamin [40]. Whether or not Arf6GTP, in analogy with its cousin Arf1 at the Golgi, interacts
with clathrin coat components remains somewhat controversial [25,41,42]. PI(4,5)P2 synthesized via Arf6-dependent
PIPK stimulation, however, does not appear to be exclusively
targeted to the clathrin pathway. In fact, in many cell types
including HeLa cells, overexpression of Arf6-GTP results in
the formation of PI(4,5)P2 -enriched membrane ruffles and a
stimulation of clathrin-independent macropinocytosis [40].
Moreover, Arf6-dependent PIPKIγ activation has been implicated in the fusion of secretory vesicles in neuroendocrine
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Figure 2 Arf6 directly stimulates the activity of endogenous or
recombinant PIPKIγ
(A) Liposomes containing PI(4)P (6%, w/w) as a substrate were
incubated for 10 min at 37◦ C with brain cytosol and recombinant myristoylated Arf6 mutants or wild-type (wt) (200 nM) as indicated in the
presence of neomycin, GTP and [γ -32 P]ATP. Lipids were extracted and
separated by HPTLC. Results are means ± S.D. for four independent
experiments. Values were normalized to the amount of PIP2 generated in
the absence of Arf6 proteins (fold stimulation). (B) Recombinant PIPKIγ
(4 ng) was preincubated for 20 min at 4◦ C with recombinant myristoylated Arf6 proteins, GTP and total brain liposomes. Reactions were started
by the addition of [γ -32 P]ATP and after a 7 min incubation (37◦ C),
lipid products were extracted, separated by TLC as described in [35],
analysed by autoradiography (see bottom panel for a typical experiment)
and quantified (phosphoimager). The top panel shows the quantification
of three independent experiments (means ± S.D.); the data presented
are normalized to the activity of PIPKIγ in the absence of exogenously
added Arf6 proteins (fold stimulation). Reproduced from The Journal of
Cell Biology, 2003, vol. 162, pp. 113–124, by copyright permission from
The Rockefeller University Press.
[49] as well as the small GTPase Rac may also affect 5phosphatase activity under certain conditions [6].
At synapses, PI(4,5)P2 levels appear to be under activitydependent control. Both synaptojanin and PIPKIγ undergo
activity-dependent dephosphorylation and presumably interact with regulatory proteins in a manner influenced by
synaptic activity. In hippocampal neurons, a retrograde signalling pathway has been discovered that leads to elevated
PI(4,5)P2 levels within the presynaptic compartment following NMDA (N-methyl-D-aspartate) receptor activationdependent postsynaptic production of nitric oxide. Increased
PI(4,5)P2 levels facilitate presynaptic vesicle cycling [50],
perhaps by stimulating clathrin-mediated endocytosis of presynaptic vesicle components. How exactly NO regulates
PI(4,5)P2 concentrations is unknown but pharmacological
inhibitor studies suggest an involvement of soluble guanylate
cyclase and cGMP [50]. We are thus only now beginning to
understand the spatiotemporal control of PI metabolism and
its relationship with cellular signalling pathways.
I am grateful to members of my laboratory and to my collaborators,
in particular S. Höning, D. Owen, S. Moss, J. Kittler, R. Jahn, E. Neher,
J. Sorensen, K. Takei and P. De Camilli. Work in our laboratory was
supported by the EMBO Young Investigator Programme (YIP Award
2003), the Deutsche Forschungsgemeinschaft (SFB449, SFB366 and
HA2686/1-1) and the Fonds der Chemischen Industrie (FCI).
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Received 5 May 2005
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