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Phosphoinositides: Regulators of membrane traffic and protein function
FEBS Letters 581 (2007) 2105–2111
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Phosphoinositides: Regulators of membrane traffic and protein function Michael Krauß, Volker Haucke* Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universita¨t Berlin, Takustraße 6, 14195 Berlin, Germany Received 13 December 2006; revised 30 January 2007; accepted 31 January 2007 Available online 12 February 2007 Edited by Thomas So¨llner
Abstract Phosphoinositides serve as important spatio-temporal regulators of intracellular trafficking and cell signalling events. In addition to their recognition by specific phosphoinositide binding domains present within cytoplasmic adaptor proteins or membrane integral channels and transporters phosphoinositides may affect membrane transport by eliciting conformational changes within proteins or by regulating enzymatic activities. During adaptor-mediated membrane traffic phosphoinositides form part of coincidence detection systems that aid in targeting pools of specific phosphoinositides to select intracellular transport pathways. In this review, we discuss potential mechanisms for conferring selectivity onto the phosphoinositide code as well as possible avenues for future research. Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Phosphoinositides; Membrane traffic; Adaptor proteins; Phosphoinositide binding domains; Coincidence detection
1. Introduction Over the past decade phosphatidylinositol and its phosphorylated derivatives (referred to as phosphoinositides, PIs) have been implicated in numerous membrane trafficking and cellular signalling events (reviewed in [1–4]). Different phosphoinositide species have been shown to exhibit distinct characteristic subcellular distribution patterns [3,4]: Whereas Golgi membranes have been reported to be enriched in phosphatidylinositol 4phosphate [PI(4)P], higher phosphorylated derivatives, such as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], are found predominantly in the inner leaflet of the plasma membrane. Following growth factor receptor activation PI(3,4,5)P3 may also accumulate on endomembranes. Endosomal organelles are characterized by the presence of phosphatidylinositol 3phosphate [PI(3)P] and derived lipid species. Based on these observations it has been suggested, that PIs contribute to the generation of organelle identity, and do so by recruiting specific sets of proteins that harbour appropriate PI binding domains [2–4]. Strikingly, different PI species can be reversibly interconverted through the regulated activities of specific kinases and phosphatases, resulting in short-term alterations of the protein * Corresponding author. Fax: +49 30 838 56919. E-mail address: [email protected] (V. Haucke).
binding properties and functions of a given membrane domain. These plastic properties render PIs ideally suited for integrating membrane trafficking and cell signalling events. Correspondingly, PI metabolizing enzymes appear to be constituents of complex regulatory networks that have been the subject of intense research. Regulators of PI metabolizing enzymes have been identified that control PI turnover in time and space and thereby serve as triggers for the generation of local PI pools dedicated to distinct (sub-) cellular functions. Progress in this area has been the subject of excellent recent reviews [3–6]. Despite of the large progress made we still poorly understand how one and the same PI species depending on the physiological context can trigger distinct events. In this review, we will discuss possible mechanisms by which specificity of PI-mediated functions can be achieved. 2. Phosphoinositides promote specific membrane recruitment of cytosolic protein subsets PIs can directly associate with a variety of different proteins including cytoplasmic factors, such as membrane trafficking and cell signalling adaptors as well as membrane integral channels and transporters. In most cases binding is achieved by electrostatic interactions involving the negatively charged PI headgroups and patches of positive charges within the target protein. PI binding sites can be contained within unstructured regions harboring clusters of basic residues, as is the case for MARCKS and related proteins [7], or within folded domains that may recognize distinct arrangements of PI-exposed phosphate residues by preformed binding grooves or surfaceexposed fingers [8,9]. Sometimes binding is supported by additional hydrophobic amino acid residues located in close proximity to the basic patch, which may steer into the lipid bilayer. A variety of structurally different PI binding domains have been identified, including PH (pleckstrin homology; [10]), FYVE (named after four proteins in which it has been initially identified: Fab1p, YOTB, Vac1p and EEA1), PX (phox homology; [11]), E/ANTH (epsin/AP180 N-terminal homology) [12–14], C2, PTB, and FERM domains [8] (Fig. 1). These display marked differences in specificity and affinity, with some of them promoting exclusive binding to one type of PI, and others being more promiscuous by recognizing more or less closely related PI species. PH domains, for instance, have been identified in more than 250 proteins, rendering this the largest family of PI binding modules [10]. Sequence homology between different PH domain family members is relatively low; nonetheless, the tertiary structure of this approximately 120 amino acid long domain appears to be highly conserved,
0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.01.089
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Fig. 1. Structures of phosphoinositide binding domains and their ligands. Structures of the PI(4,5)P2-specific PH domain of phospholipase Cd in complex with inositol (1,4,5)-triphosphate (PDB: 1MAI), the inositol (1,3)-bisphosphate complexed FYVE domain of early endosomal antigen 1 (EEA1; PDB: 1HYI), the PI(3)-binding Phox-homology (PX) domain of p40Phox (PDB: 1H6H), and the A/ENTH domains derived from the endocytic proteins CALM (an AP180-homologue; PDB: 1HFA) or epsin 1 (PDB: 1H0A) with their respective phosphoinositide ligands.
exhibiting a b-sandwich structure flanked by an a-helical stretch at its carboxy-terminal end. Both, b-strands and loop regions contribute positively charged side chains that establish non-covalent bonds with hydroxyl and phosphate groups of the respective phosphoinositide (Fig. 1, top left). Loops represent the regions of lowest sequence conservation and probably account for the large variability in PI binding specificity. Notably, a genome-wide analysis in yeast has revealed that only few PH domains selectively recognize one particular PI, and that most bind PIs with low affinity [15]. Therefore, specific membrane-targeting must be achieved by additional factors. Binding of proteins to PH domains has been described in a few cases of which the association between Gbc and the PH domain of GRK2 may represent the best documented example [16]. This represents a potential caveat for the use of PH domains as PI-selective probes in live cell imaging studies [17]. By contrast to PH domains FYVE domains display an exclusive specificity for PI(3)P. These domains are relatively small units (about 70 amino acids in length) composed of one a-helix and four short b-strands being held together by two cysteinecoordinated Zn2+-ions (Fig. 1, top middle) [18]. Specificity for PI(3)P is achieved by a selective network of electrostatic interactions and hydrogen bonds between the lipid headgroup and conserved amino acid side chains contributed by three signature motifs. PX domains (Fig. 1, top right), which have been found in numerous proteins with a wide spectrum of functions including vesicular traffic, and phospholipid metabolism display a much broader PI specificity than FYVE domains. A stretch of about 120 amino acids is folded into a three-stranded b-sheet and six helical regions, which together form a positively charged binding pocket for the cognate PI. Binding of
PX and FYVE domains to the membrane surface might be assisted by additional regions exposing hydrophobic residues, which are presumably capable of penetrating the membrane.
