Article |
Address correspondence to Volker Haucke, Zentrum für Biochemie und Molekulare Zellbiologie, Dept. of Biochemistry II, University of Göttingen, Humboldtallee 23, Göttingen D-37073, Germany. Tel.: 49-551-39-98-54. Fax: 49-551-39-12-198. E-mail: vhaucke{at}gwdg.de
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Abstract |
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Key Words: ARF; clathrin coat assembly; PIPK I; PIP2 formation; endocytosis
* Abbreviations used in this paper: ARF, ADP-ribosylation factor; DTSP, 3,3'-Dithio-bis(propionic acid N-hydroxysuccinimide ester); GEF, guanine nucleotide exchange factor; LP2, lysed synaptosomal membrane fraction; PH, pleckstrin homology; PI(4,5)P2, phosphatidylinositol (4,5)-bisphosphate; PIPKI
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Introduction |
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Growing evidence implicates membrane lipids, in particular phosphoinositides, in the regulation of clathrin-mediated endocytosis (Jost et al., 1998; Cremona and De Camilli, 2001). Several endocytotic proteins such as the and µ2 subunits of heterotetrameric AP-2 complexes (Gaidarov and Keen, 1999; Collins et al., 2002; Rohde et al., 2002), the ENTH domains of AP180 and epsin (Ford et al., 2001; Itoh et al., 2001; Mao et al., 2001), and the pleckstrin homology (PH) domain of the large GTPase dynamin (Barylko et al., 1998) can interact directly with phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). Phosphatidylinositol 4-phosphate 5-kinase type I
(PIPKI
) and synaptojanin 1, the major brain PI(4)phosphate 5-kinase and PI(4,5)P2 polyphosphoinositide phosphatase, respectively, are concentrated at synapses, where they undergo activity-dependent dephosphorylation (McPherson et al., 1996; Ishihara et al., 1998; Wenk et al., 2001). Moreover, elevated PI(4,5)P2 levels induced by genetic inactivation of synaptojanin 1 result in the accumulation of clathrin-coated vesicles at the synapse (Cremona et al., 1999; Harris et al., 2000). These data suggest that clathrin-dependent retrieval of SV membranes may at least in part depend on phosphoinositide metabolism. Given the rapid turnover of phosphoinositides, it seems likely that PI(4,5)P2 synthesis is under tight regulatory control (Cremona and De Camilli, 2001), thereby linking it to the exo-endocytotic cycling of SV membranes.
The formation of clathrin-coated endocytotic intermediates has been reconstituted from lysed nerve terminal membranes incubated with brain cytosol and nucleotides (Takei et al., 1996). Detailed morphometric analysis by EM revealed that the presence of clathrin/AP-2coated buds on native synaptic membranes was potently stimulated by ATP and GTPS, a nonhydrolyzable analogue of GTP (Takei et al., 1996). This effect may be contributed to some extent by an inhibition of clathrin-coated vesicle fission induced by the GTP
S block of dynamin function (Takei et al., 1996). However, by analogy to other vesicular transport events, one might also speculate that GTP
S could act in part by locking a small GTPase in the GTP-bound conformation (Springer et al., 1999). More specifically, ADP-ribosylation factor (ARF) family members have been implicated in clathrin-coated budding events at the Golgi complex and at the cell periphery (Stamnes and Rothman, 1993; Traub et al., 1993; West et al., 1997). ARF has been shown to trigger assembly of vesicle coats onto membranes by directly interacting with coat proteins (Donaldson et al., 1992; Stamnes and Rothman, 1993; Traub et al., 1993; Zhao et al., 1997; Austin et al., 2002) or by stimulating phospholipase D (PLD) activity (Ktistakis et al., 1996; West et al., 1997; Arneson et al., 1999). Additionally, ARF family members have been found to recruit and activate PI kinases, which mediate PI(4,5)P2 synthesis (Godi et al., 1999; Honda et al., 1999). However, so far, the physiological role of ARF proteins in the recruitment of clathrin/AP-2 coats at the plasma membrane (Robinson and Kreis, 1992; West et al., 1997) and in SV recycling has remained unclear. Among the different ARF family members known to date, only ARF6 is localized to the plasma membrane, where it has been implicated in regulating actin dynamics and membrane turnover (D'Souza-Schorey et al., 1998; Randazzo et al., 2000; Brown et al., 2001). ARF6 has also been demonstrated to regulate clathrin-mediated endocytosis from the apical (Altschuler et al., 1999) and basolateral surface (Palacios et al., 2002) of polarized MDCK cells. Consistent with a putative role of ARF6 in SV recycling, mSec7, a brefeldin Ainsensitive ARF6-specific guanine nucleotide exchange factor (GEF) has been demonstrated to function at the synapse (Ashery et al., 1999). Here, we show that activated ARF6 facilitates clathrin/AP-2coated pit nucleation from synaptic membranes via the stimulation of PIP2 production mediated by PIPKI
activation.
