© The Rockefeller University Press, 0021-9525/2002/7/209 $5.00
The Journal of Cell Biology, Volume 158, Number 2, July 22, 2002 209-214


Report

A phosphatidylinositol (4,5)-bisphosphate binding site within µ2-adaptin regulates clathrin-mediated endocytosis

Gundula Rohde1, Dirk Wenzel2 and Volker Haucke1

1 Zentrum für Biochemie and Molekulare Zellbiologie, Department of Biochemistry II, University of Göttingen, D-37073 Göttingen, Germany
2 Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany

Address correspondence to Volker Haucke, Zentrum für Biochemie and Molekulare Zellbiologie, Department of Biochemistry II, University of Göttingen, Humboldtalle 23, D-37073 Göttingen, Germany. Tel.: 49-55-139-9854. Fax: 49-551-39 1-2198. E-mail: vhaucke{at}gwdg.de


   Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within µ2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant µ2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant µ2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to µ2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.

Key Words: clathrin; endocytosis; adaptor; µ2; phosphoinositides


   Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Clathrin-mediated endocytosis is a vesicular transport process by which eukaryotic cells take up nutrients, internalize growth factor receptors (Mellman, 1996; Marsh and McMahon, 1999), and recycle synaptic vesicles after exocytotic activity (Hannah et al., 1999; Brodin et al., 2000; Slepnev and De Camilli, 2000; Jarousse and Kelly, 2001). Clathrin is the major structural component of coated pits (Hirst and Robinson, 1998; Kirchhausen, 2000; Robinson and Bonifacino, 2001) that coassembles with the heterotetrameric adaptor complex AP-2 and the monomeric coat protein AP180/CALM into clathrin-coated pits (Marsh and McMahon, 1999; Kirchhausen, 2000). This process is aided by several accessory proteins including amphiphysin, eps15, syndapin, endophilin, intersectin, and epsin (for reviews see Brodin et al., 2000; Slepnev and De Camilli, 2000). The AP-2 adaptor complex (composed of four subunits, {alpha}, ß2, µ2, and {sigma}2) executes two key functions in the initial stages of clathrin-coated pit nucleation: it recruits clathrin to the membrane and, via its µ2 subunit, selects specific cargo proteins (Robinson and Bonifacino, 2001). A crucial question is how AP-2 assembly and cargo recognition are regulated at endocytotic "hot spots" at the plasmalemma. Phosphoinositide lipids, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2),* can directly interact with several endocytotic proteins, including {alpha}-adaptin (Gaidarov and Keen, 1999), thereby facilitating clathrin-mediated endocytosis (Jost et al., 1998; Arneson et al., 1999; Cremona et al., 1999; Wenk et al., 2001). Here we report on the identification and characterization of a novel phosphoinositide binding site within µ2-adaptin.


   Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
The endocytotic proteins {alpha}-adaptin, AP180, and epsin have been shown to interact with phosphoinositides via clusters of basic residues (Gaidarov and Keen, 1999; Ford et al., 2001; Itoh et al., 2001; Mao et al., 2001). Because µ2-adaptin harbors a major membrane binding site within AP-2 (Page and Robinson, 1995), we analyzed the primary sequence of µ2-adaptins from various species. Human, mouse, rat, fly, and nematode µ2-adaptins all shared a common sequence motif containing several conserved lysine residues reminiscent of the phosphoinositide binding site of AP-180 (Ford et al., 2001; Mao et al., 2001) (Fig. 1 A). Medium chains of AP complexes targeted to intracellular locations other than the plasma membrane, such as µ1- or µ3-adaptins, did not contain the putative phosphoinositide binding motif. This lysine cluster represents a surface-exposed positively charged patch within subdomain B of the crystallized cargo recognition domain of µ2 (Owen and Evans, 1998; Nesterov et al., 1999) (Fig. 1 B). To test whether µ2 can interact with phosphoinositides, we purified recombinant wild-type µ2 (aa 157–435) or a mutant in which three conserved lysine residues (K345, K354, and K356) had been replaced by glutamates (Fig. 1 C). We then prepared unilamellar liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) supplemented with 10% PI(4,5)P2, PI (4)-phosphate (PI[4]P), or PC and assayed the ability of µ2 to interact with the liposomal membrane. Wild-type µ2 cosedimented only with PI(4,5)P2-containing liposomes but not with PI(4)P or PC. The µ2 mutant (KKK–EEE) displayed a greatly reduced affinity for PI(4,5)P2 (Fig. 1, C and D). Among different phosphoinositides, the strongest interaction was seen with PI(4,5)P2, whereas both PI(3,5)P2 and PI(3,4)P2 were less effective in recruiting µ2. To our surprise, no specific interaction with PI (3,4,5)-trisphosphate (PI[3,4,5]P3) or any other lipid tested was detectable (Fig. 1 E).