3. Binding to phosphoinositides can be associated with conformational changes The ENTH of epsin [12] and the ANTH domain of AP180 [13,14] associate selectively with PI(4,5)P2. Despite forming homologous solenoid structures, both proteins use different mechanisms to bind to PI(4,5)P2. Whereas the ANTH domain exposes a basic patch of three lysine and one histidine residue at its surface (Fig. 1, bottom left), the ENTH domain embeds the PI(4,5)P2 headgroup in a deep, positively charged pocket (Fig. 1, bottom right). Strikingly, the N-terminal ENTH a-helix (termed ‘helix 0’) is involved in the formation of this pocket. Folding of the ‘0-helix’ is induced upon the approximation of the ENTH domain to a PI(4,5)P2-enriched membrane surface. Thus, in the case of epsin PI(4,5)P2 binding triggers a conformational change (i.e. folding) of a distinct portion of the protein. The newly formed helix is amphipathic in nature; positively charged residues on one side of the helix are engaged in lipid headgroup binding, whereas hydrophobic residues on the other face insert into the cytoplasmic face of the lipid bilayer. Binding of the epsin ENTH domain to PI(4,5)P2-rich plasmalemmal sites may drive acquisition of negative curvature by insertion of hydrophobic moieties into one leaflet of the underlying membrane (Fig. 2A) [12]. A similar PI(4)P-induced conformational change has been proposed to occur in the epsin family member enthoprotin/Clint/epsinR [19] to induce
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Fig. 2. Examples of phosphoinositide-induced conformational changes. (A) PI(4,5)P2 (yellow)-induced folding of an N-terminal helix (helix 0; purple) within the ENTH domain of epsin 1 (green) drives membrane curvature acquisition by insertion of hydrophobic residues within this helix into the cytoplasmic leaflet of the plasma membrane. (B) In the absence PI(4,5)P2 of voltage-gated K+-channels (KV) are inactive due to plugging of the pore by an N-terminal globular inactivation domain. PI(4,5)P2 (yellow) was suggested to counteract the inactivation mechanism by sequestering the inactivation domain at the membrane surface, thereby preventing it from clogging the pore.
bending of trans-Golgi network (TGN)-derived membranes. Moreover, some members of the BAR domain family of membrane curvature-sensors and inducers [20] also contain an amphipathic, potentially membrane-active helix or interact with small GTPases that could exhibit similar activities. Membrane penetrating activity has also been suggested for synaptotagmin 1, a type I membrane protein implicated in Ca2+-triggered exo–endocytosis of synaptic vesicles at neuronal synapses. Synaptotagmin 1 contains two cytoplasmic, Ca2+-sensing C2-domains, termed C2A and C2B. The C2B domain also harbors a high-affinity binding site for PI(4,5)P2. It has been suggested, that the C2B domain pre-absorbs to PI(4,5)P2-containing membranes, but is reoriented upon Ca2+-binding such that it inserts hydrophobic residues into the lipid bilayer [21]. The binding site for PI(4,5)P2 partially overlaps with residues associating with the heterotetrameric clathrin adaptor protein AP-2 (comprising a, b2, l2, and r2 subunits) [22]. The precise interplay between all three factors still remains unclear, although one might expect that the interactions of synaptotagmin 1 with PI(4,5)P2 and AP-2 are mutually exclusive and thus need to be precisely regulated. AP-2 itself contains two patches of basic amino acid residues within its a- and l-subunits and has been proposed to adopt two interconvertible conformations. In its closed, presumably inactive conformation the binding site for cargo proteins displaying tyrosine-based endocytic motifs is buried by side chains from b2 as the soluble carboxy-terminal domain of l2 folds back onto the AP-2 core [23]. In this conformational state, the two PI(4,5)P2 binding sites are located on perpendicular faces of the AP-2 complex with only one of them being
able to access the membrane at any given time. Phosphorylation of l2 at a single threonine residue within a hinge region connecting its N- and C-terminal domains by the serine/threonine kinases AAK1 or GAK/auxilin 2 facilitates a dramatic conformational change. As a result of this change the soluble domain of l2 is flipped out, so that both PI(4,5)P2 binding sites are situated in one plane with the binding site for tyrosinebased endocytic motifs being accessible now. In this still somewhat hypothetical scenario the presence of PI(4,5)P2 in the plasma membrane appears to shift the equilibrium towards the ‘open’ conformation thereby increasing the affinity for cargo membrane proteins (compare also Fig. 3, further discussed below) [24]. It is likely that similar mechanisms facilitate cargo recognition by AP-1 [25], AP-3, or AP-4 adaptor complexes at TGN or endosomal membranes. Ion channels and transporters constitute another large group of proteins that undergo conformational changes upon binding to PIs [26]. PI-sensitivity has been initially reported for + 2+ ATP-sensitive Kþ ATP -channels and Na –Ca -exchangers, but the number of channels regulated by PIs is rapidly increasing, including voltage-gated K+- and Ca2+-as well as sensory transduction channels, especially members of the TRP family [27]. Only in a few cases putative binding sites for PIs have been identified with their modes of action remaining poorly characterized. Based on site-directed mutagenesis experiments clusters of basic and hydrophobic residues within the cytoplasmic domain have been suggested to contribute to the PI(4,5)P2-sensitivity of the afore-mentioned inward-rectifying K+-channels (Kir). Increasing PI(4,5)P2-levels in excised patches impede rundown of these channels by allosteric
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Fig. 3. Coincidence detection of PI(4,5)P2 by trafficking adaptor proteins. Following ligand-induced phosphorylation of G-protein coupled receptors (GPCRs) b-arrestins (left) become recruited to the cytoplasmic domain of the receptor and concomitantly associate with PI(4,5)P2 or PI(3,4,5)P3. In addition they undergo a conformational change exposing binding sites for clathrin and AP-2 (not depicted). Clathrin adaptor complexes AP-2 or AP1 (center) recognize tyrosine-based sorting signals within cargo membrane proteins only in the context of PI(4,5)P2 or PI(4)P, respectively. AP-1 in addition requires binding to the small GTPase Arf1. The endosomal sorting adaptor Hrs (right) sorts ubiquitinated cargo at early endosomal membranes by interacting with PI(3)P via its FYVE domain.
