- Email: [email protected]
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Feeley, J. C., Gibson, R. J., Gorman, G. W., Langford, N. C., Rasheed, J. K., Mackel, D. C., and Blaine, W. B. (1979). Charcoal‐yeast extract agar: Primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10, 437–441. Horwitz, M. A. (1983a). Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J. Exp. Med. 158, 1319–1331. Horwitz, M. A. (1983b). The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome lysosome fusion in human monocytes. J. Exp. Med. 158, 2108–2126. Joiner, K. A., Fuhrman, S. A., Miettinen, H. M., Kasper, L. H., and Mellman, I. (1990). Toxoplasma gondii: Fusion competence of parasitophorous vacuoles in Fc receptor‐ transfected fibroblasts. Science 249, 641–646. Kagan, J. C., and Roy, C. R. (2002). Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat. Cell Biol. 4, 945–954. Kagan, J. C., Stein, M. P., Pypaert, M., and Roy, C. R. (2004). Legionella subvert the functions of Rab1 and Sec22b to create a replicative organelle. J. Exp. Med. 199, 1201–1211. Luo, Z. Q., and Isberg, R. R. (2004). Multiple substrates of the Legionella pneumophila Dot/ Icm system identified by interbacterial protein transfer. Proc. Natl. Acad. Sci. USA 101, 841–846. Nagai, H., Kagan, J. C., Zhu, X., Kahn, R. A., and Roy, C. R. (2002). A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295, 679–682. Roy, C. R., and Tilney, L. G. (2002). The road less traveled: Transport of Legionella to the endoplasmic reticulum. J. Cell Biol. 158, 415–419. Roy, C. R., Berger, K., and Isberg, R. R. (1998). Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol. Microbiol. 28, 663–674. Segal, G., Purcell, M., and Shuman, H. A. (1998). Host cell killing and bacterial conjugation require overlapping sets of genes within a 22‐kb region of the Legionella pneumophila genome. Proc. Natl. Acad. Sci. USA 95, 1669–1674. Vogel, J. P., Andrews, H. L., Wong, S. K., and Isberg, R. R. (1998). Conjugative transfer by the virulence system of Legionella pneumophila. Science 279, 873–876. Yamamoto, Y., Klein, T. W., Newton, C. A., Widen, R., and Friedman, H. (1988). Growth of Legionella pneumophila in thioglycolate‐elicited peritoneal macrophages from A/J mice. Infect. Immun. 56, 370–375.
[8] Reconstitution of Rab4‐Dependent Vesicle Formation In Vitro By ADRIANA PAGANO and MARTIN SPIESS Abstract
We have developed an in vitro assay to reconstitute the formation of endosomal recycling vesicles. To achieve specificity for endosomes as the donor organelle, cells are surface‐biotinylated and allowed to endocytose for 10 min, after which the remaining surface‐biotin is stripped off. The cells are then permeabilized and the cytosol washed away. Upon addition METHODS IN ENZYMOLOGY, VOL. 403 Copyright 2005, Elsevier Inc. All rights reserved.
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of exogenous cytosol and energy, sealed vesicles containing biotinylated recycling receptors are produced. Modification of the cytosol, for example, by immunodepletion or addition of purified proteins, allows the identification of proteins involved in vesicle formation. The results show that recycling is mediated by AP‐1/clathrin‐coated vesicles, requires Rab4, and is negatively regulated by rabaptin‐5/rabex‐5. Introduction
Transport receptors like the transferrin receptor, the low‐density lipoprotein (LDL) receptor, and the asialoglycoprotein (ASGP) receptor cycle continuously between the plasma membrane and early endosomes (Spiess, 1990; Trowbridge et al., 1993). Upon internalization, endocytic vesicles fuse to sorting endosomes. The receptors exit into tubular membranes that form recycling endosomes (or endocytic recycling compartment, ERC), whereas released ligands with the main fluid volume form endosomal carrier vesicles/multivesicular bodies (ECVs/MVBs) to late endosomes (Maxfield and McGraw, 2004). There appear to be two main recycling pathways from early endosomes to the plasma membrane, directly from sorting endosomes or via recycling endosomes (Hao and Maxfield, 2000; Sheff et al., 1999; van Dam et al., 2002). Rab4 and its interactor rabaptin‐5 were implicated in regulating recycling to the cell surface in nonpolarized and to the apical surface in polarized cells based on overexpression and depletion studies in vivo (de Wit et al., 2001; Deneka et al., 2003; Mohrmann et al., 2002; van der Sluijs et al., 1992). We have reconstituted the formation of recycling vesicles in vitro using permeabilized cells (Pagano et al., 2004). The role of candidate proteins can be tested using immunodepleted cytosol or cytosol supplemented with purified proteins or inhibitors. Since the formation of a single cohort of vesicles is analyzed, indirect effects on the organization of endosomes are less likely to affect the results than in in vivo studies. In vitro formation of endosomal vesicles containing the recycling ASGP receptor H1 was found to require Rab4 and to be inhibited by its effector rabaptin‐5/rabex‐5. In Vitro Reconstitution of Endosomal Recycling Vesicles
Overview The basic procedure of the assay is summarized schematically in Fig. 1. Since permeabilized cells, rather than purified endosomes, are used for reconstitution, the specificity for the donor organelle has to be introduced
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FIG. 1. Overview of the procedure to reconstitute the formation of recycling vesicles. For a description see the text. Black membranes contain biotinylated proteins. Recycling receptors are indicated by short lines spanning the membrane (I). G, Golgi; LE, late endosome; Ly lysosome; PM, plasma membrane; RE, recycling endosome; SE, sorting endosome. Other organelles are omitted for simplicity.
