COMMENTARY
1041
Phosphoinositides in membrane traffic at the synapse
Ottavio Cremona1 and Pietro De Camilli2
1Department
2Department
of Medical Sciences, Università del Piemonte Orientale ‘A. Avogadro’, Via Solaroli 17, 28100 Novara, Italy
of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT
06510, USA
e-mail: [email protected]; [email protected]
Journal of Cell Science 114, 1041-1052 © The Company of Biologists Ltd
Summary
Inositol phospholipids represent a minor fraction of
membrane phospholipids; yet they play important
regulatory functions in signaling pathways and membrane
traffic. The phosphorylated inositol ring can act either as
a precursor for soluble intracellular messengers or as a
binding site for cytosolic or membrane proteins. Hence,
phosphorylation-dephosphorylation of phosphoinositides
represents a mechanism for regulation of recruitment to
the membrane of coat proteins, cytoskeletal scaffolds or
signaling complexes and for the regulation of membrane
proteins. Recent work suggests that phosphoinositide
metabolism has an important role in membrane traffic at
the synapse. PtdIns(4,5)P2 generation is implicated in the
Introduction
Ca2+-regulated exocytosis of neurotransmitters is the primary
mechanism by which neurons communicate with each other
and with effector cells. Prior to secretion, neurotransmitters
are stored in two distinct classes of vesicle. Non-peptide
neurotransmitters, including all fast-acting neurotransmitters
(e.g. glutamate, acetylcholine, γ-aminobutyric acid and
glycine), accumulate in small vesicles – the synaptic vesicles
(SVs) – which represent specialized synaptic organelles.
Peptide neurotransmitters, by contrast, are stored in larger
vesicles – the dense-core vesicles (DCVs) – which might also
contain biogenic amines (e.g. dopamine, norepinephrine,
serotonine and histamine). DCVs represent the major secretory
organelles of neuroendocrine cells. SVs and DCVs differ in a
variety of properties, including stimulus-secretion coupling
and biogenesis. However, fundamental mechanisms of
exocytosis are common to the two organelles (De Camilli and
Jahn, 1990; Sudhof, 1995; Calakos and Scheller, 1996; Hannah
et al., 1999).
The availability of procedures to purify SVs, the
development of powerful in vitro assays to reconstitute
vesicular transport reactions, the identification of targets of
clostridial neurotoxins, and advancements in forward and
reverse genetics in a variety of organisms have led to the
identification of many proteins essential for neuronal secretion.
These studies have revealed that the protein machinery
involved in exocytosis/endocytosis at the synapse is
evolutionary highly conserved and similar to that which
participates in exocytosis/endocytosis in all eukaryotic cells
(Rothman and Warren, 1994; Schekman, 1994; Wu and Bellen,
1997; Geli and Riezman, 1998; Slepnev and De Camilli, 2000).
More generally, membrane fusion and budding mechanisms
secretion of at least a subset of neurotransmitters.
Furthermore, PtdIns(4,5)P2 plays a role in the nucleation
of clathrin coats and of an actin-based cytoskeletal scaffold
at endocytic zones of synapses, and PtdIns(4,5)P2
dephosphorylation accompanies the release of newly
formed vesicles from these interactions. Thus, the
reversible phosphorylation of inositol phospholipids may
be one of the mechanisms governing the timing and
vectorial progression of synaptic vesicle membranes during
their exocytic-endocytic cycle.
Key words: Phosphoinositide, Synaptic vesicle, Neurotransmitter
release, Clathrin-mediated endocytosis
are fundamentally similar throughout the secretory and
endocytic pathways. Thus, the exocytic/endocytic cycle of SVs
has become a powerful model system in the field of membrane
traffic.
Growing evidence indicates that membrane lipids have not
only a structural role but also an important regulatory function
in membrane traffic. Phosphoinositides, in particular, are key
regulatory molecules (De Camilli et al., 1996; Martin, 1998;
Odorizzi et al., 2000). The reversible phosphorylation of their
inositol ring generates a series of stereoisomers that can bind
to cytosolic and membrane proteins with variable affinities and
specificities. Thus, membrane phosphoinositides can nucleate
cytoskeletal scaffolds, vesicle coats and signaling complexes.
Proteins bind to phosphoinositides by interacting either with
protein modules – e.g. PH, SH2, PTB, FYVE and C2 domains
– or, less specifically, with clusters of positively charged
residues (Janmey, 1994; Bottomley et al., 1998; Martin, 1998;
Corvera et al., 1999; Sechi and Wehland, 2000). Here, we focus
on the many connections between phosphoinositides and
membrane dynamics at the synapse.
Phosphoinositides in neurotransmitter release
Almost fifty years ago, Hokin and Hokin (Hokin and Hokin,
1953) observed that stimulation of secretion from pancreatic
acinar cells results in increased phosphorylation of
phospholipids. Further experiments demonstrated that this
effect is limited to a minor fraction of these lipids, the inositol
phospholipids (Larrabee et al., 1963), and that it occurs in most
secretory systems, including brain synaptosomes (Durell and
Sodd, 1966). These findings led to the hypothesis that
phosphoinositide turnover is closely linked to the SV cycle
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JOURNAL OF CELL SCIENCE 114 (6)
(Hawthorne and Pickard, 1979; Fig. 1). However, the
subsequent discovery that products of PtdIns(4,5)P2 hydrolysis
are potent intracellular messengers shifted attention to the role
of phosphoinositide turnover in intracellular signaling. Only in
the past decade has the role of phosphoinositide metabolism in
membrane trafficking, including trafficking at the synapse,
returned to center stage, owing to a convergence of information
obtained from biochemistry, cell-free assays and yeast genetics
(Eberhard et al., 1990; Herman and Emr, 1990; Hay and
Martin, 1993; Schu et al., 1993; Hay et al., 1995; McPherson
et al., 1996). These studies have shown that some of the effects
of phosphoinositides are direct – i.e. not mediated by the their
cleavage products – and have revealed important physiological
roles of novel phosphoinositide species.
DCV exocytosis
The first piece of evidence for a role for PtdIns(4,5)P2
in vesicular neuroendocrine secretion, independent of its
phospholipase-C-mediated cleavage, came from studies of
regulated exocytosis of catecholamines from DCVs of broken
chromaffin cells (Holz et al., 1989; Eberhard et al., 1990). Holz
et al. showed that an ATP-dependent priming step precedes
Ca2+-triggered secretion (Holz et al., 1989). Furthermore,
PtdIns(4,5)P2-degrading or -masking agents (phospholipase C
and neomycin, respectively) produced the same effects as ATP
removal, which suggested that ATP is required, at least in part,
for PtdIns(4,5)P2 synthesis (Eberhard et al., 1990). A search
for the cytosolic factors required for the ATP-dependent
priming step led to the identification of two enzymes involved
in phosphoinositide metabolism: a phosphatidylinositol
transfer protein (PITPα) (Hay and Martin, 1993) and a type I
PIP kinase, which functions primarily as a PIP(4)P 5-kinase
(Hay et al., 1995). These findings, together with the
identification of a DCV-associated PtdIns 4-kinase essential
for DCV exocytosis (Wiedemann et al., 1996), led to the
hypothesis that generation of PtdIns(4,5)P2 from PtdIns is an
important step preceding DCV exocytosis.
PITPα catalyzes the ATP-independent exchange of PtdIns
between membrane bilayers in vitro (Wirtz, 1991; Sha and
Luo, 1999). Its identification as a factor required for an ATPdependent step may reflect its reported role in PtdIns(4,5)P2
synthesis. PITP might replenish substrates for PtdIns(4,5)P2
generation at specific membrane microdomains at which
PtdIns(4,5)P2 generation is needed for secretion. Alternatively,
or in addition, it might play a more direct role in
phosphoinositide synthesis by presenting the lipid substrate to
lipid kinases (Cunningham et al., 1995; Kearns et al., 1998).
Whereas PI 4-kinase is tightly bound to DCV membranes
(Wiedemann et al., 1996; Gasman et al., 1998), PIP 5-kinase
activity appears to be primarily associated with the plasma
membrane (Wiedemann et al., 1998). Furthermore, recent
studies have suggested that the PtdIns(4,5)P2 pool involved in
exocytosis is localized in the plasma membrane (Holz et al.,
2000). Thus, it remains to be established whether the
PtdIns(4)P generated by the DCV-associated kinase represents
the precursor of PtdIns(4,5)P2 needed for exocytosis. In
principle, plasma-membrane-associated PIP 5-kinase could
phosphorylate ‘in trans’ DCV-membrane PI(4)P after DCVs
have docked at the plasma membrane. Alternatively, another
PI(4) kinase, present on the plasma membrane, may come
into play. The precise function of PtdIns(4,5)P2 in DCV
docking/fusion reactions remains unknown. Generation of
Ins(1,4,5)P3 and diacylglycerol from PtdIns(4,5)P2 is not
required for exocytosis, since phospholipase C treatment of
broken cell preparations even reverses the priming reaction
(Eberhard et al., 1990). Furthermore, PI 3-kinase inhibitors do
not affect regulated exocytosis, which indicates that
PtdIns(3,4,5)P3 generation from PtdIns(4,5)P2 is not required
for this process (Martin et al., 1997). Hence, PtdIns(4,5)P2 is
likely to act directly on a specific target.
After ATP-dependent priming, progression of DCVs to
Exocytosis
Endocytosis
ATP ADP
PtdIns
PtdIns(4)P
PI4K
Pi
ATP ADP
PtdIns(4)P
PtdIns(4,5)P2
PtdIns(4,5)P2
Pi
PtdIns(4)P
PtdIns
synaptojanin1
PIP5K
?
PtdIns(4,5)P2
PtdIns(4)P
PtdIns(4,5)P2
dynamin
clathrin coat
actin
Fig. 1. A putative link between membrane traffic at the synapse and PtdIns(4,5)P2 synthesis/dephosphorylation. The model proposes the
existence of a phosphoinositide cycle nested within the synaptic vesicle cycle. PI4-K, PI 4-kinase; PIP5-K, PIP 5-kinase.
PIs in membrane traffic at the synapse
fusion with the plasma membrane requires Ca2+ and cytosolic
factors (Walent et al., 1992). In broken PC12 cells (a
neuroendocrine cell line), a 145 kDa protein – CAPS (Ca2+dependent Activator Protein for Secretion) – can substitute for
cytosol in Ca2+-triggered exocytosis (Walent et al., 1992). This
protein is abundantly expressed in tissues of neuroectodermic
origin. It binds to Ca2+ with moderate affinity (Ann et al., 1997)
and to PtdIns(4,5)P2 with high specificity but relatively low
affinity in a variety of assays (Loyet et al., 1998). In vivo, a
large fraction of CAPS is bound to the plasma membrane and
DCVs. CAPS is the orthologue of the product of the nematode
unc-31 gene, and its critical role in neurosecretion is supported
by defects in serotonin release observed in unc-31 mutants
(Avery et al., 1993). These findings make CAPS a potential
major effector of PtdIns(4,5)P2 in regulated secretion of
DCVs. However, its precise role in exocytosis is unknown.
