1 Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
2 Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Author for correspondence (e-mail: histol3{at}post.tau.ac.il)
Accepted 20 January 2005
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Summary |
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Key words: Endocytic recycling compartment (ERC), Endocytosis, Synaptotagmin, Protein kinase C, RBL-2H3 mast cells
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Introduction |
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Materials and Methods |
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Reagent
Texas red-conjugated human transferrin (Tfn) was obtained from Molecular Probes (Eugene, OR). Brefeldin A and glutathione-Sepharose were from Sigma-Aldrich and 12-O-tetradecanoylphorbol-13-acetate (TPA) from Calbiochem.
Cell culture
RBL cells and CHO cells were maintained in adherent cultures in DMEM supplemented with 10% FCS (for RBL cells) and 10% FCS and proline (for CHO cells) in a humidified atmosphere of 5% CO2 at 37°C. RBL cells stably transfected with T7-Syt IX (RBL-Syt IX+) or Flag-Syt IX-GFP cDNA were described previously (Haberman et al., 2003).
DNA constructs
T7-Syt IX, Flag-Syt IX-GFP, GST-Syt IX-C2A and GST-Syt IX-C2B cDNA were described previously (Haberman et al., 2003).
Cell transfection
For stable transfection CHO cells (8 x106) were transfected with 20 µg of either recombinant vector (pcDNA3-Syt IX, or pShooter-FLAG-Syt IX-GFP) by electroporation [250 V (0.25), 960 µF]. Cells were immediately replated in tissue culture dishes containing growth medium (supplemented Dulbecco's modified Eagle's medium; DMEM), and stable clones were obtained by selection in the presence of G418 (1 mg/ml), added 24 hours after transfection.
For transient transfection, RBL cells (6 x107) were electroporated [350 V (0.35), 1500 µF] in the presence of 40 µg of the desired plasmids and immediately replated in tissue culture dishes containing supplemented DMEM for the desired time periods.
Affinity chromatography on GST fusion proteins
GST, GST-Syt IX-C2A or GST-Syt IX-C2B (20 µg) were incubated for 4 hours at 4°C with RBL cell lysates (500 µg) prepared in buffer A [50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1% Triton X-100, 1 mM phenylmethylsulphonylfluoride (PMSF) and a cocktail of protease inhibitors (Boehringer Mannheim, Germany)]. At the end of the incubation period, beads were sedimented by centrifugation at 5,000 g for 4 minutes at 4°C, washed four times with buffer A and finally suspended in 1 x Laemmli sample buffer, boiled for 5 minutes and subjected to SDS-PAGE and immunoblotting.
Immunofluorescence microscopy
RBL cells (2 x105 cells/ml) were grown on 12-mm round glass coverslips. For immunofluorescence processing cells were washed twice with phosphate-buffered saline (PBS) and fixed for 15 minutes at room temperature in 3% paraformaldehyde/PBS. Cells were subsequently washed three times with PBSCM (PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2) and permeabilized on ice for 5 minutes with 100 µg/ml digitonin. After two washes with PBSCM, cells were permeabilized for an additional 15 minutes at room temperature with 0.1% saponin in PBSCM. Cells were subsequently incubated for 1 hour at room temperature with the primary antibodies diluted in PBSCM/5% FCS/2% BSA, washed 3 times in PBSCM/0.1% saponin and incubated for 30 minutes in the dark with the appropriate secondary antibody (Cy3- or FITC-conjugated donkey anti-rabbit or anti-mouse IgG, at 1/200 dilution in PBSCM/5% FCS/2% BSA). Coverslips were subsequently washed in PBSCM/0.1% saponin and mounted with Gel Mount mounting medium (Biomedica Corp. Foster city, CA). Samples were analyzed using a Zeiss laser confocal microscope (Oberkochen, Germany).