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Figure 1. Phosphoinositide binding of µ2-adaptin. (A) Sequence alignment of µ-adaptins from various species. The conserved lysine residues within the putative phosphoinositide binding site are shaded. (B) Structural model of µ2 (aa 157–435) complexed with a tyrosine-based endocytosis signal (yellow) (modified from Owen and Evans, 1998). The three conserved lysine residues (red) form a basic patch at the surface of subdomain B. (C) Purified wild-type or mutant µ2 (2 µg) were incubated with liposomes containing 10% PC, PI(4)P, or PI(4,5)P2. After reisolation of the liposomes, samples were analyzed by SDS-PAGE and staining with Coomassie blue. 50% Std, 50% of the protein sample added to the assay. (D) Quantification of the binding of wild-type or mutant µ2 to PI(4,5) P2-containing liposomes from three independent experiments and plotted as mean ± SE. (E) Binding of µ2 to liposomes containing 10% of the indicated lipid. PS, phosphatidylserine; PA, phosphatidic acid. The experiment was performed as described in A.

 
To see whether the mutant protein had retained the structural characteristics of wild-type µ2, we used limited proteolysis. Incubation of wild-type µ2 with increasing concentrations of trypsin generated two protease-resistant fragments, f1 and f2, similar to those seen with µ2 incorporated into AP-2 complexes (Aguilar et al., 1997). The mutant protein yielded identical proteolytic products upon limited digestion (Fig. 2 A), suggesting that it had retained native folding characteristics. An important functional property of µ2-adaptin is to recognize endocytosis signals of transmembrane cargo receptors (Mellman, 1996; Marsh and McMahon, 1999; Robinson and Bonifacino, 2001). We therefore analyzed the ability of the mutant protein to interact with tyrosine-based endocytosis signals. To this aim, we coupled peptides harboring the tyrosine-based endocytosis signal YQRL or a nonfunctional variant (AQRL) to beads (Rapoport et al., 1997) and assayed the ability of wild-type and mutant µ2 to interact specifically with these motifs. Both wild-type and mutant µ2 displayed an equal ability to recognize the functional YQRL peptide, whereas both of them failed to bind to the mutant AQRL variant (Fig. 2, B and C). Thus, mutation of the triple lysine cluster does not impair the ability of µ2 to recognize tyrosine-based endocytosis signals, which is in agreement with structural studies (Owen and Evans, 1998).



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Figure 2. Effects of the KKK–EEE mutation on the interaction of µ2 with tyrosine-based endocytosis signals and synaptotagmin. (A) µ2 (157–435; 2 µg) was incubated with the indicated concentrations of trypsin for 15 min at RT. Samples were analyzed by 12% SDS-PAGE and staining with Coomassie blue. The asterisk denotes trypsin. (B) Mutant and wild-type µ2 (157–435; 2 µg) were incubated for 1 h at 4°C with peptides bearing the tyrosine-based endocytosis motif of TGN38 (YQRL) or its AQRL mutant immobilized on beads. Beads were reisolated, washed, and analyzed by SDS-PAGE and staining with Coomassie blue. Std, 50% of the protein added to the assay. (C) Quantification of the results shown in B. Data from three independent experiments were analyzed and plotted as mean ± SE. Binding of wild-type µ2 to the YQRL peptide was set as 100%.