Microinjection of anti-CAPS antibodies in melanotrophs has
shown that CAPS acts at a late stage in the secretory pathway
(Rupnik et al., 2000). Binding of CAPS to PtdIns(4,5)P2 results
in the partial penetration of this protein into the lipid bilayer
(Loyet et al., 1998). Furthermore, CAPS changes its 3-D
structure in response to PtdIns(4,5)P2 binding, and this effect
is reversed by increases in Ca2+ concentration to levels found
in stimulated secretory cells (Loyet et al., 1998). Altogether,
these data suggest that CAPS is part of the Ca2+-sensing
machinery implicated in the fusion of DCVs with the plasma
membrane.
SV exocytosis
Evidence for a requirement for PtdIns(4,5)P2 synthesis in SV
secretion is not as compelling as in DCV secretion, and
conflicting reports have been published. Wiedemann et al.
showed that, as in the case of regulated secretion of
catecholamines from DCVs, incubation of synaptosomes with
inhibitors of inositol phospholipid phosphorylation impairs
glutamate release (Wiedemann et al., 1998). However, these
drugs might have additional effects, which thus limits the
conclusions that can be drawn from their use. Furthermore,
similar experiments conducted in another laboratory led to
opposite results: norepinephrine secretion was dependent on
phosphoinositide phosphorylation, whereas glutamate and
GABA secretion was not (Khvotchev and Sudhof, 1998). The
CAPS protein described above was reported to be selectively
localized on DCVs and to be absent from SVs. Indeed two
groups have shown that CAPS is not required for glutamate
release from SVs in assays involving semi-lysed synaptosomes
(Berwin et al., 1998; Tandon et al., 1998). Studies in
nematodes, however, revealed that unc-31 mutants not only
accumulate serotonin (Desai et al., 1988), which can be costored with neuropeptides in DCVs, but also have striking
locomotion defects (Avery et al., 1993), resistance to the
acetylcholinesterase inhibitor aldicarb and sensitivity to the
acetylcholine agonist levamisole (Miller et al., 1996). These
observations may implicate nematode CAPS in regulated
secretion from SVs as well.
Several proteins that have been implicated in
neurotransmitter release, including release from SVs, bind to
phosphoinositides in vitro and thus provide indirect evidence
for some role for these lipids in SV exocytosis. C2 domains,
which are Ca2+- and acidic-phospholipid-binding modules
1043
(Rizo and Sudhof, 1998), are present in SV-associated proteins
(e.g. synaptotagmin, rabphilin, DOC and Munc13) and in
proteins of the cytoskeletal scaffold that anchors SVs to the
plasma membrane (e.g. rim) (Wang et al., 1997) and piccolo
(Fenster et al., 2000). Two of these proteins, synaptotagmin and
rabphilin, have been shown to bind to PtdIns(4,5)P2 directly
(Schiavo et al., 1996; Chung et al., 1998). Synaptotagmin, an
intrinsic SV membrane protein, has a putative role in the Ca2+
regulation of neurotransmitter release (Geppert and Sudhof,
1998), and one of the effects of Ca2+ on synaptotagmin in vitro
is to act as a switch regulating its relative preference for
PtdIns(4,5)P2 or PtdIns(3,4,5)P3 (Schiavo et al., 1996). The
functional relevance of this binding for exocytosis is underlined
by results of experiments at the squid giant synapse.
Microinjection of antibodies directed against the C2B domain
of synaptotagmin prevents the inhibition of neurotransmitter
release induced by co-injection of inositol high-polyphosphates
(IPPs), which function as competitive inhibitors of
phosphoinositides (Fukuda et al., 1995; Mochida et al., 1997).
Rabphilin is an effector for the SV-associated GTPase Rab3a
and is recruited to SVs by Rab3a-GTP. Like synaptotagmin, it
binds PtdIns(4,5)P2 by means of its C2B domain. A peptide
from the phosphoinositide-binding region of rabphilin inhibits
DCV exocytosis from permeabilized chromaffin cells (Chung
et al., 1998). However, rabphilin-knockout mice do not show
any obvious defect in neurotransmission (Schluter et al., 1999).
Recently, Augustin et al. reported a dramatic reduction of
the readily releasable pool of glutamate in mice lacking another
C2-domain-containing presynaptic protein, Munc13-1, and
have suggested a role for Munc13-1 in SV priming (Augustin
et al., 1999). Munc13-1 could therefore be a target for the
action of PtdIns(4,5)P2 in the priming reaction of exocytosis,
but it has not yet been shown to bind phosphoinositides.
An interesting class of membrane-associated PtdIns(4,5)P2binding protein at the pre-synapse is the Mint family. Mint1
and Mint2 participate in SV exocytosis by interacting with
Munc18-1/N-Sec1 (Okamoto and Sudhof, 1997) and in the
structural organization of synaptic junctions by interacting with
the CASK-neurexin complex (Butz et al., 1998). Munc18-1, a
syntaxin-binding protein, has a fundamental function in
SNARE-mediated fusion, because SV exocytosis, both
spontaneous and evoked, is completely absent in Munc18-1knockout mice in spite of the absence of major defects in
synaptic organization and structure (Verhage et al., 2000).
Exocytosis in Drosophila and yeast
In spite of evidence supporting a role for phosphoinositides
in neurosecretion, genetic studies in Drosophila and yeast
caution about concluding that inositol phospholipids play a
fundamental, rather than regulatory, role in exocytosis. In
Drosophila, disruption of a PIP 5-kinase gene – skittles –
produces embryonic lethality and major alterations in the
cytoskeletal organization of neurons but no defects in either
SV or DCV secretion, as assessed by electrophysiological
recordings at the Drosophila larval body-wall neuromuscular
junction (Hassan et al., 1998). However, at least one other
homologue of mammalian PIP 5-kinases (i.e. type I PIP
kinases) is present in Drosophila (gene CG3682 at
http://flybase.bio.indiana.edu), which could compensate for
loss of skittles.
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JOURNAL OF CELL SCIENCE 114 (6)
In yeast, disruption of MSS4, which encodes the only PI(4)P
5-kinase in this organism, produces defects in the actin
cytoskeleton but not in secretion (Desrivieres et al., 1998;
Homma et al., 1998). However, inactivation of the yeast PIK1,
which encodes a PI 4-kinase, is lethal, and temperaturesensitive mutants of this gene have severely impaired secretion
and endocytosis at restrictive temperature (Hama et al., 1999;
Walch-Solimena and Novick, 1999; Audhya et al., 2000). The
yeast orthologue of frequenin, a Ca2+-binding regulatory
protein present in nerve terminals, is an essential co-factor
for Pik1p (Hendricks et al., 1999). Strikingly, frequenin
overexpression in Drosophila results in chronic facilitation of
transmitter release at the larval neuromuscular junction and
multiple firing of action potentials (Pongs et al., 1993; AngautPetit et al., 1998).
Phosphoinositides in SV endocytosis
After exocytosis, SV membranes are rapidly internalized and
reused for the generation of new SVs. A major pathway for
their reformation involves a specialized form of clathrinmediated endocytosis (Heuser and Reese, 1973; GonzalesGaitan and Jackle, 1997; De Camilli et al., 2000), and
phosphoinositides appear to play an important role at several
steps of this pathway.
Clathrin coat recruitment and invagination
Clathrin-mediated endocytosis is thought to start with the
binding of the heterotetrameric clathrin adaptor complex AP2 and of the accessory clathrin adaptor protein AP180 to both
protein and lipids in the plasma membrane. Subsequently, the
adaptors recruit clathrin and promote its assembly to form the
coat (Cremona and De Camilli, 1997; McMahon, 1999; Brodin
et al., 2000; Kirchhausen, 2000).
Studies on Ca2+ signaling provided the first evidence for
a role for phosphoinositides in clathrin coat recruitment.
The identification of the Ins(1,4,5)P3 receptor and the
characterization of biosynthetic pathways that generate other
inositol polyphosphates (IPPs) led to a search for novel IPP
interactors. Unexpectedly, components of the synaptic clathrin
coat, namely AP-2 and AP180, and synaptotagmin were
isolated as major IPP-binding proteins (Beck and Keen, 1991;
Timerman et al., 1992; Voglmaier et al., 1992; Fukuda et al.,
1994; Niinobe et al., 1994; Ye et al., 1995). Subsequent studies
demonstrated that these proteins also bind phosphoinositides,
suggesting that, at least in the case of adaptors, these
interactions may function in coat recruitment to the bilayer
(Fig. 2; Gaidarov et al., 1996; Hao et al., 1997; Rapoport et al.,
1997). Ye et al. mapped the phosphoinositide-binding site to a
cluster of positive residues in the N-terminal ENTH-like
domain of AP180 (Ye et al., 1995) and the N-terminal region
of the α-adaptin subunit of AP-2 (Gaidarov et al., 1996;
Gaidarov et al., 1999). As discussed above, binding of
phosphoinositides to synaptotagmin is mediated by its C2B
domain, which also acts as a main membrane-docking site for
the α-adaptin subunit of AP-2 (Zhang et al., 1994; Haucke and
De Camilli, 1999).
The physiological role of these interactions is supported by
a variety of studies. PITP is required for clathrin-dependent SV
biogenesis in a cell-free assay (Schmidt and Huttner, 1998).
Furthermore, expression in wild-type fibroblastic cells of an αadaptin that has mutations in its phosphoinositide-binding site
results in the assembly of AP-2 complexes that do not bind to
the plasma membrane and, as a result, have dominant negative
effect on clathrin-mediated transferrin uptake (Gaidarov et al.,
1999). Masking of PtdIns(4,5)P2 by exogenous molecules
inhibits early steps of clathrin-mediated endocytosis (Jost et al.,
1998). Finally, the critical importance of PtdIns(4,5)P2
synthesis in the recruitment of AP-2/clathrin coats to a
physiological membrane was demonstrated in cell-free studies
using endosomal and lysosomal membranes as templates (West
et al., 1997; Arneson et al., 1999). Although AP-2 has a higher
affinity for PtdIns(3,4,5)P3 than PtdIns(4,5)P2 (Gaidarov et al.,
1996), there is no evidence so far for a role of PtdIns(3,4,5)P3
in synaptic vesicle recycling. It has been shown, however, that
a PI 3-kinase isoform (class II phosphoinositide 3-kinase C2α)
is associated with clathrin-coated vesicles from bovine brain.
This raises the possibility that PtdIns(3,4,5)P3 plays a role in
some clathrin-dependent budding reactions (Domin et al.,
2000).