Subcellular fractionation of RBL cells
Cells were fractionated as previously described (Baram et al., 1999). Briefly, RBL cells (7 x107) were washed with PBS and suspended in homogenization buffer [0.25 M sucrose, 1 mM MgCl2, 800 U/ml DNase I (Sigma-Aldrich), 10 mM Hepes, pH 7.4, 1 mM PMSF, and a cocktail of protease inhibitors (Boehringer Mannheim, Germany)]. Cells were subsequently disrupted by three cycles of freezing and thawing followed by 20 passages through a 21-gauge needle and 10 passages through a 25-gauge needle. Unbroken cells and nuclei were removed by centrifugation for 10 minutes at 500 g and the supernatants subjected to sequential filtering through 5 µm and 2 µm filters (Poretics Co.). The final filtrate was then loaded onto a continuous, 0.45-2.0 M sucrose gradient (10 ml), which was layered over a 0.3 ml cushion of 70% (wt/wt) sucrose and centrifuged for 18 hours at 100,000 g.
Cell lysates
RBL cells (1 x107) were lysed in lysis buffer comprising 50 mM Hepes pH 7.4, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.1% SDS, 50 mM NaF, 10 mM NaPPi, 2 mM NaVO4, 1 mM PMSF and a cocktail of protease inhibitors (Boehringer Mannheim, Germany). Following 10 minutes incubation on ice, lysates were cleared by centrifugation at 9,000 g for 15 minutes at 4°C. The cleared supernatants were mixed with 5 x Laemmli sample buffer, boiled for 5 minutes, and subjected to SDS-PAGE and immunoblotting.
Data presentation
Data represent one of at least three separate experiments.
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Results |
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Syt IX binds the clathrin adaptor complex AP-2
The finding that Syt IX passes through the plasma membrane on its route to the ERC implies that Syt IX internalizes from the plasma membrane in order to reach its final internal destination. Syt I, the closely related homologue of Syt IX (Fukuda and Mikoshiba, 2000) was previously shown to bind the clathrin adaptor complex AP-2 (Zhang et al., 1994
). Therefore, we investigated whether Syt IX could also bind AP-2. To this end we evaluated the ability of GST fusion proteins consisting of the C2A or C2B domain of Syt IX to pull down
-adaptin from RBL cell lysates. Indeed, GST-C2B, but not GST-C2A or control GST, effectively bound AP-2 (Fig. 3). Inclusion of Ca2+ allowed modest binding by the C2A domain, but reduced binding to the C2B domain (Fig. 3). Therefore, AP-2 binding by Syt IX does not appear to require Ca2+. This result is consistent with previous findings demonstrating that AP-2 binding by Syt I is Ca2+ independent (Chapman et al., 1998
).
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Antibody induced-internalization of Syt IX
The finding that a fraction of overexpressed Syt IX remains at the plasma membrane suggests that internalization from the plasma membrane might serve as a rate-limiting factor in the biosynthesis of Syt IX. Therefore, we investigated whether exogenous antibodies, directed against the N-terminal tag, might enhance endocytosis and facilitate targeting of membrane bound Flag-Syt IX-GFP to the ERC. Indeed, incubation of Flag-Syt IX-GFP-expressing cells with anti-Flag antibodies for increasing periods of time resulted in the internalization of both Syt IX and the exogenously added antibodies. Following 30 minutes of internalization, both protein and antibodies were detected at scattered intracellular vesicles (Fig. 4A). However, neither Flag-Syt IX-GFP, nor the internalizing antibodies have reached a perinuclear localization reminiscent of the ERC (Fig. 4A). Both Syt IX and the anti-Flag antibodies were found to remain colocalized at intracellular vesicles for up to 5 hours (Fig. 4A). To examine whether these vesicles were endosomes, cells were allowed to internalize Tfn for 10 minutes to label the early endosomes. However, only a partial colocalization was detected between Flag-Syt IX-GFP and internalized Tfn (Fig. 4B). Therefore, while Flag-Syt IX-GFP passes through early endosomes during the course of antibody-induced internalization, both Syt IX and the internalizing antibodies are subsequently delivered to vesicles that are distinct from endosomes, but whose nature is presently unknown. Therefore these findings have strongly suggested that additional signals are required to direct internalized Syt IX to the ERC.