mechanisms, whereas manipulations reducing PI(4,5)P2 levels have opposite effects. The nature of PI(4,5)P2 effects on voltage-gated K+-channels (KV) appears much clearer: These channels contain a cytoplasmic ‘inactivation ball’ at the amino-terminal end of one of their subunits which facilitates rapid channel inactivation by plugging the open channel pore. The ball contains a patch of positively charged and hydrophobic amino acids and is directed into the channel pore by negatively charged residues. PI(4,5)P2 has been suggested to cause relieve from inactivation by sequestering the ball domain into a lipid complex at the membrane surface, thereby preventing it from entering the ion channel (Fig. 2B). Thereby, the presence of PI(4,5)P2 dramatically alters gating characteristics and function of this channel. N-type Ca2+-channels display similar behaviour in response to the manipulation of intracellular PI(4,5)P2 levels [28]. Residues implicated in lipid binding have not yet been identified, but a cluster of basic amino acids is present in an intracellular loop, which might mediate the observed effects. It should be noted here that some clusters of positively charged residues have been shown to constitute unconventional endocytic motifs, which can be recognized by the AP-2 adaptor complex [22]. It is therefore conceivable that PI binding affects not only gating behavior of ion channels but also their intracellular trafficking.
4. Phosphoinositides as regulators of enzymatic activity A number of enzymes involved in membrane traffic have been shown to be directly or indirectly regulated by PIs. While it is well established that members of the ADP-ribosylation factor (Arf) family of membrane trafficking small GTPases regulate PI metabolism, e.g. by activating PI 4- and PI(4)P 5-kinases, PIs in turn may affect Arf function [29]. Arf1 is able to associate with PI(4,5)P2 and this in turn appears to promote nucleotide exchange by increasing the rate of GDP dissociation from Arf [30]. Similar observations have been made for the actin regulatory small GTPase Cdc42. Nucleotide cycling on Arf is also influenced by PIs indirectly. The Arf-specific
exchange factor (Arf-GEF) ARNO is recruited to membranes by PH-domain mediated association with PIs including PI(4,5)P2 and PI(3,4,5)P3 and this is required for efficient nucleotide exchange on Arf. Binding of positively charged residues within the Arf N-terminal domain may cooperate with ARNO to achieve efficient Arf activation [31]. Similar mechanisms exist for other Arf-GEFs. GTP hydrolysis by Arf and Arf-like proteins is facilitated by Arf GTPase activating proteins (Arf-GAPs), an expanding group of multidomain proteins belonging to several different families. Some Arf-GAPs have been found to associate with PI-binding coat proteins including AP-1, and CALM/AP180. The Arf-GAPs ASAP1 [32], AGAP1 [33], and ACAP4 [34] all contain PI(4,5)P2 binding PH domains that physically and functionally interact [35] with the GAP to promote nucleotide hydrolysis on Arf. An interesting feedback loop has been observed for the activity of the phosphatidylinositol phosphate phosphatase PTEN, an important tumor suppressor gene. A PI(4,5)P2-binding motif within the N-terminal domain of PTEN allosterically activates phosphatase activity towards 3-phosphate-containing phosphoinositides such as PI(3,4,5)P3, providing an important means for compartmentalization of enzymatic activity downstream of receptor activation [36]. Other factors regulated by PI(4,5)P2 are phospholipase D (PLD) isozymes. PLDs contain phosphoinositide binding PH and PX domains which are required for membrane localization and catalytic activity. Moreover, the PX domain of PLD has recently been observed to function as a GAP for the PI(4,5)P2-binding endocytic fission protein dynamin during growth factor-induced receptor internalization [37].
5. Conferring specificity to the system: coincidence detection mechanisms Strikingly, most proteins display relatively low affinities for their cognate PI substrates, usually in the micromolar range, which alone may not account for the PI-specific recruitment of a given protein to the membrane. The stability of individual
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protein-membrane complexes can be greatly enhanced by the introduction of additional binding sites for further membrane-resident proteins or other lipid species [4,9]. EEA1, for instance, forms homodimers and thereby increases affinity for PI(3)P [38]. PI-mediated recruitment of vesicle-associated adaptor proteins probably provide the best-studied examples for how the generation of complex protein–protein and protein–lipid interaction networks can ensure specific high affinity binding to the correct membrane (Fig. 3) [4,8,9,39]. Adaptor proteins serve to concentrate cargo proteins destined for vesicular transport at different subcellular localizations. The heterotetrameric clathrin adaptor complexes AP-1 and AP-2 recognize almost identical sets of tyrosine- or dileucine motif-containing membrane cargo proteins, yet function at different intracellular sites [19]. Whereas AP-2 serves to concentrate endocytic cargo during the formation of clathrin-coated vesicles at the plasma membrane, AP-1 plays a similar role in the sequestration of secretory/recycling cargo at perinuclear TGN/endosomal membranes. As outlined above plasmalemmal recruitment of AP-2 involves two binding sites for PI(4,5)P2 at the N-terminus of AP-2a and within its cargorecognizing l2 subunit. Efficient cargo recognition strictly depends on the presentation of endocytic sorting motifs in the context of a PI(4,5)P2-rich membrane, thus restricting AP-2 function to the plasmalemma (Fig. 3, center left) [24]. Highaffinity membrane binding of AP-2 therefore depends on the simultaneous presence of both PI(4,5)P2 and cargo membrane proteins as parts of a coincidence detection mechanism. The csubunit of the closely related TGN/endosomal AP-1 complex exposes a patch of basic residues similar to those present in AP-2a, suggesting that it may operate based on similar principles. However, the number of positive charges within the Nterminal region of AP-1c is reduced [25]. Accordingly, it has been shown that AP-1 binds to the less negatively charged lipid PI(4)P (although another contradicting study [40] had suggested association of AP-1 with PI(4,5)P2). In line with a role for PI(4)P in AP-1 recruitment, depletion of a Golgi-resident PI 4-kinase by RNA interference reduces membrane localization of AP-1 in living cells [41]. A second binding site for PI(4)P within AP-1l has not been identified. Instead, recruitment of AP-1 to perinuclear membranes is promoted by activated Arf1 (Fig. 3, center right). Its plasma membrane localized homologue Arf6 has been suggested to play a