Liposomes alone can support the assembly of endocytic
clathrin coats, which provides direct evidence for interactions
between lipids and coats. In these experimental conditions,
acidic phospholipids are strictly required for coat formation,
and the presence of phosphoinositides enhances coat
recruitment (Takei et al., 1998; Cremona et al., 1999). The
interactions of adaptors with membrane proteins or membrane
lipids are likely to be synergistic. Examples of positive
cooperativity between the two types of interaction are the
reported effect of 3′-phosphorylated phosphoinositides on the
binding of endocytic motifs to AP-2 and the effect of
phospholipase D – possibly mediated by phosphatidic acid or
PtdIns(4,5)P2 – on the recruitment of AP-2 to synaptotagmin
(Rapoport et al., 1997; Haucke and De Camilli, 1999).
Cooperativity of lipid- and protein-binding sites on the
membrane might play a critical role in vivo in the regulation
of clathrin coat nucleation in time and space. The generation
of phosphoinositides at a specific membrane location could
trigger coat assembly at a protein binding site which, by itself,
is insufficient to nucleate assembly.
Besides AP-2 and AP-180, other proteins implicated in
clathrin-mediated membrane internalization bind to
phosphoinositides. Among these factors are the ubiquitous
non-visual arrestins (β-arrestins), a family of proteins that
mediate G-protein-coupled receptor (GPCR) sequestration to
endosomes. β-Arrestins bind to clathrin and AP-2, as well as
to IPPs, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, with high affinity
(Gaidarov et al., 1999). Expression of β-arrestin mutants
lacking phosphoinositide-binding properties results in marked
inhibition of β-adrenergic receptor internalization (Gaidarov et
al., 1999). So far, there is no evidence for a role of β-arrestin
in SV recycling. However, these findings suggest that
phosphoinositide binding represents a common theme in
clathrin-adaptor recruitment to the plasma membrane
(Gaidarov and Keen, 1999).
Two well-characterized clathrin accessory proteins that
participate in SV recycling at the synapse are amphiphysin and
endophilin (Slepnev and De Camilli, 2000). Both proteins bind
to lipids, and amphiphysin affinity for membranes is enhanced
by the presence of phosphoinositides (Cremona et al., 1999).
Amphiphysin also binds to clathrin (McMahon et al., 1997;
PIs in membrane traffic at the synapse
1045
PtdIns(4,5)P2
µ
ασ β
AP-2
AP180
clathrin
dynamin
Fig. 2. Schematic representation of the
clathrin coat and of some of the
interactions involving its components
(De Camilli et al., 2000).
Ramjaun et al., 1997; Slepnev et al., 1998), AP-2 (Wang et al.,
1995; Slepnev et al., 1998), dynamin (David et al., 1996) and
another clathrin-accessory protein, synaptojanin 1 (McPherson
et al., 1996) (see below). Thus, it might function as an adaptor
that coordinates the binding of other endocytic proteins to the
membrane. Endophilin is critical for the biogenesis of SVs in
a cell-free assay (Schmidt et al., 1999) and plays an essential
role in the invagination of clathrin-coated pits in vivo (Ringstad
et al., 1999). Like amphiphysin, endophilin binds dynamin and
synaptojanin and might be involved in their recruitment to sites
of endocytosis (de Heuvel et al., 1997; Ringstad et al., 1997).
Endophilin also functions as a lysophosphatidic acid acyltransferase, mediating the synthesis of phosphatidic acid
(Schmidt et al., 1999). Potential roles of this enzymatic activity
include a direct effect on membrane curvature – given the
different geometry of lysophosphatidic acid and phosphatidic
acid in the plane of the membrane (Schmidt et al., 1999) – but
also a stimulatory effect on PtdIns(4,5)P2 production, through
stimulation of PIP 5-kinases by phosphatidic acid (Jenkins et
al., 1994).
Fission of endocytic pits
Constriction and fission of deeply invaginated buds to generate
free vesicles requires the GTPase dynamin, which is a
phosphoinositide-binding protein (the properties and function
of this protein have been extensively reviewed; De Camilli and
Takei, 1996; Hinshaw, 1999; McNiven et al., 2000; Sever et
al., 2000). Dynamin forms a scaffold around the vesicle stalk.
This scaffold may function in fission either directly, via a GTPhydrolysis-dependent conformational change that cleaves the
vesicle neck, or indirectly, by recruiting and/or regulating other
proteins (Kosaka and Ikeda, 1983; Hinshaw and Schmid, 1995;
Takei et al., 1995; Sweitzer and Hinshaw, 1998; Takei et al.,
1998). However, the presence of dynamin on growing clathrin
buds (Takei et al., 1996) and the absence of clathrin coats in
Drosophila mutants of dynamin (Kosaka and Ikeda, 1983)
suggest additional roles of this molecule in coat formation
and/or stabilization.
The phosphoinositide-binding region of dynamin is
localized in its PH domain, which binds several
phosphoinositides, in particular PtdIns(4,5)P2 (Barylko et al.,
1998). Phosphoinositides considerably boost GTPase activity
of dynamin (Zheng et al., 1996) and show cooperativity in this
synaptotagmin
function with SH3-domain-containing proteins (Barylko et al.,
1998) and with conditions that promote dynamin assembly
(Klein et al., 1998). However, regulation of GTPase activity is
unlikely to be the only or the main effect of phosphoinositides
on dynamin. Transfection of dynamin mutants lacking the PH
domain or of point mutants that exhibit impaired PtdIns(4,5)P2
binding inhibits receptor-mediated endocytosis (Barylko et al.,
1998; Lee et al., 1999; Vallis et al., 1999). This inhibition can
be rescued by deletion of the proline-rich region of the
molecule, which plays a critical role in targeting dynamin to
its sites of action, possibly by interactions with SH3-domaincontaining proteins. Thus, it would appear that binding of
dynamin to PtdIns(4,5)P2 is crucial after its targeting to
endocytic sites (Vallis et al., 1999).
Phosphoinositides could stabilize the anchoring of dynamin
to membranes after its recruitment by SH3-domain-containing
proteins; alternatively, the PH domain of dynamin could recruit
phosphoinositides to the neck of invaginated buds to favor
membrane bending and/or fission. Evidence suggests that
amphiphysin and endophilin are functional partners of
dynamin in the fission reaction (Schmidt and Huttner, 1998;
Takei et al., 1999). At least some of the actions of these two
proteins may also be mediated by their direct interactions with
lipids (Takei et al., 1999; Farsad et al., 2000).
Clathrin uncoating
The hypothesis that phosphoinositides play an important role
in the recruitment and function of endocytic proteins at the
synapse has recently been supported by the identification and
characterization of the polyphosphoinositide phosphatase
synaptojanin 1. Synaptojanin 1 is enriched at nerve terminals
and interacts with a variety of proteins of the endocytic
machinery (McPherson et al., 1996; Roos, 1998; de Heuvel et
al., 1997; Haffner et al., 1997; Ringstad et al., 1997; Sakisaka
et al., 1997; Yamabhai et al., 1998; Qualmann et al., 1999). It
hydrolyzes several phosphoinositide species, including
PtdIns(4,5)P2 and PtdIns(3,4,5)P3 (McPherson et al., 1996;
Chung et al., 1997; Woscholski et al., 1997; Cremona et al.,
1999; Guo et al., 1999). Given its enzymatic activity,
synaptojanin 1 is expected to be a negative regulator of coatmembrane interactions during uncoating, and this prediction
was confirmed genetically (Cremona et al., 1999).
Nerve terminals of synaptojanin-1-knockout mice (Cremona
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JOURNAL OF CELL SCIENCE 114 (6)
et al., 1999) contain an increased number of clathrin-coated
vesicles in the actin-rich area that surrounds SV clusters.
Furthermore, cell-free studies on brain cytosol of wild-type and
synaptojanin-1-knockout mice have revealed that the knockout
cytosol is more potent in promoting coat recruitment to
liposomes under conditions in which phosphoinositides
undergo turnover. This effect correlates with an accumulation
of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 on these artificial
membranes. However, the major phosphoinositide species
increased in neurons of knockout animals is PtdIns(4,5)P2
(Cremona et al., 1999). Consistent with these observations,
disruption of synaptojanin 1 recruitment and/or function by
antibody and peptide microinjection at lamprey giant synapses
produces an accumulation of clathrin-coated vesicles and pits
(Gad et al., 2000). Finally, an increased number of clathrincoated structures is observed in nerve terminals of C. elegans
unc26 mutants, which harbor mutations in the only
synaptojanin-like gene of this organism (Harris et al., 2000).
Besides an increase in clathrin coats, a hypertrophy of the
actin-based cytoskeleton that surrounds the active zone was
very evident in injected lamprey synapses (Gad et al., 2000)
(see below). A plausible explanation for the accumulation of
clathrin-coated membranes is that, in vivo, synaptojanin 1
plays a role in clathrin uncoating by decreasing the affinity of
the adaptors for the plasma membrane. Such a hypothesis fits
with the observation that the ATPase Hsc70, which is critically
required for clathrin uncoating, is not sufficient to remove the
adaptors (Hannan et al., 1998). Other morphological alterations
clearly evident in the lamprey and/or C. elegans model systems
(e.g. defects in fission and an enhanced actin cytoskeleton)
might reflect the pleiotropic roles of PtdIns(4,5)P2 and its
important action in actin nucleation (see below and Sakisaka
et al., 1997); alternatively, they might be secondary effects due
to the sequestration of coat components and their accessory
factors on membranes.
A phosphoinositide cycle nested within a GTPase
cycle?
Given the hypothesis that phosphoinositides play a role in the
nucleation of clathrin-coated vesicles, these phospholipids
might undergo regulated synthesis prior to endocytosis. An
attractive possibility is that the same pool of phosphoinositides
generated in preparation for exocytosis (see above and Fig. 3)
participates in the coating reaction. This pool might then be
supplemented by additional phosphoinositide synthesis
occurring after exocytosis is completed.
Studies of budding reactions on a variety of intracellular
membranes have implicated small GTPases as critical switches
for the initiation of coat recruitment; more specifically, ARF
family members appear to be involved in the nucleation of
clathrin coats (Springer et al., 1999). Some of the effects of
ARF, in turn, are mediated by phosphoinositides. Current
models predict that coat recruitment starts when a guanine
nucleotide exchange factor (GEF) for ARF (the signature for
which is the zpresence of a Sec7-homology domain) generates
ARF-GTP (Serafini et al., 1991; Goldberg, 1998; Mossessova
et al., 1998), ARF-GTP binds to membranes and promotes
coating by a series of independent but synergistic mechanisms,
including direct binding to coat proteins – as shown for the
COP1 coat (Goldberg, 1998) – or the recruitment and/or
activation of enzymes that enhance production of
phosphoinositides. For example, ARF binds to and activates
phospholipase D (Roth et al., 1999), which in turn generates
phosphatidic acid, a potent activator of PIP 5-kinases (Jenkins
et al., 1994). ARF proteins can also recruit PI 4- and PIP 5kinases (Godi et al., 1999; Honda et al., 1999; Jones et al.,
2000). Phosphoinositides generated by these reactions, in turn,
may participate in a positive feed-forward loop to stimulate
phospholipase D further (Liscovitch and Cantley, 1995; Singer
et al., 1997) or to stimulate ARF itself through additional
recruitment of ARF GEFs that contain phosphoinositidebinding PH domains (Jackson and Casanova, 2000). These
results and considerations suggest the existence of a
phosphoinositide phosphorylation/dephosphorylation cycle
that acts downstream of the GTP/GDP cycle of ARF-family
GTPases (Fig. 3).