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biochemically. (1) The plasma membrane of the cell is biotinylated using a membrane‐impermeant reagent at 4 . (2) Labeled proteins are then allowed to internalize at 37 for 10 min, which limits access to early endosomes. (3) Back at 4 , surface biotin is released with reduced glutathione. Only endocytosed proteins in early endosomes remain labeled. (4) Cells are permeabilized by hypotonic swelling and scraping, and cytosol and small vesicles are washed away. To remove peripherally attached proteins, the membranes are washed with high‐salt buffer. (5) To reconstitute vesicle formation, the permeabilized cells are incubated with exogenous cytosol and energy at 37 . (6) Newly formed vesicles are recovered in the supernatant after pelleting the broken cells and organelles. Upon membrane solubilization, biotinylated proteins, which must have originated from early endosomes, are isolated by avidin precipitation. Among this material, the protein of interest is detected by immunoblot analysis. In the case of a recycling protein like the ASGP receptor, the vesicles that contained them were destined to recycle to the plasma membrane. Protocols Buffer Solutions Buffer A: 0.1 M MES/NaOH, pH 6.6, 0.5 mM MgCl2, 1 mM ethyleneglycoltetraacetic acid (EGTA), 0.2 mM dithiothreitol (DTT). PBSþþ: phosphate‐buffered saline (PBS) with 0.7 mM CaCl2, 0.25 mM MgCl2. Sulfo‐NHS‐SS‐biotin solution: 1 mg/ml sulfosuccinimidyl‐2‐ (biotinamido)‐ethyl‐1,3‐dithiopropionate (Pierce) in PBSþþ, to be prepared immediately before use. Glycine solution: 50 mM glycine in PBSþþ. MEM/HEPES: serum‐free minimal essential medium containing 20 mM HEPES/NaOH, pH 7.2. Glutathione solution: 50 mM reduced glutathione (Sigma), 75 mM NaCl, 75 mM NaOH, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1% bovine serum albumin (Sigma). Dissolve glutathione immediately before use. Iodoacetamide solution: 5 mg/ml iodoacetamide in PBSþþ. Swelling buffer: 15 mM HEPES/KOH, pH 7.2, 15 mM KCl. Transport buffer, 10‐fold concentrated stock solution: 200 mM HEPES/KOH, pH 7.2, 900 mM KOAc, 20 mM Mg(OAc)2. Stripping buffer: 20 mM HEPES/KOH, pH 7.2, 500 mM KOAc, 2 mM Mg(OAc)2.
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Protease inhibitor cocktail, 500‐fold concentrated stock solution: 5 mg/ml benzamidine, 1 mg/ml antipain (both from Sigma), 1 mg/ml pepstatin A, 1 mg/ml leupeptin, 1 mg/ml chymostatin (all from Applichem) in 40% dimethyl sulfoxide (DMSO) and 60% ethanol. Energy regenerating system, four separate stock solutions: 0.1 M ATP; 0.2 M GTP; 0.6 M creatine phosphate; 8 mg/ml creatine kinase (all from Roche Diagnostics). Cytosol Preparation. Cytosol was obtained from calf brain as the high‐ speed supernatant after homogenization as a side product in the purification of clathrin‐coated vesicles (Campbell et al., 1984). The entire procedure is performed at 4 . Calf brains fresh from the slaughterhouse are cleaned of meninges using paper towels and rinsed with PBS. One volume of brain is homogenized with an equal volume of buffer A in a Waring blender (three times 8 s at medium speed) and centrifuged in a Sorvall GS3 rotor at 7000 rpm for 30 min. The supernatant is centrifuged in a Kontron TFT45.94 rotor at 40,000 rpm for 80 min. The clear supernatant is stored frozen in aliquots at 80 to be used as cytosol. Cell Culture. Madin–Darby canine kidney (MDCK) cell line (strain II) stably expressing the ASGP receptor subunit H1 with a C‐terminal myc‐tag (Leitinger et al., 1995) is grown in minimal essential medium supplemented with 2 mM L‐glutamine, 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin, and 0.5 mg/ml G418 sulfate (all from Life Technologies) at 37 with 7.5% CO2. The assay was also used successfully with A431, NIH/3T3 cells, and baby hamster kidney (BHK) cells. For the experiment, 15‐cm cell culture plates are coated by incubation for 30 min at 37 with 10 mg/ml poly‐L‐lysine (10,000–20,000; Fluka) in PBS followed by rinsing twice with PBS. Cells are seeded to be semiconfluent the next day. Surface Biotinylation, Endocytosis, and Surface Stripping. Four 15‐cm plates of semiconfluent cells are washed three times with ice‐cold PBSþþ and biotinylated at 4 for 30 min with 7 ml/plate of freshly prepared sulfo‐ NHS‐SS‐biotin solution. The reaction is quenched by rinsing the cells with cold PBSþþ, by a 5‐min incubation with glycine solution, and by another two rinses with PBSþþ. The cells are then incubated in prewarmed MEM/HEPES for 10 min at 37 to allow endocytosis. The cells are rinsed twice with ice‐cold PBSþþ, incubated twice for 20 min with 7 ml/plate of freshly prepared glutathione solution to release surface biotin, rinsed twice with PBSþþ, incubated for 5 min at 4 with 5 mg/ml iodoacetamide solution to quench any residual glutathione, followed by another two rinses with PBSþþ. Permeabilization and Cytosol Removal. The cells are permeabilized by addition of 10 ml/plate of swelling buffer at 4 for 15 min, scraped with a rubber policeman into 10 ml/plate of transport buffer, pooled, and