 
Clathrin/AP-2–coated pits can assemble efficiently on liposomal membranes (Takei et al., 1998). We therefore investigated whether purified µ2 could compete with clathrin/AP-2 recruitment to liposomes. We incubated liposomes containing 10% PI(4,5)P2 with rat brain cytosol and ATP/GTP{gamma}S, conditions under which clathrin-coated pits are abundantly formed in vitro (Takei et al., 1996, 1998), in the presence or absence of purified µ2. After reisolation, clathrin/AP-2 association of the liposomes was analyzed by SDS-PAGE and staining with Ponceau S (Fig. 3 A) and immunoblotting (Fig. 3 B). Wild-type µ2 inhibited the recruitment of both clathrin and AP-2 to PI(4,5)P2-containing liposomes, whereas the mutant version was an inefficient competitor (Fig. 3, A and B). Tubulin–liposome association was not affected. Inhibition was dose dependent, requiring low micromolar concentrations of µ2 (Fig. 3, C and D). This suggests that binding of PI(4,5)P2 to µ2-adaptin may contribute to clathrin/AP-2 association with liposomes.



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Figure 3. Recruitment of clathrin/AP-2 to PI(4,5)P2-containing liposomes. (A and B) PI(4,5)P2-containing liposomes were incubated with cytosol, ATP, GTP{gamma}S, and 4 µM wild-type or mutant µ2 (157–435). Liposomes were reisolated, washed, and analyzed by SDS-PAGE and staining with Ponceau S (A) or immunoblotting (B). Bands were visualized with 125I-protein A and quantified by phosphoimage analysis. (C) Dose dependence of µ2-mediated inhibition of clathrin/AP-2 recruitment to liposomes. The experiment was done as described in A, using the indicated concentrations of wild-type or mutant µ2. (D) Quantification of clathrin recruitment onto liposomes in the presence of different concentrations of µ2. Data are plotted as mean (±SE) from several experiments. The amount of clathrin recruited to liposomes in the absence of added µ2 was taken as 100%.

 
Next we analyzed whether PI(4,5)P2 binding to µ2 might facilitate clathrin/AP-2 recruitment to native membranes. We first investigated the effect of neomycin, a drug that sequesters PI(4,5)P2, on adaptor recruitment to isolated presynaptic LP2 membranes (Takei et al., 1996). In agreement with earlier observations, we found that neomycin inhibited clathrin/AP-2 recruitment (unpublished data; West et al., 1997; Jost et al., 1998). Likewise, neomycin partially inhibited the association of µ2 with synaptic membranes (Fig. 4 A). Recruitment of µ2 to LP2 membranes could also be inhibited by adding phospholipase C{delta}1, an enzyme that specifically cleaves PI(4,5)P2 (Fig. 4 B). These data suggest that phosphoinositides may aid targeting of µ2 to synaptic membranes. When we compared the ability of wild-type or mutant µ2 to bind to LP2 membranes, we noticed that only the wild-type, not the mutant, protein became efficiently recruited to the membrane (Fig. 4 C). We then incubated synaptosomal LP2 membranes with cytosol and ATP plus GTP{gamma}S in the presence of wild-type or mutant µ2. We found that only wild-type µ2, not its mutated counterpart, could effectively compete clathrin/AP-2 recruitment (Fig. 4, D and E), whereas membrane binding of hsc70 was not affected. An intact PI(4,5)P2 binding site therefore is required for the ability of µ2-adaptin to compete clathrin/AP-2 recruitment to the plasma membrane.