Although ARF6 was proposed to have a role in clathrinmediated endocytosis (D’Souza-Schorey et al., 1995), it is
unclear whether ARF family members function in clathrinmediated SV recycling. So far, no ARF proteins have been
found to be concentrated at the synapse, and brefeldin A, which
blocks a subset of ARF-GEFs, does not block clathrinmediated SV recycling (Mundigl et al., 1993; Shi et al., 1998).
However, proteins that act upstream or downstream of ARF are
present in nerve terminals, and recently at least a brefeldin-Aindependent ARF-GEF, Msec7, has been identified in this
compartment (Ashery et al., 1999; Neeb et al., 1999). Msec71 is the rat homologue of human cytohesin-1 and belongs to a
subset of brefeldin-A-insensitive ARF-GEFs that includes
ARNO, GRP1/ARNO3 and GBF1. Notably, these proteins
have been shown to bind to phosphoinositides and to be
recruited to the plasma membrane upon local production of
PtdIns(3,4,5)P3 (Jackson and Casanova, 2000). Thus, the
existence of an ARF-like protein in nerve terminals is
plausible. Furthermore, components of the endocytic
machinery, namely AP180 and amphiphysin, inhibit
phospholipase D (Lee et al., 1997; Lee et al., 2000) and might
help to shut down the positive feedback loop depicted in Fig.
3. Synaptojanin 1, which cleaves PtdIns(4,5)P2, could also
function as a very powerful inhibitor of this feedback loop by
acting as a switch-off signal at the end of the endocytic
reaction. Finally, the involvement of an ARF-like GTPase in
SV endocytosis is supported by the strong stimulatory effect
of GTPγS on clathrin coat recruitment (Takei et al., 1995;
Gustaffson et al., 1998). However, in these experiments it is
difficult to discriminate between a bona fide stimulatory effect
of GTPγS on coating mediated by an ARF-like protein or other
G proteins and an indirect stabilization of clathrin coats due to
inhibition of the fission reaction. An important priority for
future studies will be the precise identification of the enzymes
responsible for phosphoinositide biosynthesis in nerve
terminals and their regulation by GTPases.
Regulation of actin dynamics in the presynaptic
compartment
Many proteins that function in the regulation of actin dynamics
bind to phosphoinositides, and local production of
phosphoinositides on membranes is a mechanism to generate
focal nucleation of actin (Janmey, 1994; Martin, 1998; Sechi
and Wehland, 2000). A major pathway through which
PIs in membrane traffic at the synapse
1047
ARF GEF
Fig. 3. Diagram illustrating a positive feedback loop through which
activation of the GTPase ARF leads to enhanced PtdIns(4,5)P2
generation, which in turn further activates ARF. Some of the
effectors of ARF are enzymes that lead to PtdIns(4,5)P2 synthesis.
PtdIns(4,5)P2 not only regulates effectors such as the actin
cytoskeleton, vesicle coats and signaling scaffolds but further
stimulates formation of ARF-GTP through its recruitment of PHdomain-containing ARF guanine nucleotide exchange factors
(GEFs). Synaptojanin 1 acts as a major ‘off’ switch for this feedback
loop. This model was derived from data obtained in a variety of
systems, including coating reactions on Golgi complex membranes,
where ARF-GTP and PtdIns(4,5)P2 cooperate in the recruitment of at
least on type of coat, COP1. It is still unclear whether an ARF or
ARF-like protein is involved in synaptic vesicle recycling.
phosphoinositides can promote actin nucleation involves their
cooperation with Rho-type GTPases in recruiting WASP
family members to the plasma membrane (Rohatgi et al.,
1999; Zigmond, 2000). A convergence of biochemical and
morphological studies have recently suggested that the actin
cytoskeleton has an important role in the SV cycle (Gustaffson
et al., 1998; De Camilli et al., 2000; Qualmann et al., 2000)
and therefore raised the possibility that phosphoinositide
turnover plays a critical role in the control of this actin pool.
Strands of actin have been detected within the SV cluster
(Hirokawa et al., 1989), which is consistent with the ability of
synapsin, a major SV-associated protein (De Camilli et al.,
1990), to bind to actin. However, the bulk of presynaptic actin
is localized at the periphery of the vesicle cluster. This
localization is particularly evident at large synapses, such as
the frog neuromuscular junction or the synapses of the giant
reticulospinal axon of the lamprey, where filamentous actin can
be visualized with a sufficient level of resolution by light
microscopy using fluorescent phalloidin (Brodin, 1999;
Dunaevsky and Connor, 2000; Gad et al., 2000). Genetic
studies in C. elegans and Drosophila have shown that these
actin-rich zones, which surround ‘active zones’ of secretion,
play an important role in defining the structure of the synapse
(Schaefer et al., 2000; Wan et al., 2000; Zhen et al., 2000).
Furthermore, they are the sites at which the majority of
clathrin-coated vesicles form (Heuser and Reese, 1973; Roos
and Kelly, 1999; Gad et al., 2000). It is therefore interesting
that some of the proteins involved in SV endocytosis also
appear to have a role in actin function (Mundigl et al., 1998;
Witke et al., 1998; Ochoa et al., 2000; Qualmann and Kelly,
2000). Significantly, in yeast, all forms of endocytosis are
critically dependent on actin (Geli and Riezman, 1998).
Synaptojanin 1 function is strongly linked to actin dynamics.
As discussed above, disruption of synaptojanin 1 function at
the synapse results not only in an impairment of the SV cycle
but also in accumulation of actin around active zones; this
suggests that an equilibrium between polymerization and
depolymerization of actin has been altered (Sakisaka et al.,
1997; Gad et al., 2000). These effects can be explained by a
corresponding imbalance between PtdIns(4,5)P2 synthesis and
dephosphorylation. Studies of the giant lamprey synapse under
a variety of experimental conditions have shown that this actin
pool is highly dynamic and that its polymerization is enhanced
by nerve terminal stimulation (Gustaffson et al., 1998; Brodin,
1999). The precise role of actin in the endocytosis reaction is
coats
actin cytoskeleton
ARF
signaling scaffolds
phospholipase D
PI 4-kinase
PtdIns
synaptojanin 1
phosphatidic
acid
PIP 5-kinase
PtdIns(4) P
PtdIns(4,5) P2
still unclear. One of its roles may be to propel the nascent
vesicle away from the membrane back to the vesicle cluster.
Fate of the vesicle after endocytosis
In the classical model of clathrin-mediated endocytosis, an
endosomal sorting station is the target for endocytic vesicles
after uncoating. Phosphoinositide metabolites, in particular
PtdIns(3)P, which binds to FYVE domains, play a critical role
in endosomal function in a variety of systems (Corvera et al.,
1999; Stenmark and Aasland, 1999; Wurmser et al., 1999).
However, the involvement of endosomes, and therefore of
PtdIns(3)P, in SV recycling is questionable. There is evidence
that an endosomal sorting station can be bypassed during SV
reformation and that newly uncoated vesicles are targeted
directly to the SV cluster (Takei et al., 1996; Murthy and Stevens,
1998). The few bona fide endosomes present at the synapse
might mediate housekeeping membrane recycling and sorting –
possibly via the AP3-dependent pathway (Shi et al., 1998) – of
the fraction of SV proteins that escapes direct recycling. From
these endosomes, some proteins are targeted to the axon for
retrograde flow in multivescicular bodies. It will be of interest
to determine whether homologues of the yeast Fab protein (a
PI(3)P 5-kinase) participate in the biogenesis of these organelles,
as Emr and co-workers have proposed for the biogenesis of
homologous organelles in yeast (Odorizzi et al., 1998).
Endosome-like compartments do accumulate in nerve
terminals after massive stimulation, but they are thought to
form by bulk endocytosis from the plasma membrane (Takei et
al., 1996). Thus, they may function in parallel with the plasma
membrane as ‘donors’ of clathrin-coated vesicles rather than
as ‘acceptors’ of newly uncoated vesicles. Bulk endocytosis is
thought to be an actin-dependent reaction (Schmalzing et al.,
1995), and is thus another process that might involve
phosphoinositide-mediated actin polymerization.
General considerations about phosphoinositides
and membrane traffic
Several features make phosphoinositides powerful modulators
of membrane traffic and membrane-cytoskeleton interactions,
in addition to their well-established roles as precursors of
intracellular second messengers. First, the reversible
phosphorylation of their inositol group can function in the
regulated and reversible recruitment of cytosolic proteins to
1048
JOURNAL OF CELL SCIENCE 114 (6)
membranes, much like tyrosine phosphorylation of membrane
proteins. Second, the phosphorylated state of the entire
population of inositol phospholipids in a membrane
microdomain could in principle be changed very rapidly owing
to positive feedback loops built into phosphoinositide
metabolic pathways (such as that involving ARF,
phospholipase D and phosphoinositide kinases; see Fig. 3).
Third, one can predict from structural properties of
phosphoinositide-metabolizing enzymes that at least some of
them act processively – that is, they undergo several cycles of
catalysis without leaving the membrane (Scott et al., 1990;
Scott et al., 1994; Rao et al., 1998). In the case of a vesicle,
such a mechanism could allow the modification of the
composition of the entire vesicle membrane by one or few
enzyme molecules. Fourth, owing to the synergistic roles of
phosphoinositides and membrane proteins, generation of
specific phosphoinositide species can be used to temporally
and spatially regulate the recruitment of cytosolic proteins.
Because of these properties, phosphoinositides can function
as very powerful signals to tag a membrane for a given fate.
Thus, much like GTPases, phosphoinositides can function in
regulating membrane traffic and in defining the vectoriality of
transport. Not surprisingly, a reciprocal relationship between
these two regulatory mechanisms has been identified.
Concluding remarks
Here, we have summarized growing evidence linking
phosphoinositide metabolism to the control of neurosecretion
and the SV cycle. An important role for PtdIns(4,5)P2 has
clearly emerged both in the cascades of reactions leading to
exocytosis and in the compensatory endocytic mechanisms.
Other phosphoinositides probably play roles in presynaptic
function. The dual effects of phosphoinositides on coat
formation and actin nucleation strongly suggest that these two
processes are linked. Some protein targets for the actions of
phosphoinositides have been identified, and genetic disruption
of normal phosphoinositide metabolism has been shown to
affect the vesicle cycle. It remains to be understood whether
phosphoinositides act as essential switches or simply as critical
regulatory components. The mild phenotype observed in yeast
after disruption of PtdIns(4,5)P2-synthesizing enzymes speaks
against a fundamental role of this phosphoinositide species in
the exocytic reaction. However, further use of genetics in
higher organisms will be required if we are to answer this
question. Given the high cytosolic levels of certain IPP species,
it will be important to understand the potential physiological
role of competition between IPPs and phosphoinositides in
binding to specific proteins and the possible independent roles
of IPPs. It will also be important to define how the effects of
phosphoinositides on actin and membrane traffic discussed
here impact on intracellular signaling pathways controlled by
phosphoinositide metabolites. Although, we anticipate that
these two actions of phosphoinositides are interrelated, this
remains an unexplored area of research.