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TPA induced-internalization of Syt IX
Multiple lines of evidence implicate protein kinase C (PKC) in down-regulating a variety of cell surface receptors as well as membrane transporters. Moreover, this process is primarily achieved by enforcing redistribution of the receptors or transporters from the plasma membrane to endosomal compartments (Peng et al., 2002; Becker and Hannun, 2003
; Loder et al., 2003; Le et al., 2002
). Therefore, we examined whether the phorbol ester TPA, which directly activates PKC, could affect the steady state localization of Flag-Syt IX-GFP. TPA treatment for 30 minutes, which is sufficient to activate PKC causing its translocation to the plasma membrane (Peng et al., 2002
), resulted in the complete relocation of Flag-Syt IX-GFP to a perinuclear intracellular localization (Fig. 5A). Moreover, a complete colocalization between Flag-Syt IX-GFP and internalized Tfn could be observed, therefore confirming that TPA treatment results in the internalization of Syt IX in early endosomes and subsequent targeting to its correct intracellular destination at the ERC (Fig. 5B). Furthermore, inclusion of 0.45 M sucrose in the medium, a treatment that was previously shown to block clathrin-coated pit formation (Heuser and Anderson, 1989
), markedly reduced Syt IX redistribution (Fig. 5C). Quantitative analyses of the results obtained in these experiments revealed that the steady state distribution of Flag-Syt IX-GFP in untreated cells included exclusive plasma membrane localization in approximately 20% of the cells and both plasma membrane and ERC in the remaining cells (Fig. 5D). In contrast, in TPA-treated cells, Flag-Syt IX-GFP was localized to the membrane in only 1% of the cells, it was distributed between the plasma membrane and the ERC in 18% and in the remaining 80%, Flag-Syt IX-GFP was solely at the ERC (Fig. 5D). Inclusion of hypertonic sucrose resulted in exclusive membrane localization of Flag-Syt IX-GFP in 55% of cells and distribution between the plasma membrane and the ERC in the remaining 45% of cells (Fig. 5C,D). In fact, in the presence of hypertonic sucrose, the steady state amount of intracellular Flag-Syt IX-GFP was also significantly diminished whereby an intracellular localization of Flag-Syt IX-GFP was detected only in 50% of cells, while in the remaining cells, Flag-Syt IX-GFP was solely at the plasma membrane (Fig. 5C,D). These results therefore support the notion that clathrin and presumably AP-2 mediate Syt IX internalization.
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To confirm that the effect of TPA is mediated by PKC, we also exposed the Flag-Syt IX-GFP-expressing RBL cells to TPA in the presence of two inhibitors of PKC: GF109203X, an inhibitor that specifically blocks PKC, ß1, ß2,
,
and
(Way et al., 2000
) and Go 6976, an inhibitor that selectively blocks the classical Ca2+-dependent PKC isozymes: PKC
and PKCß (Martiny-Baron et al., 1993
). These experiments have demonstrated that both inhibitors (GF109203X and Go 6976) prevent the delivery of Flag-Syt IX-GFP from the plasma membrane to the ERC (Fig. 5E). Specifically, in the presence of either PKC inhibitor, Flag-Syt IX-GFP was distributed between the plasma membrane and the ERC in
50% of cells, while in the remaining 50% it was only at the plasma membrane (Fig. 5D). Therefore phosphorylation by a Ca2+-dependent PKC isozyme(s) is required for the correct delivery of Flag-Syt IX-GFP from its plasma membrane localization to the ERC.
Because antibody-induced internalization of Flag-Syt IX-GFP failed to direct Syt IX to the ERC, we also examined the fate of Flag-Syt IX-GFP in cells exposed to the combination of both anti-Flag antibodies and TPA. Indeed, under these conditions, both Flag-Syt IX-GFP and the endocytosed anti-Flag antibodies were targeted to the ERC (Fig. 5F). These results therefore corroborated the notion that phosphorylation by cPKC(s) provides a dominant sorting signal for the delivery of Syt IX to the ERC.