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Figure 4. Recruitment of clathrin/AP-2 and µ2 to synaptic LP2 membranes. (A) Membrane association of µ2 is inhibited by neomycin. Carbonate-washed LP2 membranes (10 µg) were incubated with cytosol (0.4 mg/ml), ATP, GTP{gamma}S, and µ2 (157–435; 1.5 µg) in the presence or absence of 2 mM neomycin. LP2 membranes were reisolated, washed, and analyzed by Western blotting and staining with Ponceau S. Std, 50% of the µ2 added to the assay. (B) Membrane association of µ2 is inhibited by phospholipase C{delta}1. Recruitment of µ2 was assayed as described in A in the presence or absence of 5 µg purified phospholipase C{delta}1 or BSA. (C) Membrane recruitment of µ2 requires an intact PI(4,5)P2-binding site. Recruitment of wild-type or mutant µ2 was analyzed as in A, except that the samples were analyzed by staining with Ponceau S to detect bound µ2 and immunoblotting for synaptotagmin I as a membrane marker. (D) Clathrin/AP-2 recruitment to LP2 membranes can be competed by wild-type but not KKK–EEE mutant µ2. LP2 membranes (20 µg) were incubated with cytosol, ATP, GTP{gamma}S, and 2 or 4 µM of wild-type or mutant µ2 (157–435). Membranes were reisolated, washed, and analyzed by staining with Ponceau S (top) or immunoblotting (bottom) for clathrin heavy chain (HC), {alpha}-adaptin, hsc70, and synaptotagmin I. 1/4 cyt, 25% of the cytosol used in the experiment. (E) Quantification of clathrin recruitment as shown in D. The amount of clathrin recruited to LP2 in the absence of µ2 was taken as 100%. Data are plotted as mean (±SE) from three independent experiments.

 
Finally, we were interested to see whether mutant µ2 defective for PI(4,5)P2 binding and incorporated into AP-2 complexes would affect AP-2 localization and receptor-mediated endocytosis in living cells. To this aim, we transiently expressed epitope-tagged versions of full-length wild-type or mutant µ2 bearing an internal hemagglutinin (HA) epitope. In agreement with previous studies (Nesterov et al., 1999), we found that both µ2 variants were incorporated into AP-2 complexes, as judged by specific coimmunoprecipitation of {alpha}-adaptin with HA–µ2 (Fig. 5 A). Whereas a large fraction of the expressed wild-type HA–µ2 was associated with membranes, most of the mutant version localized to the soluble fraction (Fig. 5 B). At low expression levels, both µ2 variants displayed a punctate distribution that colocalized with endogenous {alpha}-adaptin. High-level expression of mutant, but not wild-type, HA–µ2 resulted in a decreased association of {alpha}-adaptin with coated pits (Fig. 5 C). These data suggest that phosphoinositide binding to µ2-adaptin contributes to localizing AP-2 to clathrin-coated pits in vivo.



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Figure 5. Expression of KKK–EEE mutant µ2 inhibits clathrin-mediated endocytosis in CHO cells. (A) CHO cells (10 x 106) transiently transfected with HA-tagged wild-type or mutant µ2 were lysed and subjected to immunoprecipitations with monoclonal antibodies against the HA tag. Samples were analyzed by SDS-PAGE and immunoblotting for {alpha}-adaptin and hsc70. Extract, 10% of the total extracted proteins used for the experiment. (B) Transfected CHO cells (as in A) were fractionated into membrane (M) and cytosol (C). Samples were analyzed by SDS-PAGE and immunoblotting for HA-tagged µ2 or hsc70. (C) HA-tagged wild-type or mutant µ2 were transiently expressed in CHO cells. 48 h after transfection, cells were methanol fixed and immunostained with antibodies against {alpha}-adaptin or HA. Bar, 10 µm. (D) HA-tagged wild-type or mutant µ2 was transiently expressed in CHO cells (see C) and analyzed for their ability to internalize Texas red–labeled transferrin (2.5 µg/ml; 10 min at 37°C) by immunofluorescence microscopy. Transfected cells are indicated by an arrow. The results are representative of three independent transfection experiments in which 85% of the cells expressing elevated levels of mutant µ2 displayed strongly reduced transferrin uptake. (E) HA-tagged wild-type or mutant µ2 were transiently expressed in HeLa cells and analyzed for the ability to internalize Texas red–labeled EGF (2 µg/ml; 3 min at 37°C) by immunofluorescence microscopy.