We thank Drs Markus Wenk, Niels Ringstad, Gilbert Di Paolo and
Lorenzo Pellegrini for discussion and critical review of the
manuscript. Work reviewed in this commentary and carried out in the
lab. of the authors was supported in part by grants NIH NS36251 and
CA46128 to PDC and by grants from Telethon (project D61 and
D111) and from MURST (COFIN97 and COFIN2000) to O.C.
References
Angaut-Petit, D., Toth, P., Rogero, O., Faille, L., Tejedor, F. J. and Ferrus,
A. (1998). Enhanced neurotransmitter release is associated with reduction
of neuronal branching in a Drosophila mutant overexpressing frequenin.
Eur. J. Neurosci. 10, 423-434.
Ann, K., Kowalchyk, J. A., Loyet, K. M. and Martin, T. F. (1997). Novel
Ca2+-binding protein (CAPS) related to UNC-31 required for Ca2+-activated
exocytosis. J. Biol. Chem. 272, 19637-19640.
Arneson, L. S., Kunz, J., Anderson, R. A. and Traub, L. M. (1999). Coupled
inositide phosphorylation and phospholipase D activation initiates clathrincoat assembly on lysosomes. J. Biol. Chem. 274, 17794-17805.
Ashery, U., Koch, H., Scheuss, V., Brose, N. and Rettig, J. (1999). A
presynaptic role for the ADP ribosylation factor (ARF)-specific GDP/GTP
exchange factor msec7-1. Proc. Nat. Acad. Sci. USA 96, 1094-1099.
Audhya, A., Foti, M. and Emr, S. D. (2000). Distinct roles for the yeast
phosphatidylinositol 4-kinases, stt4p and pik1p, in secretion, cell growth,
and organelle membrane dynamics. Mol. Biol. Cell 11, 2673-2689.
Augustin, I., Rosenmund, C., Sudhof, T. C. and Brose, N. (1999). Munc131 is essential for fusion competence of glutamatergic synaptic vesicles.
Nature 400, 457-461.
Avery, L., Bargmann, C. I. and Horvitz, H. R. (1993). The Caenorhabditis
elegans unc-31 gene affects multiple nervous system-controlled functions.
Genetics 134, 455-464.
Barylko, B., Binns, D., Lin, K. M., Atkinson, M. A., Jameson, D. M., Yin,
H. L. and Albanesi, J. P. (1998). Synergistic activation of dynamin GTPase
by Grb2 and phosphoinositides. J. Biol. Chem. 273, 3791-3797.
Beck, K. A. and Keen, J. H. (1991). Interaction of phosphoinositide cycle
intermediates with the plasma membrane-associated clathrin assembly
protein AP-2. J. Biol. Chem. 266, 4442-4447.
Berwin, B., Floor, E. and Martin, T. F. (1998). CAPS (mammalian UNC31) protein localizes to membranes involved in dense-core vesicle
exocytosis. Neuron 21, 137-145.
Bottomley, M. J., Salim, K. and Panayotou, G. (1998). Phospholipid-binding
protein domains. Biochim. Biophys. Acta 1436, 165-183.
Brodin, L. (1999). Actin-dependent steps in synaptic vesicle recycling.
Biochimie 81, S49.
Brodin, L., Low, P. and Shupliakov, O. (2000). Sequential steps in clathrinmediated synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 10, 312-320.
Butz, S., Okamoto, M. and Sudhof, T. C. (1998). A tripartite protein complex
with the potential to couple synaptic vesicle exocytosis to cell adhesion in
brain. Cell 94, 773-782.
Calakos, N. and Scheller, R. H. (1996). Synaptic vesicle biogenesis, docking,
and fusion: a molecular description. Physiol. Rev. 76, 1-29.
Chung, J. K., Sekiya, F., Kang, H. S., Lee, C., Han, J. S., Kim, S. R., Bae,
Y. S., Morris, A. J. and Rhee, S. G. (1997). Synaptojanin inhibition of
phospholipase D activity by hydrolysis of phosphatidylinositol 4,5bisphosphate. J. Biol. Chem. 272, 15980-15985.
Chung, S. H., Song, W. J., Kim, K., Bednarski, J. J., Chen, J., Prestwich,
G. D. and Holz, R. W. (1998). The C2 domains of Rabphilin3A specifically
bind phosphatidylinositol 4,5-bisphosphate containing vesicles in a Ca2+dependent manner. In vitro characteristics and possible significance. J. Biol.
Chem. 273, 10240-10248.
Corvera, S., D’Arrigo, A. and Stenmark, H. (1999). Phosphoinositides in
membrane traffic. Curr. Opin. Cell Biol. 11, 460-465.
Cremona, O. and De Camilli, P. (1997). Synaptic vesicle endocytosis. Curr.
Opin. Neurobiol. 7, 323-330.
Cremona, O., Di Paolo, G., Wenk, M. R., Luthi, A., Kim, W. T., Takei, K.,
Daniell, L., Nemoto, Y., Shears, S. B., Flavell, R. A. et al. (1999). Essential
role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99,
179-188.
Cunningham, E., Thomas, G. M., Ball, A., Hiles, I. and Cockcroft, S.
(1995). Phosphatidylinositol transfer protein dictates the rate of inositol
trisphosphate production by promoting the synthesis of PIP2. Curr. Biol. 5,
775-783.
D’Souza-Schorey, C., Li, G., Colombo, M. I. and Stahl, P. D. (1995). A
regulatory role for ARF6 in receptor-mediated endocytosis. Science 267,
1175-1178.
David, C., McPherson, P. S., Mundigl, O. and de Camilli, P. (1996). A role
of amphiphysin in synaptic vesicle endocytosis suggested by its binding to
dynamin in nerve terminals. Proc. Nat. Acad. Sci. USA 93, 331-335.
De Camilli, P. and Jahn, R. (1990). Pathways to regulated exocytosis in
neurons. Annu. Rev. Physiol. 52, 625-645.
De Camilli, P., Benfenati, F., Valtorta, F. and Greengard, P. (1990). The
synapsins. Annu. Rev. Cell Biol. 6, 433-460.
PIs in membrane traffic at the synapse
De Camilli, P. and Takei, K. (1996). Molecular mechanisms in synaptic
vesicle endocytosis and recycling. Neuron 16, 481-486.
De Camilli, P., Emr, S. D., McPherson, P. S. and Novick, P. (1996).
Phosphoinositides as regulators in membrane traffic. Science 271, 15331539.
De Camilli, P., Slepnev, V. I., Shupliakov, O. and Brodin, L. (2000).
Synaptic Vesicle Endocytosis. The Johns Hopkins University Press.
de Heuvel, E., Bell, A. W., Ramjaun, A. R., Wong, K., Sossin, W. S. and
McPherson, P. S. (1997). Identification of the major synaptojanin-binding
proteins in brain. J. Biol. Chem. 272, 8710-8716.
Desai, C., Garriga, G., McIntire, S. L. and Horvitz, H. R. (1988). A genetic
pathway for the development of the Caenorhabditis elegans HSN motor
neurons. Nature 336, 638-646.
Desrivieres, S., Cooke, F. T., Parker, P. J. and Hall, M. N. (1998). MSS4, a
phosphatidylinositol-4-phosphate 5-kinase required for organization of the
actin cytoskeleton in Saccharomyces cerevisiae. J. Biol. Chem. 273, 1578715793.
Domin, J., Gaidarov, I., Smith, M. E., Keen, J. H. and Waterfield, M. D.
(2000). The class II phosphoinositide 3-kinase PI3K-C2alpha is
concentrated in the trans-Golgi network and present in clathrin-coated
vesicles. J. Biol. Chem. 275, 11943-11950.
Dunaevsky, A. and Connor, E. A. (2000). F-actin is concentrated in
nonrelease domains at frog neuromuscular junctions. J. Neurosci. 20, 60076012.
Durell, J. and Sodd, M. A. (1966). Studies on the acetylcholine-stimulated
incorporation of radioactive inorganic orthophosphate into the phospholipid
of brain particulate preparations. II. Subcellular distribution of enzymic
activity. J. Neurochem. 13, 487-491.
Eberhard, D. A., Cooper, C. L., Low, M. G. and Holz, R. W. (1990).
Evidence that the inositol phospholipids are necessary for exocytosis. Loss
of inositol phospholipids and inhibition of secretion in permeabilized cells
caused by a bacterial phospholipase C and removal of ATP. Biochem. J. 268,
15-25.
Farsad, K., Ringstad, N., Floyd, S., Takei, K. and De Camilli, P. (2000).
Endophilin tubulates lipid bilayers alone and in co-assembly with dynamin.
Mol. Biol. Cell 11, 278a.
Fenster, S. D., Chung, W. J., Zhai, R., Cases-Langhoff, C., Voss, B.,
Garner, A. M., Kaempf, U., Kindler, S., Gundelfinger, E. D. and Garner,
C. C. (2000). Piccolo, a presynaptic zinc finger protein structurally related
to bassoon. Neuron 25, 203-214.
Fukuda, M., Aruga, J., Niinobe, M., Aimoto, S. and Mikoshiba, K. (1994).
Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of
IP4BP/synaptotagmin II. J. Biol. Chem. 269, 29206-29211.
Fukuda, M., Moreira, J. E., Lewis, F. M., Sugimori, M., Niinobe, M.,
Mikoshiba, K. and Llinás, R. (1995). Role of the C2B domain of
synaptotagmin in vesicular release and recycling as determined by specific
antibody injection into the squid giant synapse preterminal. Proc. Nat. Acad.
Sci. USA 92, 10708-10712.
Gad, H., Ringstad, N., Low, P., Kjaerulff, O., Gustafsson, J., Wenk, M.,
Di Paolo, G., Nemoto, Y., Crun, J., Ellisman, M. H. et al. (2000). Fission
and uncoating of synaptic clathrin-coated vesicles are perturbed by
disruption of interactions with the SH3 domain of endophilin. Neuron 27,
301-312.
Gaidarov, I., Chen, Q., Falck, J. R., Reddy, K. K. and Keen, J. H. (1996).
A functional phosphatidylinositol 3,4,5-trisphosphate/phosphoinositide
binding domain in the clathrin adaptor AP-2 alpha subunit. Implications for
the endocytic pathway. J. Biol. Chem. 271, 20922-20929.
Gaidarov, I. and Keen, J. H. (1999). Phosphoinositide-AP-2 interactions
required for targeting to plasma membrane clathrin-coated pits. J. Cell Biol.
