- Email: [email protected]
Clathrin‐Coated Vesicle Formation from Isolated Plasma Membranes
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CCV formation from isolated plasma membranes
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Sweitzer, S., and Hinshaw, J. (1998). Dynamin undergoes a GTP‐dependent conformational change causing vesiculation. Cell 93, 1021–1029. Takei, K., McPherson, P. S., Schmid, S. L., and De Camilli, P. (1995). Tubular membrane invaginations coated by dynamin rings are induced by GTPS in nerve terminals. Nature 374, 186–190. Takei, K., Slepnev, V. I., and De Camilli, P. (2001). Interactions of dynamin and amphiphysin with liposomes. Meth. Enzymol. 369, 478–486. Tuma, P. L., and Collins, C. A. (1994). Activation of dynamin GTPase is a result of positive cooperativity. J. Biol. Chem. 269, 30842–30847. Warnock, D. E., Hinshaw, J. E., and Schmid, S. L. (1996). Dynamin self assembly stimulates its GTPase activity. J. Biol. Chem. 271, 22310–22314.
[44] Clathrin‐Coated Vesicle Formation from Isolated Plasma Membranes By ISHIDO MIWAKO and SANDRA L. SCHMID Abstract
Endocytic clathrin‐coated vesicle (CCV) formation is a complex process involving a large number of proteins and lipids. The minimum machinery and the hierarchy of the events involved in CCV formation have yet to be defined. Here we describe an in vitro assay for CCV formation from highly purified rat liver plasma membranes. This rapid and easy assay can be used to quantitatively evaluate the different protein requirements for different endocytic receptors. Introduction
Clathrin‐coated vesicle formation is a highly regulated process in which cargo proteins are selectively sequestered. The events involved in CCV formation and the molecules required have been extensively studied and reviewed elsewhere (Conner and Schmid, 2003; Owen et al., 2004; Slepnev and De Camilli, 2000). Core components known to be important for the CCV formation are clathrin, AP2, and dynamin. In addition to these three proteins, dozens of proteins have been reported to be involved in CCV formation. Recently, it has been reported that cargo‐specific adaptors such as AP180/CALM, Dab2, ‐arrestins, autosomal recessive hypercholesterolemia (ARH) protein, epsin, and HIP1/Hip1R are also important for specific CCV formation (Robinson, 2004; Traub, 2003). In addition, there are several accessory proteins such as endophilin and Hsc70 known to be involved in CCV formation. Some of these accessory proteins including METHODS IN ENZYMOLOGY, VOL. 404 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)04044-9
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amphiphysin, intersectin, SNX9, and synaptojanin interact with dynamin through its C‐terminal proline‐rich domain. Furthermore, the involvement of actin dynamics in CCV formation through interaction with dynamin and other endocytic accessory proteins has been suggested (Schafer, 2004). However, the hierarchy of events involved in CCV formation still remains to be resolved. To untangle the complexity of the molecular mechanisms involved in CCV formation, it is important to clarify the role of each protein for endocytosis of each endocytic receptor in detail. Here we describe a simple in vitro CCV formation assay to quantitatively measure endocytosis of several endocytic receptors at the same time. The assay provides a potential means to analyze the complicated interconnected networks of adaptor and accessory proteins. Materials and Reagents
Buffers, Reagents, and Antibodies XTR transport buffer: 0.25 M sorbitol, 20 mM Hepes, pH 7.4, 150 mM potassium acetate, 1 mM magnesium acetate. XTR transport buffer is filtered through a 0.45 M filter and stored at 4 . 20‐fold ATP‐regenerating system: 16 mM ATP, pH 7.0, 100 mM creatine phosphate and 100 units/ml creatine phosphokinase stored in aliquots at 80 . GTP stock: 10–100 mM GTP in 20 mM Hepes, pH 7.4 stored in aliquots at 80 . 10% BSA. 1000‐fold protease inhibitor cocktail (Sigma‐Aldrich Co., St. Louis, MO) in DMSO stored at 20 . Antibodies: Rabbit anti‐LRP light chain was obtained from Dr. J. Herz (University of Texas Southwestern Medical Center, Dallas, TX), rabbit anti‐SR‐BI antibody from Dr. M. Krieger (Massachusetts Institute of Technology, Cambridge, MA), mouse monoclonal anti–human transferrin receptor (TfnR) antibody (H68) from Dr. I. S. Trowbridge (Salk Institute, La Jolla, CA), and mouse monoclonal anti‐asialoglycoprotein receptor (ASGPR) antibody from Daiichi Pure Chemicals Co., Ltd (Ibaraki, Japan). Preparation and Storage of Cytosol, Clathrin, AP2, and Dynamin Rat and bovine brain cytosol are prepared as previously described (Miwako et al., 2003). To prepare a Cl/AP2 and dynamin‐depleted ammonium sulfate supernatant of bovine brain cytosol, cytosol is brought to 30% (NH2)4SO4 by adding saturated (NH2)4SO4 while gently stirring at 4 .