 
Cells expressing HA-tagged µ2 were then analyzed for their ability to internalize extracellularly added Texas red–labeled transferrin or EGF. Whereas cells expressing wild-type µ2 could endocytose transferrin normally (Fig. 5 D; Nesterov et al., 1999), cells expressing the PI(4,5)P2 binding–defective mutant displayed a reduced ability to accumulate transferrin within recycling endosomes (Fig. 5 D). This defect was similar to that seen for a mutant of µ2 in which W421 was changed to alanine (unpublished data), a mutation known to impair the interaction of µ2 with the transferrin receptor (Owen and Evans, 1998; Nesterov et al., 1999). Similarly, transfected cells expressing mutant HA–µ2 were also less capable of internalizing Texas red–labeled EGF (Fig. 5 E). We conclude that the PI(4,5)P2 binding site within µ2-adaptin facilitates clathrin-mediated endocytosis in living cells.

In the present study, we have identified and functionally characterized a PI(4,5)P2 binding site within the µ2 subunit of AP-2. Structurally, the phosphoinositide binding motif within µ2 resembles the basic fingers found in AP180 (Ford et al., 2001; Mao et al., 2001), with several conserved lysine residues forming a surface-exposed positively charged patch. In contrast to {alpha}-adaptin, which preferentially recognizes PIP3 over PIP2, µ2 displays a high degree of specificity for PI(4,5)P2. This suggests that AP-2 may interact with phosphoinositides via two independent sites within its {alpha} and µ2 subunits. One possibility is that phosphoinositide binding to both subunits synergistically triggers coated pit assembly. Alternatively, different stimuli, such as growth factor activation, could result in the formation of distinct species of PI lipids (Di Fiore and De Camilli, 2001; McPherson et al., 2001). These might then exert differential effects on AP-2 with regard to coated pit assembly or cargo recognition. Lastly, it is conceivable that PI(4,5)P2 and PI(3,4,5)P3 sequentially interact with µ2 and {alpha}-adaptin during progressive coated pit invagination (Kirchhausen, 2000; Slepnev and De Camilli, 2000) or upon cargo selection and transport. Support for the latter possibility comes from the observation that 3'-phosphoinositides, which are synthesized by a clathrin-activated kinase (Gaidarov et al., 2001), stimulate binding of tyrosine-based endocytosis signals to AP-2 (Rapoport et al., 1997). It is therefore conceivable that binding of 3'-phosphoinositides to AP-2 induces a conformational change that allows µ2 to interact more efficiently with cargo protein sorting signals at the membrane perhaps assisted by the concomitant association of µ2 with PI(4,5)P2.

We favor the possibility that the temporally or spatially ordered interaction of several endocytotic proteins with PI(4,5)P2 is required for clathrin-mediated endocytosis. This view is supported by the observation that the phosphoinositide binding sites within AP180/CALM (Ford et al., 2001; Mao et al., 2001), {alpha}-adaptin (Gaidarov and Keen, 1999), and µ2 (this study) apparently cannot substitute for one another. Although binding of phosphoinositides to {alpha}-adaptin may largely function in targeting AP-2 to the plasma membrane (Gaidarov and Keen, 1999), the association of µ2 with PI(4,5)P2 could in addition help to stabilize AP-2 at sites of endocytosis or to reorient the molecule with respect to the membrane.

In conclusion, the results reported here support a model according to which the binding of phosphoinositides to µ2-adaptin, as well as to other endocytotic proteins, links the formation of clathrin/AP-2–coated pits with the selection of cargo proteins.


   Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Antibodies and lipids
All lipids used were from Sigma-Aldrich or Calbiochem. Monoclonal antibodies against clathrin (TD.1) and synaptotagmin I (Cl41.1) were gifts from Pietro De Camilli (Yale University, New Haven, CT) and Reinhard Jahn (Max-Planck Institute). Anti–{alpha}-adaptin, {alpha}-tubulin, and anti-HA antibodies were from Affinity BioReagents, Inc. and Sigma-Aldrich, respectively.