146, 755-764.
Gaidarov, I., Krupnick, J. G., Falck, J. R., Benovic, J. L. and Keen, J. H.
(1999). Arrestin function in G protein-coupled receptor endocytosis requires
phosphoinositide binding. EMBO J. 18, 871-881.
Gasman, S., Chasserot-Golaz, S., Hubert, P., Aunis, D. and Bader, M. F.
(1998). Identification of a potential effector pathway for the trimeric Go
protein associated with secretory granules. Go stimulates a granule-bound
phosphatidylinositol 4-kinase by activating RhoA in chromaffin cells. J.
Biol. Chem. 273, 16913-16920.
Geli, M. I. and Riezman, H. (1998). Endocytic internalization in yeast and
animal cells: similar and different. J. Cell Sci. 111, 1031-1037.
Geppert, M. and Sudhof, T. C. (1998). RAB3 and synaptotagmin: the yin
and yang of synaptic membrane fusion. Annu. Rev. Neurosci. 21, 75-95.
Godi, A., Pertile, P., Meyers, R., Marra, P., Di Tullio, G., Iurisci, C., Luini,
A., Corda, D. and De Matteis, M. A. (1999). ARF mediates recruitment
1049
of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4, 5)P2 on
the Golgi complex. Nature Cell Biol. 1, 280-287.
Goldberg, J. (1998). Structural basis for activation of ARF GTPase:
mechanisms of guanine nucleotide exchange and GTP-myristoyl switching.
Cell 95, 237-248.
Gonzales-Gaitan, M. and Jackle, H. (1997). Role of Drosophila alphaadaptin in presynaptic vesicle recycling. Cell 88, 767-776.
Guo, S., Stolz, L. E., Lemrow, S. M. and York, J. D. (1999). SAC1-like
domains of yeast SAC1, INP52, and INP53 and of human synaptojanin
encode polyphosphoinositide phosphatases. J. Biol. Chem. 274, 1299012995.
Gustaffson, J., Shupliakov, O., Takei, K., Low, P., De Camilli, P. and
Brodin, L. (1998). GTPgammaS induces an actin matrix associated with
coated intermediates in presynaptic neurons. Neurosci. Soc. Abstr. 327, 19.
Haffner, C., Takei, K., Chen, H., Ringstad, N., Hudson, A., Butler, M. H.,
Salcini, A. E., Di Fiore, P. P. and De Camilli, P. (1997). Synaptojanin 1:
localization on coated endocytic intermediates in nerve terminals and
interaction of its 170 kDa isoform with Eps15. FEBS Lett. 419, 175-180.
Hama, H., Schnieders, E. A., Thorner, J., Takemoto, J. Y. and DeWald, D.
B. (1999). Direct involvement of phosphatidylinositol 4-phosphate in
secretion in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 274, 3429434300.
Hannah, M. J., Schmidt, A. A. and Huttner, W. B. (1999). Synaptic vesicle
biogenesis. Annu. Rev. Cell Dev. Biol. 15, 733-798.
Hannan, L. A., Newmyer, S. L. and Schmid, S. L. (1998). ATP- and cytosoldependent release of adaptor proteins from clathrin-coated vesicles: A dual
role for Hsc70. Mol. Biol. Cell 9, 2217-2229.
Hao, W., Tan, Z., Prasad, K., Reddy, K. K., Chen, J., Prestwich, G. D.,
Falck, J. R., Shears, S. B. and Lafer, E. M. (1997). Regulation of AP-3
function by inositides. Identification of phosphatidylinositol 3, 4, 5trisphosphate as a potent ligand. J. Biol. Chem. 272, 6393-6398.
Harris, T. W., Hartwieg, E., Horvitz, H. R. and Jorgensen, E. M. (2000).
Mutations in synaptojanin disrupt synaptic vesicle recycling. J. Cell Biol.
150, 589-600.
Hassan, B. A., Prokopenko, S. N., Breuer, S., Zhang, B., Paululat, A. and
Bellen, H. J. (1998). skittles, a Drosophila phosphatidylinositol 4-phosphate
5-kinase, is required for cell viability, germline development and bristle
morphology, but not for neurotransmitter release. Genetics 150, 1527-1537.
Haucke, V. and De Camilli, P. (1999). AP-2 recruitment to synaptotagmin
stimulated by tyrosine-based endocytic motifs. Science 285, 1268-1271.
Hawthorne, J. N. and Pickard, M. R. (1979). Phospholipids in synaptic
function. J. Neurochem. 32, 5-14.
Hay, J. C., Fisette, P. L., Jenkins, G. H., Fukami, K., Takenawa, T.,
Anderson, R. A. and Martin, T. F. (1995). ATP-dependent inositide
phosphorylation required for Ca(2+)-activated secretion. Nature 374, 173177.
Hay, J. C. and Martin, T. F. (1993). Phosphatidylinositol transfer protein
required for ATP-dependent priming of Ca(2+)-activated secretion. Nature
366, 572-575.
Hendricks, K. B., Wang, B. Q., Schnieders, E. A. and Thorner, J. (1999).
Yeast homologue of neuronal frequenin is a regulator of
phosphatidylinositol-4-OH kinase. Nature Cell Biol. 1, 234-241.
Herman, P. K. and Emr, S. D. (1990). Characterization of VPS34, a gene
required for vacuolar protein sorting and vacuole segregation in
Saccharomyces cerevisiae. Mol. Cell. Biol. 10, 6742-6754.
Heuser, J. E. and Reese, T. S. (1973). Evidence for recycling of synaptic
vesicle membrane during transmitter release at the frog neuromuscular
junction. J. Cell Biol. 57, 315-344.
Hinshaw, J. E. (1999). Dynamin spirals. Curr. Opin. Struct. Biol. 9, 260-267.
Hinshaw, J. E. and Schmid, S. L. (1995). Dynamin self-assembles into rings
suggesting a mechanism for coated vesicle budding. Nature 374, 190-192.
Hirokawa, N., Sobue, K., Kanda, K., Harada, A. and Yorifuji, H. (1989).
The cytoskeletal architecture of the presynaptic terminal and molecular
structure of synapsin 1. J. Cell Biol. 108, 111-126.
Hokin, M. and Hokin, L. (1953). Enzyme secretion and the incorporation of
P32 into phospholipides of pancreas slices. J. Biol. Chem. 203, 967-977.
Holz, R. W., Bittner, M. A., Peppers, S. C., Senter, R. A. and Eberhard,
D. A. (1989). MgATP-independent and MgATP-dependent exocytosis.
Evidence that MgATP primes adrenal chromaffin cells to undergo
exocytosis. J. Biol. Chem. 264, 5412-5419.
Holz, R. W., Hlubek, M. D., Sorensen, S. D., Fisher, S. K., Balla, T., Ozaki,
S., Prestwich, G. D., Stuenkel, E. L. and Bittner, M. A. (2000). A
pleckstrin homology domain specific for phosphatidylinositol 4, 5bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein
1050
JOURNAL OF CELL SCIENCE 114 (6)
identifies plasma membrane PtdIns-4, 5-P2 as being important in exocytosis.
J. Biol. Chem. 275, 17878-17885.
Homma, K., Terui, S., Minemura, M., Qadota, H., Anraku, Y., Kanaho,
Y. and Ohya, Y. (1998). Phosphatidylinositol-4-phosphate 5-kinase
localized on the plasma membrane is essential for yeast cell morphogenesis.
J. Biol. Chem. 273, 15779-15786.
Honda, A., Nogami, M., Yokozeki, T., Yamazaki, M., Nakamura, H.,
Watanabe, H., Kawamoto, K., Nakayama, K., Morris, A. J., Frohman,
M. A. et al. (1999). Phosphatidylinositol 4-phosphate 5-kinase alpha is a
downstream effector of the small G protein ARF6 in membrane ruffle
formation. Cell 99, 521-532.
Jackson, C. L. and Casanova, J. E. (2000). Turning on ARF: the Sec7 family
of guanine-nucleotide-exchange factors. Trends Cell Biol. 10, 60-67.
Janmey, P. A. (1994). Phosphoinositides and calcium as regulators of cellular
actin assembly and disassembly. Annu. Rev. Physiol. 56, 169-191.
Jenkins, G. H., Fisette, P. L. and Anderson, R. A. (1994). Type I
phosphatidylinositol 4-phosphate 5-kinase isoforms are specifically
stimulated by phosphatidic acid. J. Biol. Chem. 269, 11547-11554.
Jones, D. H., Morris, J. B., Morgan, C. P., Kondo, H., Irvine, R. F. and
Cockcroft, S. (2000). Type I phosphatidylinositol 4-phosphate 5-kinase
directly interacts with ADP-ribosylation factor 1 and is responsible for
phosphatidylinositol 4,5-bisphosphate synthesis in the Golgi compartment.
J. Biol. Chem. 275, 13962-13966.
Jost, M., Simpson, F., Kavran, J. M., Lemmon, M. A. and Schmid, S. L.
(1998). Phosphatidylinositol-4,5-bisphosphate is required for endocytic
coated vesicle formation. Curr. Biol. 8, 1399-1402.
Kearns, B. G., Alb, J. G. Jr and Bankaitis, V. (1998). Phosphatidylinositol
transfer proteins: the long and winding road to physiological function.
Trends Cell Biol. 8, 276-282.
Khvotchev, M. and Sudhof, T. C. (1998). Newly synthesized
phosphatidylinositol phosphates are required for synaptic norepinephrine
but not glutamate or gamma-aminobutyric acid (GABA) release. J. Biol.
Chem. 273, 21451-21454.
Kirchhausen, T. (2000). Three ways to make a vesicle. Nature Rev. Mol. Cell
Biol. 1, 187-198.
Klein, D. E., Lee, A., Frank, D. W., Marks, M. S. and Lemmon, M. A.
(1998). The pleckstrin homology domains of dynamin isoforms require
oligomerization for high affinity phosphoinositide binding. J. Biol. Chem.
273, 27725-27733.
Kosaka, T. and Ikeda, K. (1983). Possible temperature-dependent blockage
of synaptic vesicle recycling induced by a single gene mutation in
Drosophila. J. Neurobiol. 14, 207-225.
Larrabee, M. G., Klingman, J. D. and Leicht, W. S. (1963). Effects of
temperature, calcium and activity on phospholipid metabolism in a
sympathetic ganglion. J. Neurochem. 10, 549-570.
Lee, A., Frank, D. W., Marks, M. S. and Lemmon, M. A. (1999). Dominantnegative inhibition of receptor-mediated endocytosis by a dynamin-1 mutant
with a defective pleckstrin homology domain. Curr. Biol. 9, 261-264.
Lee, C., Kang, H. S., Chung, J. K., Sekiya, F., Kim, J. R., Han, J. S., Kim,
S. R., Bae, Y. S., Morris, A. J. and Rhee, S. G. (1997). Inhibition of
phospholipase D by clathrin assembly protein 3 (AP3). J. Biol. Chem. 272,
15986-15992.