similar stimulatory role during AP-2 recruitment [4,29,39]. However, the function of Arf6 in this process might be restricted to stimulation of PI(4,5)P2 synthesis and could therefore indirectly affect the localization of AP-2. A presumably weak direct association of Arf6-GTP with AP-2 has also been reported [42]. FAPP 1 and 2 proteins appear to function at TGN membranes by directing cargo proteins into constitutive carrier vesicles and tubules destined for the cell surface. Targeting of FAPP to the Golgi is dependent on the integrity of its N-terminal PH-domain, which displays high specificity for PI(4). In addition, FAPP-PH associates with GTP-bound Arf1, a mechanism that probably increases the avidity of FAPP binding [43]. Interestingly, Arf1 also binds to and stimulates PI 4-kinase type IIIb [44], thereby ensuring the generation of high local concentrations of PI(4)P that in turn are needed for adaptor recruitment. The two monomeric adaptors epsin 1 and Hrs/Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate) form a
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similar pair of proteins that recognize the same cargo, but function at different subcellular membranes. Both adaptors possess binding sites for mono-ubiquitinated membrane proteins, and are recruited to the plasma membrane or to early endosomal membranes, respectively [45]. Their differential distribution is due to the selective binding of epsin 1 to PI(4,5)P2containing membranes (see above), and the specificity of Hrs/ Vps27 for PI(3)P conferred by its FYVE domain. As for the recruitment of heterotetrameric adaptor complexes, efficient membrane translocation of Hrs/Vps27 requires coincidence detection of both PI(3)P and ubiquitinated cargo proteins (Fig. 3, right) which subsequently triggers association with the ESCRT-1 complex protein Tsg101 and cargo delivery into multivesicular bodies. Arrestins serve as adaptors for the internalization of G-protein coupled receptors (GPCR). Stimulation with agonist causes phosphorylation of serine and threonine residues in the receptor’s cytoplasmic domain. This creates a negatively charged surface that penetrates deeply into the b-arrestin core triggering a conformational change. As a result of this change binding sites for clathrin and AP-2 within b-arrestin’s C-terminal domain become accessible. An additional region adjacent to the interaction surface for the phosphorylated GPCR cytoplasmic tail is engaged in interaction with PIs [PI(3,4,5)P3 or PI(4,5)P2], thereby coupling clathrin/AP-2-mediated receptor endocytosis to PI metabolism (Fig. 3, left) [46]. Several platforms for the interaction with additional signalling molecules have been identified, i.e. Arf6, ARNO, c-Src, or Mdm2. It is therefore easy to imagine that PI-binding could modulate the interactions of arrestins with other binding partners, and vice versa.
6. Conclusion and perspectives From the above said it seems clear that the role of phosphoinositides as spatial landmarks for membrane trafficking and cell signalling [47] is well established now. We are also beginning to understand how PIs and peripheral membrane proteins may cooperate in providing spatiotemporal cues for vesicle budding and cargo transport along intracellular routes. Examples of coincidence detection are likely to be used for integrating PI-dependent protein functions in membrane traffic with cell signalling events and regulation of the cytoskeleton [20]. Given that the first high resolution structures of membrane integral ion channels and transporters have become available we may soon also know more about the role of PIs in regulating membrane integral proteins and their intracellular dynamics. The importance of PIs in cell physiology is further illustrated by the number and severity of genetic diseases related to defects in PI metabolism, frequently caused by or related to defective vesicular traffic. Another area of growing importance is the subversion of PIs and their effectors in intracellular transport and motility by bacterial pathogens [48,49]. On the other hand, we still lack insights into the dynamics of PIs and PI binding proteins in a native-like environment. This is largely owed to limitations in detecting PIs in situ [50]. Specific probes based on PI binding domain fusion proteins are available for all PIs with the exceptions of PI(5)P and phosphatidylinositol itself, and have been used to determine the subcellular distribution of distinct PIs even on an ultrastructural level in
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cryosections [51]. However, their use in living cells or organisms in vivo is limited by uncertainties regarding possible dominantnegative effects caused by PI sequestration as well as considerations regarding the availability of PI subpools in different membranes due to competition with endogenous PI binding factors. Fo¨rster resonance energy transfer (FRET)-based in vivo sensors, perhaps paired with the use of tandem PI probes may alleviate at least some of these problems and could provide important information regarding PI dynamics and their effects on adaptor-mediated membrane transport or the localization of subpools of PIs in the vicinity of ion channels and trafficking signalling receptors. Moreover, little is known about the exact chemical concentrations of PIs in different tissues or native isolated organelles. With the development of ever more sensitive mass spectrometric methods we are likely to witness progress here. Ideally, we would like to be able to quantitatively analyze in vitro and visualize in vivo PI binding adaptor proteins or transmembrane ion channels in complex with their PI ligands. How can we be certain that PIs are directly involved in the membrane trafficking processes discussed above? So far most studies pertaining to the role of PIs in vesicular transport and membrane protein function have relied on indirect and/ or long-term methods of manipulating PI levels, i.e. by genetic ablation, knock-down, or overexpression of PI metabolizing enzymes or their respective regulators causing irreversible constitutive changes in PIs. The recent development of rapidly inducible systems to manipulate the intracellular concentration of PIs including PI(3)P and PI(4,5)P2 holds great promise [52– 54] and should help us to solidify our hypotheses on how and where exactly PIs may act in a given physiological context. Likewise, fast-acting acute chemical inhibitors of PI metabolizing enzymes could provide important new insights into the role of PIs in regulating membrane traffic and protein function. Acknowledgements: The work in the authors’ laboratory is supported by grants from the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie (FCI), and the European Molecular Biology Organization (YIP Award to V.H.).