Molecular biology procedures
Constructs encoding full-length µ2-adaptin bearing an internal HA epitope tag (YPYDVDYA) between aa 236 and 237 (Nesterov et al., 1999) were generated by PCR (plasmid DNA was a gift from J.S. Bonifacino, National Institutes of Health, Bethesda, MD), subcloned into pcDNA3, and verified by DNA sequencing. Hexahistidine-tagged wild-type or mutant versions of µ2-adaptin (aa 157–435) were made by PCR, subcloned into pET28a (Novagen Inc.), and verified by DNA sequencing. Standard techniques were used for preparation of plasmid and genomic DNA, restriction analysis, PCR, and cloning of DNA fragments.

Generation of liposomes
Unilamellar liposomes were made as previously described (Takei et al., 1998), by using a mixture of defined lipids (70% PC, 20% PE, plus 10% variable lipid [wt/wt]). Liposome recovery was followed by tagging with the lipophilic dye 1,6-diphenyl-1,3,5-hexatriene ({lambda}EX = 360 nm; {lambda}EM = 430 nm) on a fluorimeter after reisolation of the liposomes.

Protein recruitment to membranes and liposomes
For biochemical analysis of protein recruitment onto synaptic LP2 membranes, published procedures were used (Takei et al., 1996, 1998). Protein binding to liposomes (Fig. 1) was done as follows: 100 µg liposomes was incubated for 30 min at RT with 2 µg purified µ2 (aa 157–435) in cytosolic buffer. Liposomes were reisolated by sedimentation, washed extensively, and analyzed by SDS-PAGE and staining with Coomassie blue. For the experiments shown in Fig. 3, liposomes (0.4 mg/ml) were incubated with rat brain cytosol (3 mg/ml) in the presence of 2 mM ATP, 200 µM GTP{gamma}S, and purified µ2 (aa 157–435) for 10 min at 37°C, chilled on ice, and reisolated as described above.

Transfection experiments and endocytosis assay
CHO (Fig. 5, A–D) or HeLa (Fig. 5 E) cells were transfected with plasmids encoding internally HA-tagged µ2-adaptin (wild type or mutant) with Lipofectamine 2000 (GIBCO BRL). 48 h after transfection, cells were washed and incubated for 2–3 h in serum-free medium. Texas red–labeled transferrin (2.5 µg/ml) or EGF (2 µg/ml) were added and cells were allowed to internalize the probe for 10 or 3 min at 37°C, respectively. Cells were acid washed, fixed, and processed for indirect immunofluorescence microscopy.

For biochemical fractionation studies, cells were harvested 40 h after transfection by scraping into isotonic buffered sucrose (10 mM Hepes, 320 mM sucrose, pH 7.4, 1 mM PMSF). Cells were homogenized using an EMBL cell cracker and centrifuged at 1,500 g. The supernatant was separated into membrane and soluble fractions by centrifugation for 30 min at 12,000 g, and samples were precipitated with 10% trichloroacetic acid and analyzed by SDS-PAGE.

Miscellaneous
Rat brain cytosol and LP2 synaptic membrane preparations have been described previously (Takei et al., 1996; 1998). Hexahistidine-tagged fusion proteins were purified from CHAPS (2%)-lysed Escherichia coli detergent extracts according to the manufacturer's instructions. Bacterially expressed histidine-tagged phospholipase C{delta}1 was purified according to Bromann et al. (1997). Standard procedures were used for indirect immunofluorescence, SDS-PAGE, and immunoblotting.


   Footnotes
 
* Abbreviations used in this paper: HA, hemagglutinin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; P, phosphate; P2, bisphosphate; P3, trisphosphate; PI, phosphatidylinositol. Back


   Acknowledgments
 
We thank Drs. Pietro De Camilli and Reinhard Jahn for antibodies and reagents, Dr. Jon Lomasney (Northwestern University, Chicago, IL) for the expression construct encoding human phospholipase C{delta}1, Dr. Peter Schu (University of Göttingen) for help with molecular modeling, Dr. Michael Krauss (University of Göttingen) for preparing cytosol and LP2 membranes and Stefanie Thiel for technical assistance.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB523, project B8) and the Fonds der Chemischen Industrie to V. Haucke.

Submitted: 25 March 2002

Revised: 29 May 2002

Accepted: 29 May 2002


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