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The suspension is centrifuged for 30 min at 100,000g rpm in a Ti60 rotor and the supernatant is collected and gel‐filtered over a G‐25 column equilibrated with XTR transport buffer to remove (NH2)4SO4. The resulting 30% (NH2)4SO4 is >80% depleted of clathrin, AP2, and dynamin, but retains >90% of total cytosolic protein. Clathrin and AP2 are purified from microsome extracts as previously described (Manfredi and Bazari, 1987; Smythe et al., 1992). Clathrin is stored in 30 mM Tris, pH 7.0 in aliquots at 80 . AP2 is stored in XTR transport buffer in aliquots at 80 . Recombinant dynamin‐1 is prepared as described (Marks et al., 2001). Isolation of Rat Liver PMs Rat liver PM sheets are isolated according to a protocol developed by Ann Hubbard and colleagues (Bartles and Hubbard, 1990; Hubbard et al., 1983; Scott et al., 1993) with slight modifications (Miwako et al., 2003). Six male Sprague‐Dawley rats (120–170g) are fasted for 24 h. One at a time the rats are placed in an anesthetizing chamber infused with isoflurane gas. When fully unconscious, the rat is opened surgically to locate the heart. An 18‐gauge needle is inserted into the left ventricle and 100 ml ice‐cold 0.154 M NaCl is injected to perfuse the liver. The liver is excised, weighed (6 g/liver), and treated at 0–4 throughout the subsequent procedures. The liver is cut with a razor blade into slivers and transferred into a 40‐ml glass dounce. Four volumes of 0.25 M STM containing 0.25 M sucrose, 5 mM Tris HCl, pH 7.4, 0.5 mM MgCl2, protease inhibitor cocktail (Sigma‐ Aldrich Co.) is added and the liver is homogenized using 10 strokes with a loose‐fitting pestle. The homogenate of all the liver tissue is collected in a 250‐ml cylinder and adjusted to 20% (liver wet weight to total volume) with 0.25 M STM. The homogenate is filtered through four layers of cheesecloth moistened with 0.25 M STM. The filtrate is transferred to a 50‐ml tube (25–30 ml per tube) and centrifuged at 280g (1100 rpm in Beckman Allegra‐6R) for 5 min. The supernatant is saved and the pellet is resuspended with 3 strokes of a loose dounce in ½ original homogenate volume of 0.25 M STM. The suspension is again centrifuged as above. The first and second supernatants are combined and centrifuged at 1500g for 10 min (25–30 ml per 50‐ml tube). The resulting pellets are pooled and resuspended with 3 strokes of a loose dounce in 1–2 ml of 0.25 M STM per gram initial wet weight liver. 2.0 M STM is added to obtain a density of 1.42 M, determined using a refractometer. Sufficient 1.42 M STM is added to bring the volume to approximately twice that of the original homogenate. Samples are added to cellulose nitrate tubes (30 ml/tube) and overlaid with 2–4 ml of 0.25 M sucrose. The samples are centrifuged at
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82,000g (25,000 rpm, Beckman L7‐65, SW‐28 rotor, no brake) for 60 min. The pellicle at the interface is collected and resuspended with 3 strokes of a loose dounce. The density of the suspension is adjusted to 1.42 M by adding 2.0 M STM. The sample is transferred to ultracentrifuge tubes (30 ml/ tube). 1.42 M STM is added to adjust volume if necessary. 2–4 ml of 0.25 M sucrose is overlaid and the floating procedure is repeated again. The pellicle at the interface is collected and resuspended with 3 strokes of a loose dounce. Distilled H2O is slowly added to obtain a sucrose density of 0.25 M. This suspension is centrifuged at 1500g for 10 min and the final pellet is resuspended with 3 strokes of a loose dounce in 0.25 M sucrose to obtain a final concentration of 2 mg/ml. At each step, the enrichment of the PMs and the protein concentration is measured to calculate the yield and the fold‐purification of the PMs. The enzymatic activity of alkaline phosphodiesterase is used as a PM marker, and measured using a colorimetric assay (Hubbard et al., 1983). 50 l of 2 mg/ml thymidine 50 ‐monophosphate p‐nitrophenyl ester (Sigma‐ Aldrich Co.) in 0.1 M Tris, 40 mM CaCl2, pH 9.0 is added to 75 l of the 1001000‐fold diluted sample. The sample is incubated for 1 h at 37 . Then the reaction is stopped by adding 1 ml of 200 mM Na2CO3, 100 mM glycine, and absorbance at 400 nm is determined. Protein concentration is measured using Coomassie Protein Assay Kit (23200, Pierce Biotechnology, Inc., Rockford, IL). After the first flotation, PMs are obtained in 12– 25% yield and 10–15‐fold enrichment After the second flotation, the yield is about 10–15% and the enrichment is 15–20‐fold. The final PMs have the yield of 10–15% and 20–40‐fold enrichment. 100 or 200 l aliquots of 2 mg/ml PMs in 0.25 M sucrose are frozen in liquid nitrogen and stored at 80 . PMs stored in 0.5 M or 0.75 M sucrose in the presence or absence of 10% DMSO at 80 have similar selectivity and activity of LDL receptor‐related protein (LRP) uptake in our CCV formation assay; however, PMs stored in XTR buffer have low efficiency of LRP uptake. Methods
In Vitro CCV Formation Assay from Isolated PMs Our in vitro CCV formation assay is diagrammed in Fig. 1A. 50 g of 2 mg/ml rat liver PMs are used for each assay. PMs stored at 80 are thawed quickly and collected by centrifugation in a refrigerated Eppendorf centrifuge at 20,000g for 1 min. The pellet is resuspended with XTR transport buffer to a final concentration of 4 mg/ml. Then the sample is centrifuged again to obtain the pellet containing PMs. This procedure
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FIG. 1. (A) Diagram of an in vitro assay for CCV formation from PMs. LSP: low‐speed pellet. LSS: low‐speed supernatant. HSP: high‐speed pellet. HSS: high‐speed supernatant. (B) Temperature‐dependent recruitment of endocytic receptors from LSPs to HSPs in the presence of ATP, GTP, and cytosol. Scavenger receptor class B, type I (SR‐BI) is a PM marker that is independent of clathrin‐mediated endocytosis. (Figures reproduced from Miwako et al., 2003.)