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Results |
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Clathrin/AP-2 recruitment to synaptic membranes is not inhibited by brefeldin A
ARF family members have been shown to facilitate vesicle budding by stimulating recruitment of coat components to the membrane (Springer et al., 1999). Therefore, we tested the effect of brefeldin A (BFA) on clathrin/AP-2 recruitment in our assay. BFA inhibits nucleotide exchange factors acting on ARF family members except most of those specific for ARF6 (Randazzo et al., 2000). Consistent with earlier observations on AP-2 association with the plasma membrane of intact cells (Robinson and Kreis, 1992), no effect on the ATP- and GTPS-dependent recruitment of AP-2 and AP180 to LP2 membranes (Fig. 2) was seen. When isolated Golgi membranes were used instead of synaptic membranes, addition of BFA completely blocked clathrin binding to the Golgi complex (unpublished data; Robinson and Kreis, 1992). In agreement with these results, it has been found that BFA does not affect SV recycling, the main traffic pathway mediated by clathrin/AP-2 coats at the synapse (Mundigl et al., 1993).
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ARF6 is enriched in synaptic plasma membrane subfractions
A role of ARF6 in presynaptic clathrin-mediated vesicle recycling is plausible given the enrichment of the ARF6-specific nucleotide exchange factor mSec7 in nerve terminals (Ashery et al., 1999). pAbs that recognize ARF6 but not ARF1 (Fig. 3 A) detect high level expression of ARF6 in several tissues, including brain, as previously reported (Cavenagh et al., 1996; Yang et al., 1998; and unpublished data). The subcellular distribution of ARF6 as well as that of other well-characterized synaptic markers was analyzed in fractions of pig brain (obtained by the procedure of Maycox et al. [1992]). ARF6 was found in synaptosomes (P2) and within synaptosomes was primarily localized to the plasma membranecontaining fraction (LP1; Fig. 3 B). The LP2 fraction, mostly comprising SV and endosomal membranes, was highly enriched in the SV protein synaptophysin, but contained only relatively low levels of ARF6. ARF6 was also present (but not enriched) in highly purified clathrin-coated vesicles isolated from nerve terminals (Fig. 3 B) or whole brain (not depicted). Consistent with these biochemical data, activated HA-tagged ARF6(Q67L) accumulated at synapses in transfected cortical neurons grown in vitro, as demonstrated by its enrichment in synaptophysin-positive structures. (Fig. 3 C). We conclude that ARF6 is present at synapses, where it is found predominantly in plasma membrane fractions from which SV recycling occurs.
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The interaction between ARF6 and PIPKI appeared to be direct because GST-ARF6 bound to radiolabeled PIPKI
synthesized by coupled transcription/translation in vitro (unpublished data). To determine the isoform specificity for different ARF family members, hexahistidine-tagged myristoylated ARF6(Q67L) or ARF1(Q71L) were compared for their ability to bind to PIPKI
or the trans-Golgi clathrin adaptor AP-1, a protein complex known to bind to both ARF1 and ARF6 (Austin et al., 2002). Although both ARF1(Q71L) and ARF6(Q67L) displayed similar affinities for AP-1, as detected by Western blotting of the affinity-purified material for the AP-1 subunit
adaptin, only ARF6 was able to effectively pull-down PIPKI
(Fig. 5 B). By contrast, a control protein (arfaptin2) did not interact with either AP-1 or PIPKI
. Together, these results show that ARF6 specifically binds to PIPKI
in vitro in a GTP-dependent manner, but not to clathrin/AP-2 coat components.
