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.
- adaptor
- ADP-ribosylation factor 6 (Arf6)
- clathrin
- endocytosis
- phosphoinositide
- trans-Golgi network
Introduction
Eukaryotic cells are compartmentalized into membrane-bounded 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 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 dual-key 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.
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.
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 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 (σ2). The two large subunits together with σ2 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 lipid-binding 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 YXXΦ (where Φ is a bulky hydrophobic residue) and dileucine motifs, which bind directly to distinct sites within the AP-2 core domain [15]. YXXΦ 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-isoxazole-propionic acid) and GABAA (γ-aminobutyric acid type A) receptors (J.T. Kittler, personal communication), the α1B-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] 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 surface-exposed 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).
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.
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 Arf-GEF (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 clathrin-mediated endocytosis. This includes Arf-mediated stimulation of phospholipase D, another potent activator of type I PIPK, Rac-induced actin rearrangements and NM23-H1-catalysed GTP formation, subsequently made available to GTPases including dynamin [40]. Whether or not Arf6-GTP, 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 PC12 cells [43], consistent with a role for PI(4,5)P2 in both the exo- and endo-cytic limbs of vesicle cycling [27,44].
(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-32P]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-32P]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.
Several enzymatic activities may contribute to PI(4,5)P2 degradation which is required for uncoating of CCVs [24]. Among these are phospholipase Cδ, inositol 5-phosphatases including synaptojanin, SHIP [SH2 (Src homology 2)-domain-containing inositol 5-phosphatase], Ocrl, 5-phosphatase II and PIPP (proline-rich inositol polyphosphate 5-phosphatase). Moreover, PI(4,5)P2 is the precursor for PtdIns(3,4,5)P3 synthesis following activation of phosphoinositide 3-kinase by ligand-bound cell signalling receptors [9]. How exactly the interplay between PI kinases and phosphatases is regulated is unknown. In the case of synaptojanin, it has been shown that the endocytic accessory protein endophilin via its SH3 domain recruits synaptojanin to endocytic sites at synapses [45–47] and stimulates its enzymatic activity. Phosphorylation of synaptojanin's proline-rich domain by proline-directed protein kinases such as Cdk5 inhibits its interaction with endophilin [48]. Other SH3 domain-containing proteins including syndapin/PACSIN (protein kinase C and CK2 substrate in neurons), amphiphysin or intersectin [49] as well as the small GTPase Rac may also affect 5-phosphatase activity under certain conditions [6].
At synapses, PI(4,5)P2 levels appear to be under activity-dependent 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 activation-dependent 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.
Acknowledgments
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).
Footnotes
Cell Architecture: from Structure to Function: A Focus Topic at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by S. Cockroft (University College London, U.K.), Y. Goda (University College London, U.K.), R. Insall (Birmingham, U.K.) and M. Wakelam (Birmingham, U.K.).
Abbreviations: 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
- © 2005 The Biochemical Society
References
October 2005
Volume: 33 Issue: 6
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