Lee, C., Kim, S. R., Chung, J. K., Frohman, M. A., Kilimann, M. W. and
Rhee, S. G. (2000). Inhibition of phospholipase D by amphiphysins. J. Biol.
Chem. 275, 18751-18758.
Liscovitch, M. and Cantley, L. C. (1995). Signal transduction and membrane
traffic: the PITP/phosphoinositide connection. Cell 81, 659-662.
Loyet, K. M., Kowalchyk, J. A., Chaudhary, A., Chen, J., Prestwich, G.
D. and Martin, T. F. J. (1998). Specific binding of phosphatidylinositol 4,
5-bisphosphate to calcium-dependent activator protein for secretion
(CAPS), a potential phosphoinositide effector protein for regulated
exocytosis. J. Biol. Chem. 273, 8337-8343.
Martin, T. F. (1998). Phosphoinositide lipids as signaling molecules: common
themes for signal transduction, cytoskeletal regulation, and membrane
trafficking. Annu. Rev. Cell. Dev. Biol. 14, 231-264.
Martin, T. F., Loyet, K. M., Barry, V. A. and Kowalchyk, J. A. (1997). The
role of PtdIns(4, 5)P2 in exocytotic membrane fusion. Biochem. Soc. Trans.
25, 1137-1141.
McMahon, H. T. (1999). Endocytosis: an assembly protein for clathrin cages.
Curr. Biol. 9, R332-335.
McMahon, H. T., Wigge, P. and Smith, C. (1997). Clathrin interacts
specifically with amphiphysin and is displaced by dynamin. FEBS Lett. 413,
319-322.
McNiven, M. A., Cao, H., Pitts, K. R. and Yoon, Y. (2000). The dynamin
family of mechanoenzymes: pinching in new places. Trends Biochem. Sci.
25, 115-120.
McPherson, P. S., Garcia, E. P., Slepnev, V. I., David, C., Zhang, X., Grabs,
D., Sossin, W. S., Bauerfeind, R., Nemoto, Y. and De Camilli, P. (1996).
A presynaptic inositol-5-phosphatase. Nature 379, 353-357.
Miller, K. G., Alfonso, A., Nguyen, M., Crowell, J. A., Johnson, C. D. and
Rand, J. B. (1996). A genetic selection for Caenorhabditis elegans synaptic
transmission mutants. Proc. Nat. Acad. Sci. USA 93, 12593-12598.
Mochida, S., Fukuda, M., Niinobe, M., Kobayashi, H. and Mikoshiba, K.
(1997). Roles of synaptotagmin C2 domains in neurotransmitter secretion
and inositol high-polyphosphate binding at mammalian cholinergic
synapses. Neuroscience 77, 937-943.
Mossessova, E., Gulbis, J. M. and Goldberg, J. (1998). Structure of the
guanine nucleotide exchange factor Sec7 domain of human arno and
analysis of the interaction with ARF GTPase. Cell 92, 415-423.
Mundigl, O., Matteoli, M., Daniell, L., Thomas-Reetz, A., Metcalf, A.,
Jahn, R. and De Camilli, P. (1993). Synaptic vesicle proteins and early
endosomes in cultured hippocampal neurons: differential effects of
Brefeldin A in axon and dendrites. J. Cell Biol. 122, 1207-1221.
Mundigl, O., Ochoa, G. C., David, C., Slepnev, V. I., Kabanov, A. and De
Camilli, P. (1998). Amphiphysin I antisense oligonucleotides inhibit neurite
outgrowth in cultured hippocampal neurons. J. Neurosci. 18, 93-103.
Murthy, V. N. and Stevens, C. F. (1998). Synaptic vesicles retain their identity
through the endocytic cycle. Nature 392, 497-501.
Neeb, A., Koch, H., Schurmann, A. and Brose, N. (1999). Direct interaction
between the ARF-specific guanine nucleotide exchange factor msec7-1 and
presynaptic Munc13-1. Eur. J. Cell Biol. 78, 533-538.
Niinobe, M., Yamaguchi, Y., Fukuda, M. and Mikoshiba, K. (1994).
Synaptotagmin is an inositol polyphosphate binding protein: isolation and
characterization as an Ins 1,3,4,5-P4 binding protein. Biochem. Biophys.
Res. Commun. 205, 1036-1042.
Ochoa, G. C., Slepnev, V. I., Neff, L., Ringstad, N., Takei, K., Daniell, L.,
Kim, W., Cao, H., McNiven, M., Baron, R. et al. (2000). A functional link
between dynamin and the actin cytoskeleton at podosomes. J. Cell Biol. 150,
377-389.
Odorizzi, G., Babst, M. and Emr, S. D. (1998). Fab1p PtdIns(3)P 5-kinase
function essential for protein sorting in the multivesicular body. Cell 95,
847-858.
Odorizzi, G., Babst, M. and Emr, S. D. (2000). Phosphoinositide signaling
and the regulation of membrane trafficking in yeast. Trends Biochem. Sci.
25, 229-235.
Okamoto, M. and Sudhof, T. C. (1997). Mints, Munc18-interacting proteins
in synaptic vesicle exocytosis. J. Biol. Chem. 272, 31459-31464.
Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Engelkamp, D., KrahJentgens, I., Lambrecht, H. G., Koch, K. W., Schwemer, J., Rivosecchi,
R. et al. (1993). Frequenin–a novel calcium-binding protein that modulates
synaptic efficacy in the Drosophila nervous system. Neuron 11, 15-28.
Qualmann, B., Roos, J., DiGregorio, P. J. and Kelly, R. B. (1999). Syndapin
I, a synaptic dynamin-binding protein that associates with the neural
Wiskott-Aldrich syndrome protein. Mol. Biol. Cell 10, 501-513.
Qualmann, B. and Kelly, R. B. (2000). Syndapin isoforms participate in
receptor-mediated endocytosis and actin organization. J. Cell Biol. 148,
1047-1062.
Qualmann, B., Kessels, M. M. and Kelly, R. B. (2000). Molecular links
between endocytosis and the actin cytoskeleton. J. Cell Biol. 150, F111-116.
Ramjaun, A. R., Micheva, K. D., Bouchelet, I. and McPherson, P. S. (1997).
Identification and characterization of a nerve terminal-enriched
amphiphysin isoform. J. Biol. Chem. 272, 16700-16706.
Rao, V. D., Misra, S., Boronenkov, I. V., Anderson, R. A. and Hurley, J.
H. (1998). Structure of type IIbeta phosphatidylinositol phosphate kinase:
a protein kinase fold flattened for interfacial phosphorylation. Cell 94, 829839.
Rapoport, I., Miyazaki, M., Boll, W., Duckworth, B., Cantley, L. C.,
Shoelson, S. and Kirchhausen, T. (1997). Regulatory interactions in the
recognition of endocytic sorting signals by AP-2 complexes. EMBO J. 16,
2240-22450.
Ringstad, N., Nemoto, Y. and De Camilli, P. (1997). The
SH3p4/Sh3p8/SH3p13 protein family: binding partners for synaptojanin and
dynamin via a Grb2-like Src homology 3 domain. Proc. Nat. Acad. Sci. USA
94, 8569-8574.
Ringstad, N., Gad, H., Low, P., Di Paolo, G., Brodin, L., Shupliakov, O.
and De Camilli, P. (1999). Endophilin/SH3p4 is required for the transition
from early to late stages in clathrin-mediated synaptic vesicle endocytosis.
Neuron 24, 143-154.
PIs in membrane traffic at the synapse
Rizo, J. and Sudhof, T. C. (1998). C2-domains, structure and function of a
universal Ca2+-binding domain. J. Biol. Chem. 273, 15879-15882.
Rohatgi, R., Ma, L., Miki, H., Lopez, M., Kirchhausen, T., Takenawa, T.
and Kirschner, M. W. (1999). The interaction between N-WASP and the
Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97,
221-231.
Roos, J. and Kelly, R. B. (1999). The endocytic machinery in nerve terminals
surrounds sites of exocytosis. Curr. Biol. 9, 1411-1414.
Roth, M. G., Bi, K., Ktistakis, N. T. and Yu, S. (1999). Phospholipase D as
an effector for ADP-ribosylation factor in the regulation of vesicular traffic.
Chem. Phys. Lipids 98, 141-152.
Rothman, J. E. and Warren, G. (1994). Implications of the SNARE
hypothesis for intracellular membrane topology and dynamics. Curr. Biol.
4, 220-233.
Rupnik, M., Kreft, M., Sikdar, S. K., Grilc, S., Romih, R., Zupancic, G.,
Martin, T. F. and Zorec, R. (2000). Rapid regulated dense-core vesicle
exocytosis requires the CAPS protein. Proc. Nat. Acad. Sci. USA 97, 56275632.
Sakisaka, T., Itoh, T., Miura, K. and Takenawa, T. (1997).
Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the
rearrangement of actin filaments. Mol. Cell. Biol. 17, 3841-3849.
Schaefer, A. M., Hadwiger, G. D. and Nonet, M. L. (2000). rpm-1, a
conserved neuronal gene that regulates targeting and synaptogenesis in C.
elegans. Neuron 26, 345-356.
Schekman, R. W. (1994). Regulation of membrane traffic in the secretory
pathway. Harvey Lect. 90, 41-57.
Schiavo, G., Gu, Q. M., Prestwich, G. D., Sollner, T. H. and Rothman, J.
E. (1996). Calcium-dependent switching of the specificity of
phosphoinositide binding to synaptotagmin. Proc. Nat. Acad. Sci. USA 93,
13327-13332.
Schluter, O. M., Schnell, E., Verhage, M., Tzonopoulos, T., Nicoll, R. A.,
Janz, R., Malenka, R. C., Geppert, M. and Sudhof, T. C. (1999).
Rabphilin knock-out mice reveal that rabphilin is not required for
rab3 function in regulating neurotransmitter release. J. Neurosci 19, 58345846.
Schmalzing, G., Richter, H. P., Hansen, A., Schwarz, W., Just, I. and
Aktories, K. (1995). Involvement of the GTP binding protein Rho in
constitutive endocytosis in Xenopus laevis oocytes. J. Cell Biol. 130, 13191332.
Schmidt, A. and Huttner, W. B. (1998). Biogenesis of synaptic-like
microvesicles in perforated PC12 cells. Methods 16, 160-169.
Schmidt, A., Wolde, M., Thiele, C., Fest, W., Kratzin, H., Podtelejnikov,
A. V., Witke, W., Huttner, W. B. and Soling, H. D. (1999). Endophilin I
mediates synaptic vesicle formation by transfer of arachidonate to
lysophosphatidic acid. Nature 401, 133-141.
Schu, P. V., Takegawa, K., Fry, M. J., Stack, J. H., Waterfield, M. D. and
Emr, S. D. (1993). Phosphatidylinositol 3-kinase encoded by yeast VPS34
gene essential for protein sorting. Science 260, 88-91.