To finally determine whether ARF6 associated with PIPKI during clathrin/AP-2 recruitment to synaptic membranes, we performed chemical cross-linking experiments. To this aim, LP2 membranes were first incubated with cytosol, ATP, ARF6(T27N)-His6 or ARF6(Q67L)-His6, and GDP or GTP, respectively. 3,3'-Dithio-bis(propionic acid N-hydroxysuccinimide ester) (DTSP), a cleavable amine-reactive cross-linking reagent was added, samples were solubilized under denaturing conditions, and ARF6-His6 was recovered by Ni-NTA affinity chromatography. ARF6(Q67L) but not its inactive GDP-bound counterpart (T27N) became efficiently cross-linked to PIPKI
(Fig. 5 C). No interaction was seen with clathrin, AP180, or any of the individual subunits of the AP-2 adaptor complex. Our combined data suggest that ARF6-GTP directly interacts with PIPKI
on synaptic membranes.
ARF6-GTP and PIPKI colocalize in transfected cells
The biochemical interaction of ARF6 with PIPKI was supported by morphological studies of cotransfected cells. Consistent with previous data (Brown et al., 2001), ARF6(Q67L)-EGFP expressed in Cos7 cells was found in peripheral plasma membrane invaginations, vacuolar structures and membrane ruffles. In these structures, it colocalized with cotransfected PIPKI
-p90 (Fig. 6 A). By contrast, little if any colocalization was seen between PIPKI
and the inactive GDP mutant of ARF6 (Fig. 6 B). As expected, PIPKI
-containing structures were highly enriched in PI(4,5)P2, as visualized by coexpression of PHPLC
-EGFP, a specific interactor for this phosphoinositide (Fig. 6 C).
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Although addition of activated ARF6 increased formation of PIP2 from total brain lipids, no effect on PIP generation was detectable (Fig. 7 C). Moreover, immunodepletion of PIPKI from cytosol almost completely abolished PIP2 formation (see also Wenk et al., 2001), and this defect could not be restored by addition of exogenous ARF6-GTP or mutants thereof (Fig. 7 C). This indicates that ARF6-mediated stimulation of PIP2 synthesis is indeed primarily due to activation of PIPKI
. In summary, these results demonstrate a direct interaction between activated ARF6 and PIPKI
in brain cytosol that results in a strong stimulation of PI(4)P 5 kinase activity and PIP2 formation.
ARF6-GTP stimulates PIP2 formation on synaptic membranes
Also, we investigated whether ARF6-mediated clathrin/AP-2 coat recruitment (Fig. 1 and Fig. 4) is accompanied by an increase of PIP2 levels in synaptic membranes. We stimulated coat recruitment to LP2 membranes with [32P]ATP and GTP in the absence or presence of the different ARF6 mutants. As shown in Fig. 7 D, 1 µM recombinant ARF6(Q67L) stimulated PIP2 synthesis up to 2.5-fold compared with the amount of PIP2 formed in the presence of cytosol alone. In contrast, ARF6(T27N) failed to affect PIP2 levels in LP2 membranes. Addition of wild-type ARF6-GTP resulted in an intermediate stimulation of PIP2 formation (Fig. 7 D). Collectively, our data suggest that ARF6-GTP stimulates PIP2 synthesis at the synapse by activating PIPK I
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Masking or degradation of PIP2 interferes with clathrin/AP-2coated pit assembly and receptor-mediated endocytosis
Finally, if the ARF6-mediated stimulation of PIPKI played a major role in mediating the effect of ATP/GTP
S on clathrin/AP-2 recruitment, one would expect that masking PIP2 with a PIP2-binding module or degradation of PIP2 by an inositol phosphatase would inhibit such recruitment to synaptic membranes or in living cells. We performed recruitment experiments in the presence of the recombinant PH domain of human PLC
1 (PHPLC
1), a specific PI(4,5)P2-binding protein. Addition of recombinant PHPLC
1 to synaptic membranes inhibited the ATP/GTP
S-induced binding of clathrin and AP-2 in a concentration-dependent manner, whereas addition of GST had no effect (Fig. 8 A). Likewise, overexpression of the prenylated HA-tagged inositol 5-phosphatase domain of synaptojanin 1 (HA-IPP1-CAAX), a PI(4,5)P2-degrading enzyme, in Cos7 cells resulted in the mislocalization of AP-2 to patch-like structures (Fig. 8 B). Similar (albeit less dramatic) effects were seen on peripheral clathrin-positive puncta. By contrast, a pool of clathrin remained associated with the trans-Golgi network in transfected cells (Fig. 8 C).