The experiments described so far, have clearly indicated that PKC-mediated phosphorylation could enable membrane bound Flag-Syt IX-GFP to reach the ERC. To confirm that PKC-mediated phosphorylation was indeed a crucial step in the biosynthetic route of ERC-localized Syt IX, we investigated whether TPA could also affect the small fraction of T7-Syt IX, which localizes to the plasma membrane in RBL-Syt IX+ cells. Indeed, following short treatment with TPA, T7-Syt IX is entirely localized to the ERC suggesting that TPA promoted the internalization of plasma membrane-localized Syt IX (Fig. 6A). To substantiate these results, we have also monitored the biosynthetic route of both T7-Syt IX and Flag-Syt IX-GFP in the presence of the PKC inhibitors Go 6976 or GF109203X. Basically, RBL cells were transfected with T7-Syt IX or Flag-Syt IX-GFP cDNA and 3 hours post-transfection cells were either left untreated or exposed to PKC inhibitors. The localization of Syt IX was then further monitored. As shown in Fig. 6, unlike T7-Syt IX, which reached the ERC in the untreated cells, Syt IX remained distributed between the plasma membrane and peripheral vesicles in cells treated with either Go 6976 (Fig. 6B) or GF109203X (data not shown). Similarly, the partial ERC localization of Flag-Syt IX-GFP disappeared in cells treated with either inhibitor (Fig. 6B and data not shown). These results therefore confirmed that PKC participates in controlling the biosynthetic trafficking of Syt IX.
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Delivery of endosomal cargo to the ERC often rescues cargo from being delivered to late endosomes and lysosomes for degradation. Indeed, we have previously shown that TPA-induced translocation of PKC to the ERC reduces the amount of down-regulated enzyme (Peng et al., 2002). However, similar amounts of Flag-Syt IX-GFP were detected in untreated cells and in cells subjected to 5 hours treatment with TPA (data not shown). This observation is consistent with the fact that in the absence of TPA, Syt IX is either directed to the ERC or alternatively remains at the plasma membrane (Fig. 5E).
Protein phosphorylation is frequently associated with a slower mobility on SDS gels. As noted above, both T7-Syt IX and Flag-Syt IX-GFP appear as a doublet on SDS-PAGE (Fig. 1B) (Haberman et al., 2003). However, while the majority (85%) of Syt IX in lysates derived from T7-Syt IX-expressing cells, corresponds to the slower migrating (50 kDa) protein, The smaller 70 kDa form of Flag-Syt IX-GFP is the major protein (Fig. 1B, Fig. 7A). Moreover, consistent with this notion, TPA increases the amount of the 80 kDa protein with a concomitant decrease in the amount of the 70 kDa form (Fig. 7A). Therefore, we next examined the intracellular distribution of the 50/40 kDa T7-Syt IX and 80/70 kDa forms of Flag-Syt IX-GFP by fractionation on continuous sucrose gradients. Consistent with our previous results (Haberman et al., 2003
), the majority of T7-Syt IX, which migrates as a protein of 50 kDa, cofractionated, at 1.25-1.45 M sucrose, with fractions 22 to 26, which also contain the ERC (Haberman et al., 2003
). The smaller fraction of T7-Syt IX, which migrates as a 40 kDa protein, could be detected in fractions 11 to 26, which contain the plasma membrane and the ERC (Fig. 7Ba). Flag-Syt IX-GFP actually appeared as three proteins of 70, 72 and 80 kDa, out of which the majority of the 70/72 kDa proteins cofractionated with the plasma membrane and to a lesser extent with the ERC, while the 80 kDa form cofractionated exclusively with the ERC (Fig. 7B,b). These results therefore confirmed that the slower migrating form of Syt IX resides exclusively with the ERC.
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Intracellular localization of Syt IX in CHO cells
We have also investigated the intracellular localization of Syt IX in non-neural/non-secretory cells, such as the CHO cells. We have chosen these cells because like the RBL cells, CHO cells possess a clearly defined ERC, which is localized in the proximity of the nucleus (Lin et al., 2002). It was also of particular interest to examine the localization of Syt IX in CHO cells, because Syt I, a highly related homologue of Syt IX (Fukuda and Mikoshiba, 2000
; Fukuda et al., 2002
), is targeted to the plasma membrane when transfected into these cells (Feany et al., 1993
). Strikingly, the pattern of distribution of Syt IX in CHO cells was identical to that observed in RBL cells. Syt IX acquired complete perinuclear localization, while Flag-Syt IX-GFP was distributed between the plasma membrane and the perinuclear region (Fig. 8). Moreover, TPA treatment relocated the plasma membrane-bound Flag-Syt IX-GFP to a perinuclear localization (Fig. 8) therefore implicating PKC in directing Syt IX to the ERC in CHO cells as well.