reduces nucleotide‐independent LRP recruitment to the high‐speed pellet (HSP). The pellet is resuspended with XTR buffer, and the PMs are transferred to another eppendorf tube containing an ATP‐regenerating system, 100 M GTP, 0.2 % BSA, and protease inhibitor cocktail in the presence or absence of cytosol or purified proteins in XTR transport buffer to make a final volume of 40 l. The samples are incubated at 37 for typically 10–20 min and then the tubes are returned to ice. The eppendorf tubes are tapped mildly to resuspend membranes and reduce trapping of newly formed vesicles. Then they are centrifuged at 20,000g for 1 min to obtain the low‐speed pellets (LSP) containing PMs. The supernatants are transferred to 1.5 ml microfuge tubes (357448, Beckman Coulter, Inc., Fullerton, CA) and further centrifuged at 200,000g for 20 min in a TLA‐ 100.3 rotor to obtain the HSPs containing newly formed vesicles. LSPs and HSPs are solubilized in sample buffer at 95 for 5 min and analyzed by SDS‐PAGE and immunoblotting with antibodies against endocytic receptors such as LRP, TfnR, and ASGPR. Endocytosis is quantitated by
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the amount of endocytic receptors recruited in the HSP relative to that remaining in the LSP. Figure 1B is a typical Western blot showing the selective internalization of endocytic receptors depending on cytosol, nucleotides, and temperature. Inhibition of Membrane Fusion Increases Efficiency The maximum efficiency of LRP uptake in our in vitro assay was about 15%, obtained in the presence of an ATP‐regenerating system, 2 mM GTP, and 200 g cytosol (Fig. 1B). But the efficiency can be enhanced to 20% by inhibiting membrane fusion using the calcium‐specific chelator, 1,2‐bis (2‐aminophenoxy) ethane‐N,N,N0 ,N0 ‐tetraacetic acid (BAPTA) (Pryor et al., 2000). In Fig. 2A, 50 g PMs are incubated in the presence of an ATP regenerating system, 100 M GTP, and 200 g bovine brain cytosol with or without 10 mM BAPTA (tetrapotassium salt, Molecular Probes, Inc., Eugene, OR) for the indicated times at 37 . Then LSPs and HSPs are analyzed by SDS‐PAGE and Western blotting with the anti‐LRP antibody. The amount of LRP recruited into the HSP is quantitated using a densitometer (Personal Densitometer SI; Amersham Biosciences, Piscataway, NJ). In the presence of BAPTA, LRP uptake is increased to 20%. Membrane recycling is also reduced by dilution because increased volume results in the decreased opportunity of vesicles encountering large membrane compartments. 50 g PMs are incubated in the presence of an ATP regenerating system, 100 M GTP, and 4 mg/ml bovine brain cytosol in 40– 200 l for 30 min at 37 (Fig. 2B). The efficiency of LRP uptake is enhanced to 20% when the volume is increased to 200 l. These data suggest that
FIG. 2. Efficiency of LRP recruitment to the HSP is increased by inhibiting membrane fusion. (A) Time course of the LRP recruitment to the HSP in the presence or the absence of 10 mM BAPTA. (B) Effect of dilution on the efficiency of LRP recruitment to the HSP.
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25% of newly formed vesicles either recycle back and fuse to the PM or fuse to each other forming larger membrane compartments (endosomes) that sediment in the LSP. Endocytosis of Different Endocytic Receptors Using this in vitro assay, internalization of several endocytic receptors can be measured at the same time to clarify different protein requirements for endocytosis. When two endocytic receptors, LRP and ASGPR, are analyzed, the same blot can be used because of the different molecular weights in an SDS‐PAGE gel (LRP light chain is about 95 kDa, ASGPR is 42 kDa). Using purified clathrin, AP2, dynamin, and 30% ammonium sulfate supernatant (AS supt) of bovine brain cytosol, cytosolic protein requirements for LRP or ASGPR uptake are examined. PMs are incubated with an ATP regenerating system, 100 M GTP, in the presence of physiological quantities of clathrin (0.2 g), AP2 (0.8 g), dynamin (2 g) and 30 g of AS supt, or in the presence ofexcess clathrin (8 g), AP2 (8 g), dynamin (20 g) alone for 20 min at 37 C (Fig. 3). In the presence of an excess amount of clathrin, AP2, and dynamin, both LRP and ASGPR are
FIG. 3. Different cytosolic protein requirements for the uptake of LRP or ASGPR. The proportion of LRP and ASGPR internalization in the presence of physiological amounts of clathrin, AP2, dynamin, and AS supt (low Cl/AP2/Dyn1/supt), or high concentration of clathrin, AP2, and dynamin (high Cl/AP2/Dyn1) compared to the maximum efficiency of the uptake obtained in the presence of 200 g bovine brain cytosol.