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Discussion |
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The strong stimulatory effect of ARF6 on PIPKI extends to this brain-enriched enzyme a property previously demonstrated for predominantly nonneuronal isoforms of PIPK (Honda et al., 1999; Skippen et al., 2002). It was also shown that PLD stimulation by ARF family proteins, including ARF6, may play a role in enhanced PI(4,5)P2 production by generating phosphatidic acid, a reported activator of PI(4)P 5-kinases (Brown et al., 1993; West et al., 1997; Arneson et al., 1999). This effect may synergize with the direct effect of ARF6 on PIPKI
in the generation of PI(4,5)P2 at the synapse. Although ARF6 appears to have a broad distribution in the nervous system, the weak immunocytochemical signal produced by available antibodies to endogenous ARF6 in brain tissue did not allow its reliable subcellular localization. However, transfected activated ARF6(Q67L) appears to be concentrated at synapses, and an ARF6-specific GEF, mSec7, regulates SV traffic (Ashery et al., 1999). Furthermore, PIPKI
is highly expressed in brain (Ishihara et al., 1998) and concentrated at synapses (Wenk et al., 2001). Thus, our findings, obtained primarily using cell-free systems, are likely to reflect a physiological process occurring in vivo.
The precise interplay between mSec7 and other brain ARF6-specific GEFs, and their role in the activation of ARF6 at the presynaptic plasma membrane remains to be investigated. It seems possible that ARF6 may synergize with the synaptically enriched focal adhesion protein talin in activating PIPKI at the synapse (Di Paolo et al., 2002; Ling et al., 2002). The finding that enhanced PI(4,5)P2 production may play a critical role in the recruitment of endocytotic clathrin coats is supported by a large body of data besides those presented here. Biochemical and structural studies have demonstrated an interaction of the clathrin adaptor AP-2, AP180, and dynamin with PI(4,5)P2 (Barylko et al., 1998; Gaidarov and Keen, 1999; Ford et al., 2001; Mao et al., 2001; Rohde et al., 2002). Genetic disruption of the polyphosphoinositide phosphatase synaptojanin (Cremona et al., 1999; Harris et al., 2000) and other manipulations that disrupt its function and therefore lead to an accumulation of PI(4,5)P2 in neurons, enhance the presence of clathrin coats on synaptic membranes both in vivo (Cremona et al., 1999; Gad et al., 2000) and in cell-free systems (Cremona et al., 1999; Wenk et al., 2001; Kim et al., 2002). Our data are also consistent with (and further support) recent reports suggesting that ARF6 (Altschuler et al., 1999; Palacios et al., 2002) and PIPK (Barbieri et al., 2001) regulate clathrin-mediated endocytosis from the plasma membrane.
As cell fractionation data indicate, neither ARF6 nor PIPKI are enriched in crude SV (Wenk et al., 2001, and this paper). Instead, ARF6 is localized in plasma membrane fractions (Cavenagh et al., 1996; Yang et al., 1998), consistent with a role in priming the membrane for coating. PIPKI
is found primarily in the cytosol (Wenk et al., 2001), but as shown by EM (Wenk et al., 2001) and biochemically (this paper), is recruited by GTP
S, and therefore most likely by ARF6-GTP, to the membranes at which clathrin-coated pits nucleate. A GTP
S-stimulated recruitment of clathrin/AP-2 coats to endosomes was demonstrated by Robinson and coworkers in nonneuronal cells (West et al., 1997). This "mislocalization" of endocytotic clathrin coats was shown to be dependent on enhanced PI(4,5)P2 production on endosomes, and has been attributed to PLD activation by an ARF GTPase. Excess PI(4,5)P2 on endosomes can also be generated by overexpression of an active form of ARF6 (Brown et al., 2001). As shown by Honda et al. (1999) and by our present results, a major mechanism through which active ARF6 may stimulate PI(4,5)P2 production is via the recruitment and activation of a PI(4)P 5-kinase activity. We speculate that in neuronal cells, PI(4,5)P2 is segregated at the plasma membrane by the coordinate action of PI(4)P 5-kinases, which are primarily localized at the cell surface, and of the phosphoinositide phosphatase synaptojanin, which cleaves PI(4,5)P2 on endocytotic membranes (Kim et al., 2002; Stefan et al., 2002). Excess active ARF6either because of GTP
S addition or because of mutation that inactivates its GTPase activitymay alter this balance and lead to an abnormal accumulation of PI(4,5)P2 on endosomes.