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Discussion |
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Exposure of Flag-Syt IX-GFP-expressing cells to exogenous anti-Flag antibodies facilitated Syt IX internalization. However, neither Syt IX nor the internalizing antibodies reached the correct ERC destination unless TPA was provided as well. These results therefore exclude the possibility of the exogenous antibodies being taken up by Syt IX during its trafficking route to the ERC, as was shown, for example, for recycling proteins such as TGN38 (Mallet and Maxfield, 1999). Rather, these results are consistent with the idea that internalization of plasma membrane-localized Syt IX is promoted upon exposure to the exogenous antibodies. Thus, presumably by inducing dimerization of Syt IX, the exogenously added antibodies accelerate Syt IX internalization, although this by itself is insufficient for correct sorting. Indeed, recent findings have implicated multimerization in playing a crucial role in Syt internalization (Grass et al., 2004
).
A series of findings indicate that phosphorylation by a classical PKC plays a key role in targeting Syt IX to its correct destination. First, inhibitors of cPKCs prevent targeting of Syt IX to the ERC and the protein is distributed between the plasma membrane and endosomes. Second, TPA treatment enables overexpressed, plasma membrane-retained Syt IX to reach the ERC. Finally, Syt IX migrates as a doublet on SDS-PAGE. However, while the major protein in T7-Syt IX-transfected cells, in which Syt IX is mainly localized to the ERC, is the slower migrating protein, the major protein in Flag-Syt IX-GFP transfected cells, in which Syt IX is mostly at the plasma membrane, is the faster migrating one. Therefore, although not proven directly here, these results are consistent with the notion that phosphorylation of Syt IX by PKC facilitates its targeting to the ERC.
Phosphorylation by PKC apparently contributes to both the internalization efficiency from the plasma membrane and the subsequent targeting to the ERC. This conclusion is based on the finding that TPA could promote internalization of overexpressed Flag-Syt IX-GFP, suggesting that sustained activation of PKC by TPA could overcome the rate-limiting step in the internalization of Syt IX.
We believe that the classical PKCs, PKC and PKCß, play a role in Syt IX targeting because Go 6976, which selectively blocks the classical Ca2+-dependent PKC isozymes, could counteract the TPA-induced delivery of Syt IX to the ERC. That these PKCs are indeed physiologically relevant is illustrated by the finding that inclusion of Go 6976 during the biosynthetic trafficking of Syt IX, prevents Syt IX from reaching the ERC. Consistent with this notion, we and others have previously shown that PKC
(Peng et al., 2002
) and PKCßII (Becker and Hannun, 2003
) move to the ERC following their activation. Furthermore, this translocation was shown to encompass functional consequences whereby activated PKC translocates to the ERC while sequestering cargo and components of the recycling endosomes (Becker and Hannun, 2003
). Specifically, PKC was shown to regulate the dynamics of endocytosis and trafficking of Tfn through its sequestration to the ERC (Becker and Hannun, 2003
). Our results are compatible with this hypothesis and implicate Syt IX as one of the cargo proteins that are internalized and delivered to the ERC and that are regulated by PKC (see model in Fig. 9). Therefore, PKC-regulated delivery to the ERC may represent a general mechanism for tuning the availability of plasma membrane proteins such as transporters, channels and proteins involved in the control of trafficking (Becker and Hannun, 2003
). The precise mechanism by which PKC regulates plasma membrane to ERC trafficking is presently unknown, however, an active kinase and phospholipase D were shown to be required (Becker and Hannun, 2004
). Finally, the finding that Syt IX also localizes to the ERC in CHO cells strongly suggests that the ERC represents the site of Syt IX localization in non-neural cells, irrespective of whether it is secretory or not. PKC-mediated targeting of Syt IX therefore seems to reflect a universal mode of regulation of non-neural Syt IX trafficking.
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Acknowledgments |
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Footnotes |
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References |
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