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internalized; however, in the presence of physiological amount of these proteins with AS supt, only LRP uptake is observed. These data show the different protein requirements for different endocytic receptors; the uptake of ASGPR requires some protein(s) excluded from AS supt, whereas clathrin, AP2, dynamin, and AS supt are sufficient for LRP uptake. Inhibition of LRP Uptake by a Kinase Inhibitor A3 Data in Fig. 4A show the effects of broad‐spectrum kinase inhibitors on LRP uptake. PMs are incubated in the presence of an ATP regenerating system, 100 M GTP, and 120 g bovine brain cytosol with or without 1 mM H‐7 (dihydrochloride, EMD Biosciences, San Diego, CA), 1 mM A3 (hydrochloride, EMD Biosciences), or 1 M staurosporine (EMD Biosciences) for 30 min on ice. Then samples are incubated for 20 min at 37 . The kinase inhibitor A3 inhibits LRP uptake from PMs, but H‐7 and staurosporine do not. These data suggest that a phosphorylation event inhibited by A3 is required for LRP uptake. It has been reported that A3 inhibits the activity of PIP5 kinase (PIP5K) (Arneson et al., 1999). Therefore, to determine whether PIP5K activity could be detected in the isolated rat liver membrane fraction, PMs are incubated in the presence of 500 M ATP containing [‐32P]ATP (25 Ci/assay, MP Biomedicals, Inc., Irvine,
FIG. 4. Effect of membrane‐bound phosphatidylinositol‐4‐phosphate 5‐kinase on the LRP uptake. (A) Effect of kinase inhibitors on the internalization of LRP. Assays were performed in the presence of 1 mM H7, 1 mM A3, or 1 M staurosporine, as indicated. (B) Effect of kinase inhibitors on the production of PI4,5P2 by isolated rat liver PMs.
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CA), 100 M GTP, 120 g bovine brain cytosol with or without 1 mM H‐7, 1 mM A3 for 10 min at 37 . Then the lipids are extracted and analyzed by TLC and the Phosphorimager SI (GE Healthcare) (Arneson et al., 1999). Indeed, PIP5K activity can be detected in isolated rat liver PMs (Fig. 4B) and it is totally inhibited by A3, but not by H‐7 (Fig. 4B). Inhibition of LRP uptake by A3 but not H‐7 or staurosporine suggests that A3 might reduce LRP uptake by inhibiting the PIPK activity. The ability to prepare and store large amounts of endocytically active PM substrate and to simultaneously assay endocytosis of several distinct receptors renders this assay useful for dissecting the complex events involved in clathrin‐mediated endocytosis. References Arneson, L. S., Kunz, J., Anderson, R. A., and Traub, L. M. (1999). Coupled inositide phosphorylation and phospholipase D activation initiates clathrin‐coat assembly on lysosomes. J. Biol. Chem. 274, 17794–17805. Bartles, J. R., and Hubbard, A. L. (1990). Biogenesis of the rat hepatocyte plasma membrane. Methods Enzymol. 191, 825–841. Conner, S. D., and Schmid, S. L. (2003). Regulated portals of entry into the cell. Nature 422, 37–44. Hubbard, A. L., Wall, D. A., and Ma, A. (1983). Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains. J. Cell Biol. 96, 217–229. Manfredi, J. J., and Bazari, W. L. (1987). Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities. J. Biol. Chem. 262, 12182–12188. Marks, B., Stowell, M. H., Vallis, Y., Mills, I. G., Gibson, A., Hopkins, C. R., and McMahon, H. T. (2001). GTPase activity of dynamin and resulting conformation change are essential for endocytosis. Nature 410, 231–235. Miwako, I., Schroter, T., and Schmid, S. L. (2003). Clathrin‐ and dynamin‐dependent coated vesicle formation from isolated plasma membranes. Traffic 4, 376–389. Owen, D. J., Collins, B. M., and Evans, P. R. (2004). Adaptors for clathrin coats: Structure and function. Annu. Rev. Cell Dev. Biol. 20, 153–191. Pryor, P. R., Mullock, B. M., Bright, N. A., Gray, S. R., and Luzio, J. P. (2000). The role of intraorganellar Ca(2þ) in late endosome‐lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles. J. Cell Biol. 149, 1053–1062. Robinson, M. S. (2004). Adaptable adaptors for coated vesicles. Trends Cell Biol. 14, 167–174. Schafer, D. A. (2004). Regulating actin dynamics at membranes: A focus on dynamin. Traffic 5, 463–469. Scott, L., Schell, M. J., and Hubbard, A. L. (1993). Isolation of plasma membrane sheets and plasma membrane domains from rat liver. Methods Mol. Biol. 19, 59–69. Slepnev, V. I., and De Camilli, P. (2000). Accessory factors in clathrin‐dependent synaptic vesicle endocytosis. Nat. Rev. Neurosci. 1, 161–172. Smythe, E., Carter, L. L., and Schmid, S. L. (1992). Cytosol‐ and clathrin‐dependent stimulation of endocytosis in vitro by purified adaptors. J. Cell Biol. 119, 1163–1171. Traub, L. M. (2003). Sorting it out: AP‐2 and alternate clathrin adaptors in endocytic cargo selection. J. Cell Biol. 163, 203–208.