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Phosphoinositides: Regulators of membrane traffic and protein function Michael Krauß, Volker Haucke* Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universita¨t Berlin, Takustraße 6, 14195 Berlin, Germany Received 13 December 2006; revised 30 January 2007; accepted 31 January 2007 Available online 12 February 2007 Edited by Thomas So¨llner
Abstract Phosphoinositides serve as important spatio-temporal regulators of intracellular trafficking and cell signalling events. In addition to their recognition by specific phosphoinositide binding domains present within cytoplasmic adaptor proteins or membrane integral channels and transporters phosphoinositides may affect membrane transport by eliciting conformational changes within proteins or by regulating enzymatic activities. During adaptor-mediated membrane traffic phosphoinositides form part of coincidence detection systems that aid in targeting pools of specific phosphoinositides to select intracellular transport pathways. In this review, we discuss potential mechanisms for conferring selectivity onto the phosphoinositide code as well as possible avenues for future research. Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Phosphoinositides; Membrane traffic; Adaptor proteins; Phosphoinositide binding domains; Coincidence detection
1. Introduction Over the past decade phosphatidylinositol and its phosphorylated derivatives (referred to as phosphoinositides, PIs) have been implicated in numerous membrane trafficking and cellular signalling events (reviewed in [1–4]). Different phosphoinositide species have been shown to exhibit distinct characteristic subcellular distribution patterns [3,4]: Whereas Golgi membranes have been reported to be enriched in phosphatidylinositol 4phosphate [PI(4)P], higher phosphorylated derivatives, such as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], are found predominantly in the inner leaflet of the plasma membrane. Following growth factor receptor activation PI(3,4,5)P3 may also accumulate on endomembranes. Endosomal organelles are characterized by the presence of phosphatidylinositol 3phosphate [PI(3)P] and derived lipid species. Based on these observations it has been suggested, that PIs contribute to the generation of organelle identity, and do so by recruiting specific sets of proteins that harbour appropriate PI binding domains [2–4]. Strikingly, different PI species can be reversibly interconverted through the regulated activities of specific kinases and phosphatases, resulting in short-term alterations of the protein * Corresponding author. Fax: +49 30 838 56919. E-mail address: [email protected] (V. Haucke).
binding properties and functions of a given membrane domain. These plastic properties render PIs ideally suited for integrating membrane trafficking and cell signalling events. Correspondingly, PI metabolizing enzymes appear to be constituents of complex regulatory networks that have been the subject of intense research. Regulators of PI metabolizing enzymes have been identified that control PI turnover in time and space and thereby serve as triggers for the generation of local PI pools dedicated to distinct (sub-) cellular functions. Progress in this area has been the subject of excellent recent reviews [3–6]. Despite of the large progress made we still poorly understand how one and the same PI species depending on the physiological context can trigger distinct events. In this review, we will discuss possible mechanisms by which specificity of PI-mediated functions can be achieved. 2. Phosphoinositides promote specific membrane recruitment of cytosolic protein subsets PIs can directly associate with a variety of different proteins including cytoplasmic factors, such as membrane trafficking and cell signalling adaptors as well as membrane integral channels and transporters. In most cases binding is achieved by electrostatic interactions involving the negatively charged PI headgroups and patches of positive charges within the target protein. PI binding sites can be contained within unstructured regions harboring clusters of basic residues, as is the case for MARCKS and related proteins [7], or within folded domains that may recognize distinct arrangements of PI-exposed phosphate residues by preformed binding grooves or surfaceexposed fingers [8,9]. Sometimes binding is supported by additional hydrophobic amino acid residues located in close proximity to the basic patch, which may steer into the lipid bilayer. A variety of structurally different PI binding domains have been identified, including PH (pleckstrin homology; [10]), FYVE (named after four proteins in which it has been initially identified: Fab1p, YOTB, Vac1p and EEA1), PX (phox homology; [11]), E/ANTH (epsin/AP180 N-terminal homology) [12–14], C2, PTB, and FERM domains [8] (Fig. 1). These display marked differences in specificity and affinity, with some of them promoting exclusive binding to one type of PI, and others being more promiscuous by recognizing more or less closely related PI species. PH domains, for instance, have been identified in more than 250 proteins, rendering this the largest family of PI binding modules [10]. Sequence homology between different PH domain family members is relatively low; nonetheless, the tertiary structure of this approximately 120 amino acid long domain appears to be highly conserved,
0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.01.089
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Fig. 1. Structures of phosphoinositide binding domains and their ligands. Structures of the PI(4,5)P2-specific PH domain of phospholipase Cd in complex with inositol (1,4,5)-triphosphate (PDB: 1MAI), the inositol (1,3)-bisphosphate complexed FYVE domain of early endosomal antigen 1 (EEA1; PDB: 1HYI), the PI(3)-binding Phox-homology (PX) domain of p40Phox (PDB: 1H6H), and the A/ENTH domains derived from the endocytic proteins CALM (an AP180-homologue; PDB: 1HFA) or epsin 1 (PDB: 1H0A) with their respective phosphoinositide ligands.
exhibiting a b-sandwich structure flanked by an a-helical stretch at its carboxy-terminal end. Both, b-strands and loop regions contribute positively charged side chains that establish non-covalent bonds with hydroxyl and phosphate groups of the respective phosphoinositide (Fig. 1, top left). Loops represent the regions of lowest sequence conservation and probably account for the large variability in PI binding specificity. Notably, a genome-wide analysis in yeast has revealed that only few PH domains selectively recognize one particular PI, and that most bind PIs with low affinity [15]. Therefore, specific membrane-targeting must be achieved by additional factors. Binding of proteins to PH domains has been described in a few cases of which the association between Gbc and the PH domain of GRK2 may represent the best documented example [16]. This represents a potential caveat for the use of PH domains as PI-selective probes in live cell imaging studies [17]. By contrast to PH domains FYVE domains display an exclusive specificity for PI(3)P. These domains are relatively small units (about 70 amino acids in length) composed of one a-helix and four short b-strands being held together by two cysteinecoordinated Zn2+-ions (Fig. 1, top middle) [18]. Specificity for PI(3)P is achieved by a selective network of electrostatic interactions and hydrogen bonds between the lipid headgroup and conserved amino acid side chains contributed by three signature motifs. PX domains (Fig. 1, top right), which have been found in numerous proteins with a wide spectrum of functions including vesicular traffic, and phospholipid metabolism display a much broader PI specificity than FYVE domains. A stretch of about 120 amino acids is folded into a three-stranded b-sheet and six helical regions, which together form a positively charged binding pocket for the cognate PI. Binding of
PX and FYVE domains to the membrane surface might be assisted by additional regions exposing hydrophobic residues, which are presumably capable of penetrating the membrane.