It seems particularly interesting that PIPKI is stimulated by ARF6, a protein previously implicated in regulating actin dynamics and membrane turnover (D'Souza-Schorey et al., 1998; Randazzo et al., 2000; Schafer et al., 2000). Invaginations of the plasma membrane that resemble those found at synapses after prolonged stimulation (Heuser and Reese, 1973; Takei et al., 1996; Gad et al., 2000) have been detected in nonneuronal cells on expression of constitutively active ARF6 (D'Souza-Schorey et al., 1998) or of an ARF6-specific exchange factor (Franco et al., 1999). ARF6 may recruit and activate PIPKI
at the synapse, thus increasing the local PIP2 concentration. This, in turn, would facilitate the formation of endocytotic structures like clathrin-coated pits and deeper membrane invaginations from which clathrin-coated vesicle budding can also occur. A number of observations suggest that clathrin-mediated endocytosis is highly interconnected to dynamics of the actin cytoskeleton (Lamaze et al., 1997). Accordingly, many accessory proteins of the clathrin pathway directly couple endocytotic coat formation to actin rearrangements (Qualmann et al., 2000; Schafer et al., 2000; Hussain et al., 2001; Lee and De Camilli, 2002; Orth et al., 2002). Endocytotic "hot spots" at synapses coincide with actin-rich zones (Kelly, 1999; Gad et al., 2000), and the accumulation of clathrin-coated vesicles induced in nerve terminals by the disruption of synaptojanin function correlates with the presence of a meshwork of actin around these vesicles (Cremona et al., 1999; Gad et al., 2000; Kim et al., 2002; Shupliakov et al., 2002). ARF6 may regulate actin dynamics through multiple cooperative mechanisms. Via the increase in PI(4,5)P2 production mediated by stimulation of PLD (Brown et al., 1993) and PIPKs (Honda et al., 1999; this paper), it enhances the recruitment and activation of actin regulatory proteins including N-WASP and small GTPases of the Rho family (Takenawa and Itoh, 2001). It can also directly regulate Rac via its binding to arfaptin 2/Por1 (Shin and Exton, 2001). Possibly through its effects on Rac and actin, ARF6 can stimulate formation of macropinosomes (Brown et al., 2001).
Thus, ARF6 appears to have a major regulatory role in endocytosis because it can control both clathrin-dependent and -independent endocytotic pathways. It will be critical to further elucidate the localization and regulation of ARF6-GEFs and the effect of synaptic activity on such regulation.
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Materials and methods |
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Recombinant proteins
Wild-type or mutant ARF6 or ARF1 GST fusion proteins were purified from bacteria according to the manufacturer's instructions (Amersham Biosciences). For production of myristoylated mutants of ARF6 or ARF1 BL21 were cotransformed with pET-21b-ARF6 and pBB131 containing yeast myristoyl transferase (Duronio et al., 1990). Proteins from lysed cell extracts were adsorbed to His-bind resin (Boehringer) and eluted with 250 mM imidazole (pH 7.4) in 50% glycerol.
Recruitment experiments
Rat brain cytosol and LP2 membranes were prepared as described previously (Haucke and De Camilli, 1999). Membranes (1 mg/ml protein) were washed at 4°C in 0.1 M sodium carbonate, pH 9.5, for 15 min, recovered by centrifugation (89,000 g, for 15 min at 4°C), and resuspended in cytosolic buffer. Recruitment experiments were performed in a total volume of 400 µl cytosolic buffer containing 60 µg/ml LP2 membranes and 1.2 mg/ml rat brain cytosol. Nucleotides and recombinant myristoylated ARF6 proteins were included where indicated. The samples were incubated for 15 min at 37°C, loaded on a cushion of 0.5 M sucrose in cytosolic buffer, and centrifuged at 150,000 g (for 1 h at 4°C). Pellets were washed with 500 µl cytosolic buffer and centrifuged at 175,000 g for 15 min. Finally, the pellet was resuspended in sample buffer and proteins recruited to LP2 membranes were analyzed by quantitative Western blots using 125I-protein A for detection and phosphoimage analysis (Image Reader 3000; Fuji).