CCV formation from isolated plasma membranes
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Sweitzer, S., and Hinshaw, J. (1998). Dynamin undergoes a GTP‐dependent conformational change causing vesiculation. Cell 93, 1021–1029. Takei, K., McPherson, P. S., Schmid, S. L., and De Camilli, P. (1995). Tubular membrane invaginations coated by dynamin rings are induced by GTPS in nerve terminals. Nature 374, 186–190. Takei, K., Slepnev, V. I., and De Camilli, P. (2001). Interactions of dynamin and amphiphysin with liposomes. Meth. Enzymol. 369, 478–486. Tuma, P. L., and Collins, C. A. (1994). Activation of dynamin GTPase is a result of positive cooperativity. J. Biol. Chem. 269, 30842–30847. Warnock, D. E., Hinshaw, J. E., and Schmid, S. L. (1996). Dynamin self assembly stimulates its GTPase activity. J. Biol. Chem. 271, 22310–22314.
[44] Clathrin‐Coated Vesicle Formation from Isolated Plasma Membranes By ISHIDO MIWAKO and SANDRA L. SCHMID Abstract
Endocytic clathrin‐coated vesicle (CCV) formation is a complex process involving a large number of proteins and lipids. The minimum machinery and the hierarchy of the events involved in CCV formation have yet to be defined. Here we describe an in vitro assay for CCV formation from highly purified rat liver plasma membranes. This rapid and easy assay can be used to quantitatively evaluate the different protein requirements for different endocytic receptors. Introduction
Clathrin‐coated vesicle formation is a highly regulated process in which cargo proteins are selectively sequestered. The events involved in CCV formation and the molecules required have been extensively studied and reviewed elsewhere (Conner and Schmid, 2003; Owen et al., 2004; Slepnev and De Camilli, 2000). Core components known to be important for the CCV formation are clathrin, AP2, and dynamin. In addition to these three proteins, dozens of proteins have been reported to be involved in CCV formation. Recently, it has been reported that cargo‐specific adaptors such as AP180/CALM, Dab2, ‐arrestins, autosomal recessive hypercholesterolemia (ARH) protein, epsin, and HIP1/Hip1R are also important for specific CCV formation (Robinson, 2004; Traub, 2003). In addition, there are several accessory proteins such as endophilin and Hsc70 known to be involved in CCV formation. Some of these accessory proteins including METHODS IN ENZYMOLOGY, VOL. 404 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)04044-9
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amphiphysin, intersectin, SNX9, and synaptojanin interact with dynamin through its C‐terminal proline‐rich domain. Furthermore, the involvement of actin dynamics in CCV formation through interaction with dynamin and other endocytic accessory proteins has been suggested (Schafer, 2004). However, the hierarchy of events involved in CCV formation still remains to be resolved. To untangle the complexity of the molecular mechanisms involved in CCV formation, it is important to clarify the role of each protein for endocytosis of each endocytic receptor in detail. Here we describe a simple in vitro CCV formation assay to quantitatively measure endocytosis of several endocytic receptors at the same time. The assay provides a potential means to analyze the complicated interconnected networks of adaptor and accessory proteins. Materials and Reagents
Buffers, Reagents, and Antibodies XTR transport buffer: 0.25 M sorbitol, 20 mM Hepes, pH 7.4, 150 mM potassium acetate, 1 mM magnesium acetate. XTR transport buffer is filtered through a 0.45 M filter and stored at 4 . 20‐fold ATP‐regenerating system: 16 mM ATP, pH 7.0, 100 mM creatine phosphate and 100 units/ml creatine phosphokinase stored in aliquots at 80 . GTP stock: 10–100 mM GTP in 20 mM Hepes, pH 7.4 stored in aliquots at 80 . 10% BSA. 1000‐fold protease inhibitor cocktail (Sigma‐Aldrich Co., St. Louis, MO) in DMSO stored at 20 . Antibodies: Rabbit anti‐LRP light chain was obtained from Dr. J. Herz (University of Texas Southwestern Medical Center, Dallas, TX), rabbit anti‐SR‐BI antibody from Dr. M. Krieger (Massachusetts Institute of Technology, Cambridge, MA), mouse monoclonal anti–human transferrin receptor (TfnR) antibody (H68) from Dr. I. S. Trowbridge (Salk Institute, La Jolla, CA), and mouse monoclonal anti‐asialoglycoprotein receptor (ASGPR) antibody from Daiichi Pure Chemicals Co., Ltd (Ibaraki, Japan). Preparation and Storage of Cytosol, Clathrin, AP2, and Dynamin Rat and bovine brain cytosol are prepared as previously described (Miwako et al., 2003). To prepare a Cl/AP2 and dynamin‐depleted ammonium sulfate supernatant of bovine brain cytosol, cytosol is brought to 30% (NH2)4SO4 by adding saturated (NH2)4SO4 while gently stirring at 4 .
