Laboratory of Cell Biology, NHLBI, National Institutes of Heath, Bethesda, MD 20892-0301, USA
* Author for correspondence (e-mail: greenel{at}helix.nih.gov)
Accepted 2 February 2005
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Summary |
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Key words: Permeabilized cells, Clathrin, Clathrin adaptors, Exchange
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Introduction |
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The observation that clathrin-exchange is a fundamental property of clathrin-coated pits both at the plasma membrane and the TGN, suggests that this exchange might be related to the structural changes that occur as clathrin-coated pits invaginate. Before they invaginate, clathrin-coated pits are planar or slightly curved structures, in which the polymerized clathrin triskelions form a hexagonal array (Crowther and Pearse, 1981). By contrast, the invaginated pits appear to be considerably more curved (Pearse et al., 2000
). Invagination may occur partially through the addition of clathrin to the edges of a growing pit, but there also appears to be an increase in curvature that would require transformation of the polymerized clathrin arrays from hexagons to pentagons (Guichet et al., 2002
). This, in turn, would require clathrin-exchange to take place as invagination occurs. Alternatively, Ehrlich et al. have suggested a mechanism, whereby clathrin coats might change their curvature without any exchange taking place (Ehrlich et al., 2004
).
We have previously observed that clathrin exchange requires ATP and that, in Caenorhabditis elegans, clathrin exchange was prevented when RNA interference was used to block the formation of auxilin (Greener et al., 2001). On this basis we proposed that, rather than clathrin passively dissociating from pits, Hsc70, auxilin and ATP are required to actively unravel individual clathrin molecules from the intricate clathrin lattice present in the pits (Wu et al., 2001
). However, previous studies found that, whereas Hsc70 was able to uncoat CVs in the presence of ATP, it was not able to uncoat clathrin-coated pits that were present on plasma membrane fragments prepared by brief sonication of tissue-culture cells (Heuser and Steer, 1989
). At the time this was done, it was not yet known that auxilin in addition to Hsc70 is required for the uncoating of CVs. In this present study, we therefore investigate, whether Hsc70 in combination with auxilin can uncoat clathrin-coated pits on the plasma membrane of permeabilized cells.
We found that a combination of Hsc70 and auxilin uncoats clathrin-coated pits in an ATP-dependent reaction in permeabilized cells. In the presence of cytosol, concomitant with clathrin-uncoating, clathrin, AP2 and epsin exchange on the pre-existing pits. Moreover, Hsc70 and auxilin are required for the clathrin-exchange. These results support our observations that clathrin and AP2 both exchange on clathrin-coated pits in vivo.
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Materials and Methods |
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Immunofluorescence
Cells were fixed with 4% paraformaldehyde before immunostaining. The following antibodies were used: anti-clathrin antibody was either X22, an IgG1 isotype (Affinity BioReagents) or CHC5.9, an IgM (Biodesign International), anti-AP2 antibody was AP.6 (Affinity BioReagents), anti-AP1 was 100/3 (Sigma), anti-GGA3 and anti-GM130 antibodies were purchased from BD Transduction Lab and anti-epsin antibody was from Santa Cruz Biotechnology, Inc. Secondary antibodies used were rhodamine-conjugated goat anti-mouse IgG antibody, rhodamine-conjugated rabbit anti-goat IgM antibody and Cy5-conjugated goat anti-mouse IgM antibody (Jackson Immunoresearch Laboratory Inc.). For the staining of nonpermeabilized cells, 0.02% saponin was added to the antibody dilution-buffer (PBS, 10% FBS, 0.02% NaN3).
Uncoating of clathrin-coated pits
Uncoating experiments were performed at room temperature and in buffer A. Uncoating of clathrin-coated pits was carried out by adding preincubated ATP-Hsc70 and auxilin onto the permeabilized cells. Unless otherwise indicated, 1 mM ATP, 2 µM Hsc70 and 0.2 µM auxilin were used. The chemical dissociation of the clathrin-coated pits was induced by 0.5 M Tris pH 7.0.
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Dissociation and rebinding of the clathrin-coated pits by brain cytosol
Brain cytosol was prepared by homogenizing bovine brains into an equal volume of homogenization buffer [0.1 M MES pH 6.5, 1 mM EGTA, 0.5 mM magnesium acetate, 3 mM NaN3, 0.2 mM phenylmethylsulphonylfluoride (PMSF)]. The homogenate was then spun at 5000 g for 1 hour and the supernatant was respun at 100,000 g for 1 hour. The supernatant was then dialyzed into buffer A, pH 7.0. The cytosol, which was used at a concentration of 10 mg/ml unless otherwise indicated, contained Hsc70, auxilin, clathrin, AP2 and many other proteins. In the dissociation experiments, the various pure proteins or cytosol were preincubated with 1 mM ATP and then added to the permeabilized cells at room temperature. Recombinant yeast YDJ1 protein and human HDJ1 protein were made according to King et al. (King et al., 1997) and the dominant-negative Hsp70 ATPase mutant (K71E) was purified according to Rajapandi et al. (Rajapandi et al., 1998
). Hsc70 was prepared according to Greene and Eisenberg (Greene and Eisenberg, 1990
) and auxilin was prepared as described in Greener et al. (Greener et al., 2000
).