3. Binding to phosphoinositides can be associated with conformational changes The ENTH of epsin [12] and the ANTH domain of AP180 [13,14] associate selectively with PI(4,5)P2. Despite forming homologous solenoid structures, both proteins use different mechanisms to bind to PI(4,5)P2. Whereas the ANTH domain exposes a basic patch of three lysine and one histidine residue at its surface (Fig. 1, bottom left), the ENTH domain embeds the PI(4,5)P2 headgroup in a deep, positively charged pocket (Fig. 1, bottom right). Strikingly, the N-terminal ENTH a-helix (termed ‘helix 0’) is involved in the formation of this pocket. Folding of the ‘0-helix’ is induced upon the approximation of the ENTH domain to a PI(4,5)P2-enriched membrane surface. Thus, in the case of epsin PI(4,5)P2 binding triggers a conformational change (i.e. folding) of a distinct portion of the protein. The newly formed helix is amphipathic in nature; positively charged residues on one side of the helix are engaged in lipid headgroup binding, whereas hydrophobic residues on the other face insert into the cytoplasmic face of the lipid bilayer. Binding of the epsin ENTH domain to PI(4,5)P2-rich plasmalemmal sites may drive acquisition of negative curvature by insertion of hydrophobic moieties into one leaflet of the underlying membrane (Fig. 2A) [12]. A similar PI(4)P-induced conformational change has been proposed to occur in the epsin family member enthoprotin/Clint/epsinR [19] to induce
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Fig. 2. Examples of phosphoinositide-induced conformational changes. (A) PI(4,5)P2 (yellow)-induced folding of an N-terminal helix (helix 0; purple) within the ENTH domain of epsin 1 (green) drives membrane curvature acquisition by insertion of hydrophobic residues within this helix into the cytoplasmic leaflet of the plasma membrane. (B) In the absence PI(4,5)P2 of voltage-gated K+-channels (KV) are inactive due to plugging of the pore by an N-terminal globular inactivation domain. PI(4,5)P2 (yellow) was suggested to counteract the inactivation mechanism by sequestering the inactivation domain at the membrane surface, thereby preventing it from clogging the pore.
bending of trans-Golgi network (TGN)-derived membranes. Moreover, some members of the BAR domain family of membrane curvature-sensors and inducers [20] also contain an amphipathic, potentially membrane-active helix or interact with small GTPases that could exhibit similar activities. Membrane penetrating activity has also been suggested for synaptotagmin 1, a type I membrane protein implicated in Ca2+-triggered exo–endocytosis of synaptic vesicles at neuronal synapses. Synaptotagmin 1 contains two cytoplasmic, Ca2+-sensing C2-domains, termed C2A and C2B. The C2B domain also harbors a high-affinity binding site for PI(4,5)P2. It has been suggested, that the C2B domain pre-absorbs to PI(4,5)P2-containing membranes, but is reoriented upon Ca2+-binding such that it inserts hydrophobic residues into the lipid bilayer [21]. The binding site for PI(4,5)P2 partially overlaps with residues associating with the heterotetrameric clathrin adaptor protein AP-2 (comprising a, b2, l2, and r2 subunits) [22]. The precise interplay between all three factors still remains unclear, although one might expect that the interactions of synaptotagmin 1 with PI(4,5)P2 and AP-2 are mutually exclusive and thus need to be precisely regulated. AP-2 itself contains two patches of basic amino acid residues within its a- and l-subunits and has been proposed to adopt two interconvertible conformations. In its closed, presumably inactive conformation the binding site for cargo proteins displaying tyrosine-based endocytic motifs is buried by side chains from b2 as the soluble carboxy-terminal domain of l2 folds back onto the AP-2 core [23]. In this conformational state, the two PI(4,5)P2 binding sites are located on perpendicular faces of the AP-2 complex with only one of them being
able to access the membrane at any given time. Phosphorylation of l2 at a single threonine residue within a hinge region connecting its N- and C-terminal domains by the serine/threonine kinases AAK1 or GAK/auxilin 2 facilitates a dramatic conformational change. As a result of this change the soluble domain of l2 is flipped out, so that both PI(4,5)P2 binding sites are situated in one plane with the binding site for tyrosinebased endocytic motifs being accessible now. In this still somewhat hypothetical scenario the presence of PI(4,5)P2 in the plasma membrane appears to shift the equilibrium towards the ‘open’ conformation thereby increasing the affinity for cargo membrane proteins (compare also Fig. 3, further discussed below) [24]. It is likely that similar mechanisms facilitate cargo recognition by AP-1 [25], AP-3, or AP-4 adaptor complexes at TGN or endosomal membranes. Ion channels and transporters constitute another large group of proteins that undergo conformational changes upon binding to PIs [26]. PI-sensitivity has been initially reported for + 2+ ATP-sensitive Kþ ATP -channels and Na –Ca -exchangers, but the number of channels regulated by PIs is rapidly increasing, including voltage-gated K+- and Ca2+-as well as sensory transduction channels, especially members of the TRP family [27]. Only in a few cases putative binding sites for PIs have been identified with their modes of action remaining poorly characterized. Based on site-directed mutagenesis experiments clusters of basic and hydrophobic residues within the cytoplasmic domain have been suggested to contribute to the PI(4,5)P2-sensitivity of the afore-mentioned inward-rectifying K+-channels (Kir). Increasing PI(4,5)P2-levels in excised patches impede rundown of these channels by allosteric
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Fig. 3. Coincidence detection of PI(4,5)P2 by trafficking adaptor proteins. Following ligand-induced phosphorylation of G-protein coupled receptors (GPCRs) b-arrestins (left) become recruited to the cytoplasmic domain of the receptor and concomitantly associate with PI(4,5)P2 or PI(3,4,5)P3. In addition they undergo a conformational change exposing binding sites for clathrin and AP-2 (not depicted). Clathrin adaptor complexes AP-2 or AP1 (center) recognize tyrosine-based sorting signals within cargo membrane proteins only in the context of PI(4,5)P2 or PI(4)P, respectively. AP-1 in addition requires binding to the small GTPase Arf1. The endosomal sorting adaptor Hrs (right) sorts ubiquitinated cargo at early endosomal membranes by interacting with PI(3)P via its FYVE domain.