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The suspension is centrifuged for 30 min at 100,000g rpm in a Ti60 rotor and the supernatant is collected and gel‐filtered over a G‐25 column equilibrated with XTR transport buffer to remove (NH2)4SO4. The resulting 30% (NH2)4SO4 is >80% depleted of clathrin, AP2, and dynamin, but retains >90% of total cytosolic protein. Clathrin and AP2 are purified from microsome extracts as previously described (Manfredi and Bazari, 1987; Smythe et al., 1992). Clathrin is stored in 30 mM Tris, pH 7.0 in aliquots at 80 . AP2 is stored in XTR transport buffer in aliquots at 80 . Recombinant dynamin‐1 is prepared as described (Marks et al., 2001). Isolation of Rat Liver PMs Rat liver PM sheets are isolated according to a protocol developed by Ann Hubbard and colleagues (Bartles and Hubbard, 1990; Hubbard et al., 1983; Scott et al., 1993) with slight modifications (Miwako et al., 2003). Six male Sprague‐Dawley rats (120–170g) are fasted for 24 h. One at a time the rats are placed in an anesthetizing chamber infused with isoflurane gas. When fully unconscious, the rat is opened surgically to locate the heart. An 18‐gauge needle is inserted into the left ventricle and 100 ml ice‐cold 0.154 M NaCl is injected to perfuse the liver. The liver is excised, weighed (6 g/liver), and treated at 0–4 throughout the subsequent procedures. The liver is cut with a razor blade into slivers and transferred into a 40‐ml glass dounce. Four volumes of 0.25 M STM containing 0.25 M sucrose, 5 mM Tris HCl, pH 7.4, 0.5 mM MgCl2, protease inhibitor cocktail (Sigma‐ Aldrich Co.) is added and the liver is homogenized using 10 strokes with a loose‐fitting pestle. The homogenate of all the liver tissue is collected in a 250‐ml cylinder and adjusted to 20% (liver wet weight to total volume) with 0.25 M STM. The homogenate is filtered through four layers of cheesecloth moistened with 0.25 M STM. The filtrate is transferred to a 50‐ml tube (25–30 ml per tube) and centrifuged at 280g (1100 rpm in Beckman Allegra‐6R) for 5 min. The supernatant is saved and the pellet is resuspended with 3 strokes of a loose dounce in ½ original homogenate volume of 0.25 M STM. The suspension is again centrifuged as above. The first and second supernatants are combined and centrifuged at 1500g for 10 min (25–30 ml per 50‐ml tube). The resulting pellets are pooled and resuspended with 3 strokes of a loose dounce in 1–2 ml of 0.25 M STM per gram initial wet weight liver. 2.0 M STM is added to obtain a density of 1.42 M, determined using a refractometer. Sufficient 1.42 M STM is added to bring the volume to approximately twice that of the original homogenate. Samples are added to cellulose nitrate tubes (30 ml/tube) and overlaid with 2–4 ml of 0.25 M sucrose. The samples are centrifuged at
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82,000g (25,000 rpm, Beckman L7‐65, SW‐28 rotor, no brake) for 60 min. The pellicle at the interface is collected and resuspended with 3 strokes of a loose dounce. The density of the suspension is adjusted to 1.42 M by adding 2.0 M STM. The sample is transferred to ultracentrifuge tubes (30 ml/ tube). 1.42 M STM is added to adjust volume if necessary. 2–4 ml of 0.25 M sucrose is overlaid and the floating procedure is repeated again. The pellicle at the interface is collected and resuspended with 3 strokes of a loose dounce. Distilled H2O is slowly added to obtain a sucrose density of 0.25 M. This suspension is centrifuged at 1500g for 10 min and the final pellet is resuspended with 3 strokes of a loose dounce in 0.25 M sucrose to obtain a final concentration of 2 mg/ml. At each step, the enrichment of the PMs and the protein concentration is measured to calculate the yield and the fold‐purification of the PMs. The enzymatic activity of alkaline phosphodiesterase is used as a PM marker, and measured using a colorimetric assay (Hubbard et al., 1983). 50 l of 2 mg/ml thymidine 50 ‐monophosphate p‐nitrophenyl ester (Sigma‐ Aldrich Co.) in 0.1 M Tris, 40 mM CaCl2, pH 9.0 is added to 75 l of the 1001000‐fold diluted sample. The sample is incubated for 1 h at 37 . Then the reaction is stopped by adding 1 ml of 200 mM Na2CO3, 100 mM glycine, and absorbance at 400 nm is determined. Protein concentration is measured using Coomassie Protein Assay Kit (23200, Pierce Biotechnology, Inc., Rockford, IL). After the first flotation, PMs are obtained in 12– 25% yield and 10–15‐fold enrichment After the second flotation, the yield is about 10–15% and the enrichment is 15–20‐fold. The final PMs have the yield of 10–15% and 20–40‐fold enrichment. 100 or 200 l aliquots of 2 mg/ml PMs in 0.25 M sucrose are frozen in liquid nitrogen and stored at 80 . PMs stored in 0.5 M or 0.75 M sucrose in the presence or absence of 10% DMSO at 80 have similar selectivity and activity of LDL receptor‐related protein (LRP) uptake in our CCV formation assay; however, PMs stored in XTR buffer have low efficiency of LRP uptake. Methods
In Vitro CCV Formation Assay from Isolated PMs Our in vitro CCV formation assay is diagrammed in Fig. 1A. 50 g of 2 mg/ml rat liver PMs are used for each assay. PMs stored at 80 are thawed quickly and collected by centrifugation in a refrigerated Eppendorf centrifuge at 20,000g for 1 min. The pellet is resuspended with XTR transport buffer to a final concentration of 4 mg/ml. Then the sample is centrifuged again to obtain the pellet containing PMs. This procedure
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FIG. 1. (A) Diagram of an in vitro assay for CCV formation from PMs. LSP: low‐speed pellet. LSS: low‐speed supernatant. HSP: high‐speed pellet. HSS: high‐speed supernatant. (B) Temperature‐dependent recruitment of endocytic receptors from LSPs to HSPs in the presence of ATP, GTP, and cytosol. Scavenger receptor class B, type I (SR‐BI) is a PM marker that is independent of clathrin‐mediated endocytosis. (Figures reproduced from Miwako et al., 2003.)