Pull-down experiments
For GST pull-downs, 0.5 mg/ml GST or GST-ARF6 bound to resin was supplemented with 1 mM GDP (GST-ARF6(T27N)) or 1 mM GTP (GST-ARF6(Q67L)) and incubated with 3 mg/ml mg rat brain extract (in 20 mM Hepes/KOH, pH 7.4, 320 mM sucrose, 2 mM MgCl2, 1% Triton X-100, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin and 100 µg/ml Pefabloc®) for 4 h at 4°C. The beads were extensively washed and finally extracted twice with 100 µl Laemmli sample buffer. Alternatively, 0.15 mg/ml His6-tagged Arfaptin 2 or myristoylated ARF6(Q67L)-His6 or ARF1(Q71L)-His6 bound to Ni2+-NTA beads were rotated end over end with 2.5 mg/ml rat brain extract supplemented with 15% glycerol for 1 h at 4°C. The resin was washed and extracted twice with 120 µl sample buffer.
Cross-linking experiments
1-mg LP2 membranes were incubated with 5 mg rat brain cytosol in the absence or presence of 200 µg myristoylated ARF6(T27N)- or ARF6(Q67L)-His6, 2 mM ATP, and 200 µM GDP or GTP in a final volume of 1.5 ml for 15 min at 37°C. Proteins were cross-linked with 0.5 mM DTSP for 1 h at 4°C. Samples were solubilized with 1% Triton X-100, centrifuged at 20,000 g (for 15 min at 4°C), and denatured in 6 M guanidinium hydrochloride for 1 h at 20°C. His6-tagged ARF6 and cross-linked partners were recovered by extracting twice with Ni2+-NTA beads in the presence of 10 mM imidazole. The collected beads were washed thoroughly and extracted with sample buffer.
Phosphoinositide kinase assays
For phosphoinositide kinase assays, liposomes (Fig. 7, AC) or LP2 membranes (Fig. 7 D) were used. Large unilamellar liposomes were prepared from cholesterol/phosphatidylcholine/phosphatidylinositol 4-phosphate (20:74:6, wt/wt/wt; Fig. 7 A) or total brain lipids (Fig. 7, B and C). 1.2 mg/ml brain cytosol was pre-incubated for 2 min at 37°C with 200 µM GTP, 100 µM neomycin, 2 mM ATP, and [32P]ATP (0.2 µCi/sample) in cytosolic buffer (25 mM Hepes/KOH, pH 7.2, 25 mM KCl, 2.5 mM magnesium acetate, and 150 mM potassium glutamate). 1 µM recombinant myristoylated ARF6 protein was included where indicated. Phosphorylation reactions (15 min at 37°C in a total volume of 500 µl) were started by adding liposomes. Lipid products were extracted and analyzed as described by Kinuta et al. (2002). For some experiments, liposomes were substituted with LP2 membranes (480 µg protein, 385 µg total phospholipids) as substrate. The activity of recombinant PIPKI
-p90 or brain PIPKI
was determined in the presence or absence of ARF6 proteins essentially as previously described using total brain lipids as a substrate (Wenk et al., 2001).
Transfection and internalization assay
Cos7 cells were transiently transfected with plasmids encoding HA-tagged PIPKI-p90, ARF6-EGFP, or HA-IPP1-CAAX using LipofectAMINETM 2000, and were assayed 24 h after transfection. For uptake assays, cells were starved in serum-free medium for 2 h before addition of 10 µg/ml Alexa® 488-Tf or 1 µg/ml Texas red-EGF for 10 min at 37°C. Surface-bound ligand was removed by a brief acid wash (pH 5.3) followed by fixation and preparation for immunofluorescence microscopy.
Nucleofection of isolated cortical neurons
Cortical neurons were isolated from newborn rats and transfected using a nucleofection system (Amaxa) according to the manufacturer's instructions.
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Acknowledgments |
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This study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB523, project B8) and the Fonds der Chemischen Industrie (to V. Haucke), the National Institutes of Health NS 36251 and DK54913 (to P. De Camilli), and by a grant-in-aid from the Ministry of Education, Sciences, Sports, and Culture of Japan, by the NOVARTIS Foundation for the Promotion of Science (Japan), and by the Japan Brain Foundation (to K. Takei and M. Kinuta).
Submitted: 3 January 2003
Revised: 19 May 2003
Accepted: 20 May 2003
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