Scott, D. L., White, S. P., Otwinowski, Z., Yuan, W., Gelb, M. H. and Sigler,
P. B. (1990). Interfacial catalysis: the mechanism of phospholipase A2.
Science 250, 1541-1546.
Scott, D. L., Mandel, A. M., Sigler, P. B. and Honig, B. (1994). The
electrostatic basis for the interfacial binding of secretory phospholipases A2.
Biophys. J. 67, 493-504.
Sechi, A. S. and Wehland, J. (2000). The actin cytoskeleton and plasma
membrane connection: PtdIns(4, 5)P(2) influences cytoskeletal protein
activity at the plasma membrane. J. Cell Sci. 113, 3685-3695.
Serafini, T., Orci, L., Amherdt, M., Brunner, M., Kahn, R. A. and
Rothman, J. E. (1991). ADP-ribosylation factor is a subunit of the coat of
Golgi-derived COP-coated vesicles: a novel role for a GTP-binding protein.
Cell 67, 239-253.
Sever, S., Damke, H. and Schmid, S. L. (2000). Garrotes, springs, ratchets,
and whips: Putting dynamin models to the test. Traffic 1, 385-392.
Sha, B. and Luo, M. (1999). PI transfer protein: the specific recognition of
phospholipids and its functions. Biochim. Biophys. Acta 1441, 268-277.
Shi, G., Faundez, V., Roos, J., Dell’Angelica, E. C. and Kelly, R. B. (1998).
Neuroendocrine synaptic vesicles are formed in vitro by both clathrindependent and clathrin-independent pathways. J. Cell Biol. 143, 947-955.
Singer, W. D., Brown, H. A. and Sternweis, P. C. (1997). Regulation of
eukaryotic
phosphatidylinositol-specific
phospholipase
C
and
phospholipase D. Annu. Rev. Biochem. 66, 475-509.
Slepnev, V. I., Ochoa, G. C., Butler, M. H., Grabs, D. and Camilli, P. D.
(1998). Role of phosphorylation in regulation of the assembly of endocytic
coat complexes. Science 281, 821-824.
1051
Slepnev, V. I. and De Camilli, P. (2000). Accesory factors in clathrindependent synaptic vesicle endocytosis. Nature Neurosci. Rev. 1, 161-172.
Springer, S., Spang, A. and Schekman, R. (1999). A primer on vesicle
budding. Cell 97, 145-148.
Stenmark, H. and Aasland, R. (1999). FYVE-finger proteins–effectors of an
inositol lipid. J. Cell Sci. 112, 4175-4183.
Sudhof, T. C. (1995). The synaptic vesicle cycle: a cascade of protein-protein
interactions. Nature 375, 645-653.
Sweitzer, S. M. and Hinshaw, J. E. (1998). Dynamin undergoes a GTPdependent conformational change causing vesiculation. Cell 93, 10211029.
Takei, K., McPherson, P. S., Schmid, S. L. and De Camilli, P. (1995).
Tubular membrane invaginations coated by dynamin rings are induced by
GTP-gamma S in nerve terminals. Nature 374, 186-190.
Takei, K., Mundigl, O., Daniell, L. and De Camilli, P. (1996). The synaptic
vesicle cycle: a single vesicle budding step involving clathrin and dynamin.
J. Cell Biol. 133, 1237-1250.
Takei, K., Haucke, V., Slepnev, V., Farsad, K., Salazar, M., Chen, H. and
De Camilli, P. (1998). Generation of coated intermediates of clathrinmediated endocytosis on protein-free liposomes. Cell 94, 131-141.
Takei, K., Slepnev, V. I., Haucke, V. and De Camilli, P. (1999). Functional
partnership between amphiphysin and dynamin in clathrin-mediated
endocytosis. Nature Cell Biol. 1, 33-39.
Tandon, A., Bannykh, S., Kowalchyk, J. A., Banerjee, A., Martin, T. F.
and Balch, W. E. (1998). Differential regulation of exocytosis by calcium
and CAPS in semi-intact synaptosomes. Neuron 21, 147-154.
Timerman, A. P., Mayrleitner, M. M., Lukas, T. J., Chadwick, C. C., Saito,
A., Watterson, D. M., Schindler, H. and Fleischer, S. (1992). Inositol
polyphosphate receptor and clathrin assembly protein AP-2 are related
proteins that form potassium-selective ion channels in planar lipid bilayers.
Proc. Nat. Acad. Sci. USA 89, 8976-8980.
Vallis, Y., Wigge, P., Marks, B., Evans, P. R. and McMahon, H. T. (1999).
Importance of the pleckstrin homology domain of dynamin in clathrinmediated endocytosis. Curr. Biol. 9, 257-260.
Verhage, M., Maia, A. S., Plomp, J. J., Brussaard, A. B., Heeroma, J. H.,
Vermeer, H., Toonen, R. F., Hammer, R. E., van den Berg, T. K., Missler,
M. et al. (2000). Synaptic assembly of the brain in the absence of
neurotransmitter secretion. Science 287, 864-869.
Voglmaier, S. M., Keen, J. H., Murphy, J. E., Ferris, C. D., Prestwich, G.
D., Snyder, S. H. and Theibert, A. B. (1992). Inositol hexakisphosphate
receptor identified as the clathrin assembly protein AP-2. Biochem. Biophys.
Res. Commun. 187, 158-163.
Walch-Solimena, C. and Novick, P. (1999). The yeast phosphatidylinositol4-OH kinase pik1 regulates secretion at the Golgi. Nature Cell Biol. 1, 523525.
Walent, J. H., Porter, B. W. and Martin, T. F. (1992). A novel 145 kd brain
cytosolic protein reconstitutes Ca(2+)-regulated secretion in permeable
neuroendocrine cells. Cell 70, 765-775.
Wan, H. I., DiAntonio, A., Fetter, R. D., Bergstrom, K., Strauss, R. and
Goodman, C. S. (2000). Highwire regulates synaptic growth in Drosophila.
Neuron 26, 313-329.
Wang, L. H., Sudhof, T. C. and Anderson, R. G. (1995). The appendage
domain of alpha-adaptin is a high affinity binding site for dynamin. J. Biol.
Chem. 270, 10079-10083.
Wang, Y., Okamoto, M., Schmitz, F., Hofmann, K. and Sudhof, T. C.
(1997). Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion.
Nature 388, 593-598.
West, M. A., Bright, N. A. and Robinson, M. S. (1997). The role of ADPribosylation factor and phospholipase D in adaptor recruitment. J. Cell Biol.
138, 1239-1254.
Wiedemann, C., Schafer, T. and Burger, M. M. (1996). Chromaffin granuleassociated phosphatidylinositol 4-kinase activity is required for stimulated
secretion. EMBO J. 15, 2094-2101.
Wiedemann, C., Schafer, T., Burger, M. M. and Sihra, T. S. (1998). An
essential role for a small synaptic vesicle-associated phosphatidylinositol 4kinase in neurotransmitter release. J. Neurosci. 18, 5594-5602.
Wirtz, K. W. (1991). Phospholipid transfer proteins. Annu. Rev. Biochem. 60,
73-99.
Witke, W., Podtelejnikov, A. V., Di Nardo, A., Sutherland, J. D., Gurniak,
C. B., Dotti, C. and Mann, M. (1998). In mouse brain profilin I and profilin
II associate with regulators of the endocytic pathway and actin assembly.
EMBO J. 17, 967-976.
Woscholski, R., Finan, P. M., Radley, E., Totty, N. F., Sterling, A. E.,
Hsuan, J. J., Waterfield, M. D. and Parker, P. J. (1997). Synaptojanin is
1052
JOURNAL OF CELL SCIENCE 114 (6)
the major constitutively active phosphatidylinositol-3,4,5-trisphosphate 5phosphatase in rodent brain. J. Biol. Chem. 272, 9625-9628.
Wu, M. N. and Bellen, H. J. (1997). Genetic dissection of synaptic
transmission in Drosophila. Curr. Opin. Neurobiol. 7, 624-630.
Wurmser, A. E., Gary, J. D. and Emr, S. D. (1999). Phosphoinositide 3Kinases and their FYVE domain-containing effectors as regulators of
vacuolar/lysosomal membrane trafficking pathways. J. Biol. Chem. 274,
9129-9132.
Yamabhai, M., Hoffman, N. G., Hardison, N. L., McPherson, P. S.,
Castagnoli, L., Cesareni, G. and Kay, B. K. (1998). Intersectin, a novel
adaptor protein with two Eps15 homology and five Src homology 3 domains.
J. Biol. Chem. 273, 31401-31407.
Ye, W., Ali, N., Bembenek, M. E., Shears, S. B. and Lafer, E. M. (1995).
Inhibition of clathrin assembly by high affinity binding of specific inositol
polyphosphates to the synapse-specific clathrin assembly protein AP-3. J.
Biol. Chem. 270, 1564-1568.
Zhang, J. Z., Davletov, B. A., Sudhof, T. C. and Anderson, R. G. (1994).
Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications
for membrane recycling. Cell 78, 751-760.
Zhen, M., Huang, X., Bamber, B. and Jin, Y. (2000). Regulation of
presynaptic terminal organization by C. elegans RPM-1, a putative guanine
nucleotide exchanger with a RING-H2 finger domain. Neuron 26, 331-343.
Zheng, J., Cahill, S. M., Lemmon, M. A., Fushman, D., Schlessinger, J.
and Cowburn, D. (1996). Identification of the binding site for acidic
phospholipids on the pH domain of dynamin: implications for stimulation
of GTPase activity. J. Mol. Biol. 255, 14-21.
Zigmond, S. H. (2000). How WASP regulates actin polymerization. J. Cell
Biol. 150, F117-F120.
Note added in proof
Recent crystallographic papers have revealed the structural
basis for the interaction of phosphoinositides with the Nterminal domain of the clathrin coat protein AP180/CALM,
now shown to have the fold of an ENTH domain (Ford et al.,
2001; Mao et al., 2001). Furthermore, the ENTH domain of
epsin, another clathrin coat accessory protein, was shown to
bind PtdIns(4,5)P2 (Itoh et al., 2001).
Ford, M. G., Pearse, B. M., Higgins, M. K., Vallis, Y., Owen, D. J., Gibson,
A., Hopkins, C. R., Evans, P. R. and McMahon, H. T. (2001).
Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the
nucleation of clathrin lattices on membranes. Science 291, 1051-1055.
Itoh, T., Koshiba, S., Kigawa, T., Kikuchi, A., Yokoyama, S. and
Takenawa, T. (2001). Role of the ENTH domain in phosphatidylinositol4,5-bisphosphate binding and endocytosis. Science 291, 1047-1051.
Mao, Y., Chen, J., Maynard, J. A., Zhang, B. and Quiocho, F. A. (2001).
A novel all helix fold of the AP180 amino-terminal domain for
phosphoinositide binding and clathrin assembly in synaptic vesicle
endocytosis. Cell 104, 433-440.