reduces nucleotide‐independent LRP recruitment to the high‐speed pellet (HSP). The pellet is resuspended with XTR buffer, and the PMs are transferred to another eppendorf tube containing an ATP‐regenerating system, 100 M GTP, 0.2 % BSA, and protease inhibitor cocktail in the presence or absence of cytosol or purified proteins in XTR transport buffer to make a final volume of 40 l. The samples are incubated at 37 for typically 10–20 min and then the tubes are returned to ice. The eppendorf tubes are tapped mildly to resuspend membranes and reduce trapping of newly formed vesicles. Then they are centrifuged at 20,000g for 1 min to obtain the low‐speed pellets (LSP) containing PMs. The supernatants are transferred to 1.5 ml microfuge tubes (357448, Beckman Coulter, Inc., Fullerton, CA) and further centrifuged at 200,000g for 20 min in a TLA‐ 100.3 rotor to obtain the HSPs containing newly formed vesicles. LSPs and HSPs are solubilized in sample buffer at 95 for 5 min and analyzed by SDS‐PAGE and immunoblotting with antibodies against endocytic receptors such as LRP, TfnR, and ASGPR. Endocytosis is quantitated by
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the amount of endocytic receptors recruited in the HSP relative to that remaining in the LSP. Figure 1B is a typical Western blot showing the selective internalization of endocytic receptors depending on cytosol, nucleotides, and temperature. Inhibition of Membrane Fusion Increases Efficiency The maximum efficiency of LRP uptake in our in vitro assay was about 15%, obtained in the presence of an ATP‐regenerating system, 2 mM GTP, and 200 g cytosol (Fig. 1B). But the efficiency can be enhanced to 20% by inhibiting membrane fusion using the calcium‐specific chelator, 1,2‐bis (2‐aminophenoxy) ethane‐N,N,N0 ,N0 ‐tetraacetic acid (BAPTA) (Pryor et al., 2000). In Fig. 2A, 50 g PMs are incubated in the presence of an ATP regenerating system, 100 M GTP, and 200 g bovine brain cytosol with or without 10 mM BAPTA (tetrapotassium salt, Molecular Probes, Inc., Eugene, OR) for the indicated times at 37 . Then LSPs and HSPs are analyzed by SDS‐PAGE and Western blotting with the anti‐LRP antibody. The amount of LRP recruited into the HSP is quantitated using a densitometer (Personal Densitometer SI; Amersham Biosciences, Piscataway, NJ). In the presence of BAPTA, LRP uptake is increased to 20%. Membrane recycling is also reduced by dilution because increased volume results in the decreased opportunity of vesicles encountering large membrane compartments. 50 g PMs are incubated in the presence of an ATP regenerating system, 100 M GTP, and 4 mg/ml bovine brain cytosol in 40– 200 l for 30 min at 37 (Fig. 2B). The efficiency of LRP uptake is enhanced to 20% when the volume is increased to 200 l. These data suggest that
FIG. 2. Efficiency of LRP recruitment to the HSP is increased by inhibiting membrane fusion. (A) Time course of the LRP recruitment to the HSP in the presence or the absence of 10 mM BAPTA. (B) Effect of dilution on the efficiency of LRP recruitment to the HSP.
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25% of newly formed vesicles either recycle back and fuse to the PM or fuse to each other forming larger membrane compartments (endosomes) that sediment in the LSP. Endocytosis of Different Endocytic Receptors Using this in vitro assay, internalization of several endocytic receptors can be measured at the same time to clarify different protein requirements for endocytosis. When two endocytic receptors, LRP and ASGPR, are analyzed, the same blot can be used because of the different molecular weights in an SDS‐PAGE gel (LRP light chain is about 95 kDa, ASGPR is 42 kDa). Using purified clathrin, AP2, dynamin, and 30% ammonium sulfate supernatant (AS supt) of bovine brain cytosol, cytosolic protein requirements for LRP or ASGPR uptake are examined. PMs are incubated with an ATP regenerating system, 100 M GTP, in the presence of physiological quantities of clathrin (0.2 g), AP2 (0.8 g), dynamin (2 g) and 30 g of AS supt, or in the presence ofexcess clathrin (8 g), AP2 (8 g), dynamin (20 g) alone for 20 min at 37 C (Fig. 3). In the presence of an excess amount of clathrin, AP2, and dynamin, both LRP and ASGPR are
FIG. 3. Different cytosolic protein requirements for the uptake of LRP or ASGPR. The proportion of LRP and ASGPR internalization in the presence of physiological amounts of clathrin, AP2, dynamin, and AS supt (low Cl/AP2/Dyn1/supt), or high concentration of clathrin, AP2, and dynamin (high Cl/AP2/Dyn1) compared to the maximum efficiency of the uptake obtained in the presence of 200 g bovine brain cytosol.