mechanisms, whereas manipulations reducing PI(4,5)P2 levels have opposite effects. The nature of PI(4,5)P2 effects on voltage-gated K+-channels (KV) appears much clearer: These channels contain a cytoplasmic ‘inactivation ball’ at the amino-terminal end of one of their subunits which facilitates rapid channel inactivation by plugging the open channel pore. The ball contains a patch of positively charged and hydrophobic amino acids and is directed into the channel pore by negatively charged residues. PI(4,5)P2 has been suggested to cause relieve from inactivation by sequestering the ball domain into a lipid complex at the membrane surface, thereby preventing it from entering the ion channel (Fig. 2B). Thereby, the presence of PI(4,5)P2 dramatically alters gating characteristics and function of this channel. N-type Ca2+-channels display similar behaviour in response to the manipulation of intracellular PI(4,5)P2 levels [28]. Residues implicated in lipid binding have not yet been identified, but a cluster of basic amino acids is present in an intracellular loop, which might mediate the observed effects. It should be noted here that some clusters of positively charged residues have been shown to constitute unconventional endocytic motifs, which can be recognized by the AP-2 adaptor complex [22]. It is therefore conceivable that PI binding affects not only gating behavior of ion channels but also their intracellular trafficking.
4. Phosphoinositides as regulators of enzymatic activity A number of enzymes involved in membrane traffic have been shown to be directly or indirectly regulated by PIs. While it is well established that members of the ADP-ribosylation factor (Arf) family of membrane trafficking small GTPases regulate PI metabolism, e.g. by activating PI 4- and PI(4)P 5-kinases, PIs in turn may affect Arf function [29]. Arf1 is able to associate with PI(4,5)P2 and this in turn appears to promote nucleotide exchange by increasing the rate of GDP dissociation from Arf [30]. Similar observations have been made for the actin regulatory small GTPase Cdc42. Nucleotide cycling on Arf is also influenced by PIs indirectly. The Arf-specific
exchange factor (Arf-GEF) ARNO is recruited to membranes by PH-domain mediated association with PIs including PI(4,5)P2 and PI(3,4,5)P3 and this is required for efficient nucleotide exchange on Arf. Binding of positively charged residues within the Arf N-terminal domain may cooperate with ARNO to achieve efficient Arf activation [31]. Similar mechanisms exist for other Arf-GEFs. GTP hydrolysis by Arf and Arf-like proteins is facilitated by Arf GTPase activating proteins (Arf-GAPs), an expanding group of multidomain proteins belonging to several different families. Some Arf-GAPs have been found to associate with PI-binding coat proteins including AP-1, and CALM/AP180. The Arf-GAPs ASAP1 [32], AGAP1 [33], and ACAP4 [34] all contain PI(4,5)P2 binding PH domains that physically and functionally interact [35] with the GAP to promote nucleotide hydrolysis on Arf. An interesting feedback loop has been observed for the activity of the phosphatidylinositol phosphate phosphatase PTEN, an important tumor suppressor gene. A PI(4,5)P2-binding motif within the N-terminal domain of PTEN allosterically activates phosphatase activity towards 3-phosphate-containing phosphoinositides such as PI(3,4,5)P3, providing an important means for compartmentalization of enzymatic activity downstream of receptor activation [36]. Other factors regulated by PI(4,5)P2 are phospholipase D (PLD) isozymes. PLDs contain phosphoinositide binding PH and PX domains which are required for membrane localization and catalytic activity. Moreover, the PX domain of PLD has recently been observed to function as a GAP for the PI(4,5)P2-binding endocytic fission protein dynamin during growth factor-induced receptor internalization [37].
5. Conferring specificity to the system: coincidence detection mechanisms Strikingly, most proteins display relatively low affinities for their cognate PI substrates, usually in the micromolar range, which alone may not account for the PI-specific recruitment of a given protein to the membrane. The stability of individual
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protein-membrane complexes can be greatly enhanced by the introduction of additional binding sites for further membrane-resident proteins or other lipid species [4,9]. EEA1, for instance, forms homodimers and thereby increases affinity for PI(3)P [38]. PI-mediated recruitment of vesicle-associated adaptor proteins probably provide the best-studied examples for how the generation of complex protein–protein and protein–lipid interaction networks can ensure specific high affinity binding to the correct membrane (Fig. 3) [4,8,9,39]. Adaptor proteins serve to concentrate cargo proteins destined for vesicular transport at different subcellular localizations. The heterotetrameric clathrin adaptor complexes AP-1 and AP-2 recognize almost identical sets of tyrosine- or dileucine motif-containing membrane cargo proteins, yet function at different intracellular sites [19]. Whereas AP-2 serves to concentrate endocytic cargo during the formation of clathrin-coated vesicles at the plasma membrane, AP-1 plays a similar role in the sequestration of secretory/recycling cargo at perinuclear TGN/endosomal membranes. As outlined above plasmalemmal recruitment of AP-2 involves two binding sites for PI(4,5)P2 at the N-terminus of AP-2a and within its cargorecognizing l2 subunit. Efficient cargo recognition strictly depends on the presentation of endocytic sorting motifs in the context of a PI(4,5)P2-rich membrane, thus restricting AP-2 function to the plasmalemma (Fig. 3, center left) [24]. Highaffinity membrane binding of AP-2 therefore depends on the simultaneous presence of both PI(4,5)P2 and cargo membrane proteins as parts of a coincidence detection mechanism. The csubunit of the closely related TGN/endosomal AP-1 complex exposes a patch of basic residues similar to those present in AP-2a, suggesting that it may operate based on similar principles. However, the number of positive charges within the Nterminal region of AP-1c is reduced [25]. Accordingly, it has been shown that AP-1 binds to the less negatively charged lipid PI(4)P (although another contradicting study [40] had suggested association of AP-1 with PI(4,5)P2). In line with a role for PI(4)P in AP-1 recruitment, depletion of a Golgi-resident PI 4-kinase by RNA interference reduces membrane localization of AP-1 in living cells [41]. A second binding site for PI(4)P within AP-1l has not been identified. Instead, recruitment of AP-1 to perinuclear membranes is promoted by activated Arf1 (Fig. 3, center right). Its plasma membrane localized homologue Arf6 has been suggested to play a similar stimulatory role during AP-2 recruitment [4,29,39]. However, the function of Arf6 in this process might be restricted to stimulation of PI(4,5)P2 synthesis and could therefore indirectly affect the localization of AP-2. A presumably weak direct association of Arf6-GTP with AP-2 has also been reported [42]. FAPP 1 and 2 proteins appear to function at TGN membranes by directing cargo proteins into constitutive carrier vesicles and tubules destined for the cell surface. Targeting of FAPP to the Golgi is dependent on the integrity of its N-terminal PH-domain, which displays high specificity for PI(4). In addition, FAPP-PH associates with GTP-bound Arf1, a mechanism that probably increases the avidity of FAPP binding [43]. Interestingly, Arf1 also binds to and stimulates PI 4-kinase type IIIb [44], thereby ensuring the generation of high local concentrations of PI(4)P that in turn are needed for adaptor recruitment. The two monomeric adaptors epsin 1 and Hrs/Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate) form a