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Results |
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If the uncoating activity of Hsc70 and auxilin is physiologically relevant, brain cytosol should have the same ability to carry out clathrin uncoating as Hsc70 and auxilin. Therefore, we tested whether brain cytosol can uncoat GFP-clathrin from the membranes of permeabilized cells in the presence of either ATP alone or ATP and GTP. Real-time imaging (Fig. 7A, panel a-c) showed that, in the presence of ATP, cytosol uncoats GFP-clathrin from clathrin-coated pits, just like it uncoats CVs (King et al., 1997). Regardless of whether GTP was present or not, the cytosol (which was diluted about 5-fold during preparation) uncoated the pits at about the same rate as observed with 2 µM Hsc70 and 0.2 µM auxilin (compare Fig. 7B and Fig. 3B). Treatment with cytosol did not lead to dissociation of the transferrin (Fig. 7A, panels d-f), which would occur if coated vesicles were budding from the membrane. Rather, the transferrin on the pits was still accessible to acid-wash or a chase by non-fluorescent transferrin after treatment with cytosol. The clathrin-coated pits from this preparation were expected not to bud because permeabilization was carried out by digitonin, which removes cholesterol from the plasma membrane (Boesze-Battaglia et al., 1990
; deDiego et al., 2002
; Hogenboom et al., 2004
). This depletion of cholesterol has been shown to inhibit clathrin-mediated endocytosis in tissue-culture cells by preventing the budding of the clathrin-coated pits (Rodal et al., 1999
; Subtil et al., 1999
).
To be certain that the uncoating activity of cytosol is due to Hsc70, we added two different J-domain proteins, either recombinant yeast YDJ1 or human HDJ1, that have been previously shown to inhibit the uncoating of CVs by Hsc70 in vitro (King et al., 1997). Clathrin uncoating was almost completely inhibited when the permeabilized cells and cytosol were pre-treated with the J-domain protein, YDJ1 (Fig. 7B). Similarly to the results of King et al. (King et al., 1997
), treatment with HDJ1 gave similar results (data not shown). In addition, a reduction in uncoating was also obtained when we added the ATPase-deficient mutant of Hsp70, Hsp70(K71E) to the cytosol (Fig. 7B). This mutant has been shown to have a dominant-negative effect on Hsc70 uncoating activity (Newmyer and Schmid, 2001
; Rajapandi et al., 1998
), and we can therefore conclude that Hsc70 in the cytosol was responsible for the clathrin uncoating.
Surprisingly, while the GFP-clathrin was uncoated by cytosol when we immunostained for clathrin, the total clathrin on the puncta remained constant (Fig. 7C). We used the same settings throughout to measure the GFP- and rhodamine-fluorescence. Furthermore, when raising the fluorescence intensity of GFP to look at the residual GFP-clathrin that remained on the pits, the rebinding of clathrin occurred on exactly those pits that had previously been uncoated (Fig. 7C inset). This suggests that the clathrin-exchange on the pits happens while cells are treated with cytosol.
To follow this exchange in greater detail, we examined concomitantly imaged GFP-clathrin and clathrin immunostaining at much higher resolution, to enable imaging of single pits or two adjacent pits. We incubated the cells with cytosol for varying times, using fixation to stop the reaction, and then immunostained for clathrin (Fig. 8A). The permeabilized cells were analyzed for both GFP-fluorescence intensity and rhodamine-conjugated antibody staining. Then, using the same settings, we imaged the GFP-fluorescence intensity (a measure of the dissociation of the initially bound GFP-clathrin) and rhodamine-conjugated antibody staining (a measure of the total amount of bound clathrin). The pits slowly lost the GFP-fluorescence intensity as the clathrin dissociated over a 5-minute period, whereas the total amount of clathrin remained unchanged. Fig. 8B shows the distribution of disappearance time of the GFP-clathrin measured for the uncoating of clathrin-coated pits by cytosol. Data were obtained from 200 puncta (five cells). More than 80% of the pits needed longer than 1 minute to disappear, whereas less that 5% of the pits disappeared within 30 seconds. As we observed with Hsc70 and auxilin (Fig. 4C), the majority of these puncta do not disappear by a catastrophic dissolution. The exchange of clathrin on clathrin-coated pits that we previously obtained in vivo (Wu et al., 2001) has now been reconstituted in this in-vitro system.
We next tested whether cytosol was able to dissociate AP2 and epsin. Real-time imaging of cells expressing GFP-AP2 and GFP-epsin showed that cytosol dissociated these proteins (Fig. 9A). Their rate of dissociation is roughly the same as that observed for clathrin but, unlike the dissociation of clathrin, that of AP2 and epsin was only slightly affected by the addition of YDJ1 to the cytosol (compare Fig. 7B and Fig. 9B). Therefore, the mechanism of dissociation of AP2 and epsin from the pits is very different from that of clathrin.