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internalized; however, in the presence of physiological amount of these proteins with AS supt, only LRP uptake is observed. These data show the different protein requirements for different endocytic receptors; the uptake of ASGPR requires some protein(s) excluded from AS supt, whereas clathrin, AP2, dynamin, and AS supt are sufficient for LRP uptake. Inhibition of LRP Uptake by a Kinase Inhibitor A3 Data in Fig. 4A show the effects of broad‐spectrum kinase inhibitors on LRP uptake. PMs are incubated in the presence of an ATP regenerating system, 100 M GTP, and 120 g bovine brain cytosol with or without 1 mM H‐7 (dihydrochloride, EMD Biosciences, San Diego, CA), 1 mM A3 (hydrochloride, EMD Biosciences), or 1 M staurosporine (EMD Biosciences) for 30 min on ice. Then samples are incubated for 20 min at 37 . The kinase inhibitor A3 inhibits LRP uptake from PMs, but H‐7 and staurosporine do not. These data suggest that a phosphorylation event inhibited by A3 is required for LRP uptake. It has been reported that A3 inhibits the activity of PIP5 kinase (PIP5K) (Arneson et al., 1999). Therefore, to determine whether PIP5K activity could be detected in the isolated rat liver membrane fraction, PMs are incubated in the presence of 500 M ATP containing [‐32P]ATP (25 Ci/assay, MP Biomedicals, Inc., Irvine,
FIG. 4. Effect of membrane‐bound phosphatidylinositol‐4‐phosphate 5‐kinase on the LRP uptake. (A) Effect of kinase inhibitors on the internalization of LRP. Assays were performed in the presence of 1 mM H7, 1 mM A3, or 1 M staurosporine, as indicated. (B) Effect of kinase inhibitors on the production of PI4,5P2 by isolated rat liver PMs.
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CA), 100 M GTP, 120 g bovine brain cytosol with or without 1 mM H‐7, 1 mM A3 for 10 min at 37 . Then the lipids are extracted and analyzed by TLC and the Phosphorimager SI (GE Healthcare) (Arneson et al., 1999). Indeed, PIP5K activity can be detected in isolated rat liver PMs (Fig. 4B) and it is totally inhibited by A3, but not by H‐7 (Fig. 4B). Inhibition of LRP uptake by A3 but not H‐7 or staurosporine suggests that A3 might reduce LRP uptake by inhibiting the PIPK activity. The ability to prepare and store large amounts of endocytically active PM substrate and to simultaneously assay endocytosis of several distinct receptors renders this assay useful for dissecting the complex events involved in clathrin‐mediated endocytosis. References Arneson, L. S., Kunz, J., Anderson, R. A., and Traub, L. M. (1999). Coupled inositide phosphorylation and phospholipase D activation initiates clathrin‐coat assembly on lysosomes. J. Biol. Chem. 274, 17794–17805. Bartles, J. R., and Hubbard, A. L. (1990). Biogenesis of the rat hepatocyte plasma membrane. Methods Enzymol. 191, 825–841. Conner, S. D., and Schmid, S. L. (2003). Regulated portals of entry into the cell. Nature 422, 37–44. Hubbard, A. L., Wall, D. A., and Ma, A. (1983). Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains. J. Cell Biol. 96, 217–229. Manfredi, J. J., and Bazari, W. L. (1987). Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities. J. Biol. Chem. 262, 12182–12188. Marks, B., Stowell, M. H., Vallis, Y., Mills, I. G., Gibson, A., Hopkins, C. R., and McMahon, H. T. (2001). GTPase activity of dynamin and resulting conformation change are essential for endocytosis. Nature 410, 231–235. Miwako, I., Schroter, T., and Schmid, S. L. (2003). Clathrin‐ and dynamin‐dependent coated vesicle formation from isolated plasma membranes. Traffic 4, 376–389. Owen, D. J., Collins, B. M., and Evans, P. R. (2004). Adaptors for clathrin coats: Structure and function. Annu. Rev. Cell Dev. Biol. 20, 153–191. Pryor, P. R., Mullock, B. M., Bright, N. A., Gray, S. R., and Luzio, J. P. (2000). The role of intraorganellar Ca(2þ) in late endosome‐lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles. J. Cell Biol. 149, 1053–1062. Robinson, M. S. (2004). Adaptable adaptors for coated vesicles. Trends Cell Biol. 14, 167–174. Schafer, D. A. (2004). Regulating actin dynamics at membranes: A focus on dynamin. Traffic 5, 463–469. Scott, L., Schell, M. J., and Hubbard, A. L. (1993). Isolation of plasma membrane sheets and plasma membrane domains from rat liver. Methods Mol. Biol. 19, 59–69. Slepnev, V. I., and De Camilli, P. (2000). Accessory factors in clathrin‐dependent synaptic vesicle endocytosis. Nat. Rev. Neurosci. 1, 161–172. Smythe, E., Carter, L. L., and Schmid, S. L. (1992). Cytosol‐ and clathrin‐dependent stimulation of endocytosis in vitro by purified adaptors. J. Cell Biol. 119, 1163–1171. Traub, L. M. (2003). Sorting it out: AP‐2 and alternate clathrin adaptors in endocytic cargo selection. J. Cell Biol. 163, 203–208.