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similar pair of proteins that recognize the same cargo, but function at different subcellular membranes. Both adaptors possess binding sites for mono-ubiquitinated membrane proteins, and are recruited to the plasma membrane or to early endosomal membranes, respectively [45]. Their differential distribution is due to the selective binding of epsin 1 to PI(4,5)P2containing membranes (see above), and the specificity of Hrs/ Vps27 for PI(3)P conferred by its FYVE domain. As for the recruitment of heterotetrameric adaptor complexes, efficient membrane translocation of Hrs/Vps27 requires coincidence detection of both PI(3)P and ubiquitinated cargo proteins (Fig. 3, right) which subsequently triggers association with the ESCRT-1 complex protein Tsg101 and cargo delivery into multivesicular bodies. Arrestins serve as adaptors for the internalization of G-protein coupled receptors (GPCR). Stimulation with agonist causes phosphorylation of serine and threonine residues in the receptor’s cytoplasmic domain. This creates a negatively charged surface that penetrates deeply into the b-arrestin core triggering a conformational change. As a result of this change binding sites for clathrin and AP-2 within b-arrestin’s C-terminal domain become accessible. An additional region adjacent to the interaction surface for the phosphorylated GPCR cytoplasmic tail is engaged in interaction with PIs [PI(3,4,5)P3 or PI(4,5)P2], thereby coupling clathrin/AP-2-mediated receptor endocytosis to PI metabolism (Fig. 3, left) [46]. Several platforms for the interaction with additional signalling molecules have been identified, i.e. Arf6, ARNO, c-Src, or Mdm2. It is therefore easy to imagine that PI-binding could modulate the interactions of arrestins with other binding partners, and vice versa.
6. Conclusion and perspectives From the above said it seems clear that the role of phosphoinositides as spatial landmarks for membrane trafficking and cell signalling [47] is well established now. We are also beginning to understand how PIs and peripheral membrane proteins may cooperate in providing spatiotemporal cues for vesicle budding and cargo transport along intracellular routes. Examples of coincidence detection are likely to be used for integrating PI-dependent protein functions in membrane traffic with cell signalling events and regulation of the cytoskeleton [20]. Given that the first high resolution structures of membrane integral ion channels and transporters have become available we may soon also know more about the role of PIs in regulating membrane integral proteins and their intracellular dynamics. The importance of PIs in cell physiology is further illustrated by the number and severity of genetic diseases related to defects in PI metabolism, frequently caused by or related to defective vesicular traffic. Another area of growing importance is the subversion of PIs and their effectors in intracellular transport and motility by bacterial pathogens [48,49]. On the other hand, we still lack insights into the dynamics of PIs and PI binding proteins in a native-like environment. This is largely owed to limitations in detecting PIs in situ [50]. Specific probes based on PI binding domain fusion proteins are available for all PIs with the exceptions of PI(5)P and phosphatidylinositol itself, and have been used to determine the subcellular distribution of distinct PIs even on an ultrastructural level in
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cryosections [51]. However, their use in living cells or organisms in vivo is limited by uncertainties regarding possible dominantnegative effects caused by PI sequestration as well as considerations regarding the availability of PI subpools in different membranes due to competition with endogenous PI binding factors. Fo¨rster resonance energy transfer (FRET)-based in vivo sensors, perhaps paired with the use of tandem PI probes may alleviate at least some of these problems and could provide important information regarding PI dynamics and their effects on adaptor-mediated membrane transport or the localization of subpools of PIs in the vicinity of ion channels and trafficking signalling receptors. Moreover, little is known about the exact chemical concentrations of PIs in different tissues or native isolated organelles. With the development of ever more sensitive mass spectrometric methods we are likely to witness progress here. Ideally, we would like to be able to quantitatively analyze in vitro and visualize in vivo PI binding adaptor proteins or transmembrane ion channels in complex with their PI ligands. How can we be certain that PIs are directly involved in the membrane trafficking processes discussed above? So far most studies pertaining to the role of PIs in vesicular transport and membrane protein function have relied on indirect and/ or long-term methods of manipulating PI levels, i.e. by genetic ablation, knock-down, or overexpression of PI metabolizing enzymes or their respective regulators causing irreversible constitutive changes in PIs. The recent development of rapidly inducible systems to manipulate the intracellular concentration of PIs including PI(3)P and PI(4,5)P2 holds great promise [52– 54] and should help us to solidify our hypotheses on how and where exactly PIs may act in a given physiological context. Likewise, fast-acting acute chemical inhibitors of PI metabolizing enzymes could provide important new insights into the role of PIs in regulating membrane traffic and protein function. Acknowledgements: The work in the authors’ laboratory is supported by grants from the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie (FCI), and the European Molecular Biology Organization (YIP Award to V.H.).
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