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We next checked that clathrin and AP2 rebound to the same clathrin-coated pits. Cells expressing GFP-clathrin were uncoated by cytosol and then immunostained for both clathrin and AP2. As shown in Fig. 10, clathrin and AP2 in the cytosol replaced the bound clathrin and AP2 on the same pits. However, no significant formation of new pits was observed, possibly for the same reason why no endocytosis occurred. Therefore, just as it has been observed in vivo (Wu et al., 2001; Wu et al., 2003
), both clathrin and AP2 exchange on the same clathrin-coated pits in permeabilized cells treated with cytosol.
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Discussion |
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Whereas Hsc70 and auxilin can dissociate clathrin from the clathrin-coated pits in an ATP-dependent reaction, they cannot dissociate AP2. Even after most of the clathrin was uncoated from the pits, both AP2 and transferrin remained associated with the same pits they were associated with before the clathrin dissociated. Similarly, neither AP1 nor GGA dissociated with the clathrin from the TGN. These results are consistent with the observation of Hannan et al., who suggested that, Hsc70 is in some way necessary for dissociation of AP2 from CVs, but it is not sufficient (Hannan et al., 1998). A cofactor in cytosol, which has recently been identified by Ghosh et al. to be a phosphatase, has been shown to be required for dissociation of AP1 from CVs (Ghosh et al., 2003). Consistent with their observations, we found that cytosol dissociated both clathrin and AP2 from the clathrin-coated pits in the permeabilized cells. Similarly, cytosol dissociated AP1 and GGA from the TGN.
Interestingly, whereas cytosol dissociated both AP2 and clathrin from the clathrin-coated pits, it did not simultaneously dissociate transferrin from the pits. Because dissociation of AP2 on the membrane by high salt caused the release of transferrin, these data indicated that clathrin and AP2 from the cytosol might be replacing the clathrin and AP2 on the pits, while being dissociated by the cytosol. This was confirmed by antibody staining which determined the total clathrin and AP2 bound to the pits. Because in the presence of ATP, the cytosol removed GFP-clathrin and GFP-AP2, they were indeed replaced by the clathrin and AP2 present in the cytosol and this replacement occurred on the same pits that had just been uncoated. This result is particularly interesting in regard to clathrin because this was the first time we were able to observe the rebinding of clathrin to membranes in vitro. The rebinding of clathrin to uncoated CVs was not previously observed, whether or not Hsc70, or nucleotide was present with the free clathrin. In fact, even when cytosol was added to the uncoated vesicles in the presence of ATP, there was no significant rebinding of clathrin in vitro (our unpublished observations). Therefore, the permeabilized cell preparation allowed us to observe not just dissociation of clathrin by Hsc70, but also rebinding of clathrin and AP2, just as it occurs in vivo during clathrin-mediated endocytosis. Moreover, actual exchange is occurring: individual cytosolic proteins are rebinding when the individual clathrin and AP2 proteins dissociate from the pits. Therefore, this in-vitro system shows the exchange of clathrin and AP2 we previously observed in vivo.
The ability of clathrin to exchange might enable the clathrin lattice to readjust its curvature to form a coated vesicle. Recently, Ehrlich et al. suggested that clathrin-coated pits initially form on the membrane as nascent pits that can dissolve before they reach maturity (Ehrlich et al., 2004). Only after binding their cargo are they committed to maturing and developing into CVs. Because the size of the cargo determines the size of the mature vesicle, the nascent pit must adjust its curvature to fit the cargo that binds to it. For example, it must adjust its size to accommodate both low density lipoprotein, which has a diameter of about 27 nm, and reovirus with a 3-fold larger diameter (Ehrlich et al., 2004
). This change in size could occur solely through distortion. However, rearrangement of the distorted clathrin-lattice through clathrin-exchange might provide a more energetically favorable way to change the curvature, compared with maintaining a distorted region of the pit after cargo has bound. Interestingly, a recent electron-cryomicroscopy study of the auxilin-clathrin basket (Fotin et al., 2004
) showed that, the binding of auxilin caused a global distortion of the clathrin basket. Distortion of the clathrin basket has also been observed in electron cryomicroscopy reconstruction of clathrin baskets that polymerized with a chimera of AP180 and auxilin upon the binding of Hsc70 at pH 6.0 (Heymann et al., 2005
). Although these structural studies certainly show that the basket can undergo global distortion, they do not show whether this distorted state is just a transient step in the uncoating process.
We demonstrated clathrin and AP2 exchange on clathrin-coated pits when cytosol and nucleotide were added to permeabilized cells; however, we did not observe a significant number of pits budding off from the membrane or detected the formation of new pits. A different method of permeabilization might preserve the membrane better than digitonin; and perhaps with a different method, the actual budding-off of clathrin-coated pits from the plasma membrane and also reformation of new pits could be observed. Nevertheless, because our present assay reconstitutes clathrin- and AP2-exchange in vitro, we should be able to identify the cytosolic factors that are required for both uncoating and rebinding of AP2 to the plasma membrane, and also the factors that facilitate the rebinding of clathrin to the uncoated pits.
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Acknowledgments |
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Footnotes |
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