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sedimented at 800g for 5 min. At this point essentially all cells are broken as judged by Trypan blue permeability. The pellet is resuspended twice for 10 min in 50 ml cold stripping buffer and centrifuged as before, and resuspended in transport buffer. The permeabilized cells are resuspended in 400 l transport buffer, which should result in a protein concentration of approximately 1 g/l. Vesicle Formation and Analysis. In siliconized Eppendorf tubes, 100 l of permeabilized cells (100 g protein) is incubated with cytosol (final protein concentration of 1.2 mg/ml) and energy‐regenerating system (1.6 mM ATP, 3.2 mM GTP, 9.6 mM creatine phosphate, and 256 g/ml creatine kinase) in a total reaction volume of 250 l with transport buffer at 37 for 30 min. In control reactions, cytosol, energy, or both are omitted. To test the requirement for nucleotide hydrolysis, the nonhydrolyzable nucleotide analogs adenylyl imidodiphosphate (AMP‐PNP; 0.16 mM) and guanylyl imidodiphosphate (GMP‐PNP; 0.32 mM) are added instead of ATP and GTP. Reactions are stopped on ice and the cells sedimented at 800g for 5 min. The supernatants are carefully aspirated and solubilized with lysis buffer (1% Triton X‐100, 0.5% deoxycholate in PBS) containing protease inhibitor cocktail for 1 h at 4 . Insoluble material is removed by centrifugation in a microcentrifuge at 14,000 rpm for 10 min. Supernatants are recovered and rotated end‐over‐end for 1 h at 4 with 40 l avidin‐ Sepharose (Pierce). The beads are washed three times with lysis buffer and boiled in SDS‐gel sample buffer containing 100 mM dithiothreitol. Proteins are separated by SDS‐gel electrophoresis and transferred to a polyvinylidene fluoride membrane that is then processed for immunoblot analysis using anti‐myc antibody (9E10; ATCC). Antibody is detected using horseradish peroxidase‐conjugated anti‐mouse secondary antibody (Sigma) and the ECL kit (Amersham Biosciences). Experimental Results Figure 2A shows the result of a typical reconstitution experiment. No H1‐containing vesicles were generated upon incubation of the permeabilized cells at 37 without additions or when incubated with nucleotides and cytosol at 4 . In the presence of ATP, GTP, and 1.2 mg/ml cytosol at 37 , typically 10% of biotinylated H1 in the starting material was recovered in the supernatant. With increased concentrations of up to 10 mg/ml cytosol, vesicle formation could be further stimulated 2‐fold (Pagano et al., 2004). Using a limiting amount of cytosol provides higher sensitivity to detect effects of depletion or addition of individual components as described below. In the absence of added cytosol, nucleotides supported a basal release of H1 into the supernatant of typically 20% of that in the
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FIG. 2. Temperature‐, energy‐, and cytosol‐dependent in vitro formation of endosomal vesicles containing H1. (A) Surface‐biotinylated cells were incubated at 37 for 10 min to allow internalization, chilled in ice, and surface biotin was stripped with reduced glutathione. The cells were then broken and incubated for 30 min at 4 or 37 , with or without cytosol, or nucleotides and energy‐regenerating system as indicated. Cells were pelleted and the supernatant was lysed and analyzed by avidin precipitation of biotinylated proteins, SDS‐gel electrophoresis, and immunoblotting using anti‐myc antibody. Values were normalized to the amount of total labeled H1 by analyzing 10% of a sample before centrifugation as a standard (10% std). Average and standard deviation of at least four independent determinations are shown. (B) Vesicle formation was assayed in the presence of cytosol (12.5 mg/ml) with GTP or GMP‐PNP (GN), and ATP or AMP‐PNP (AN), or without nucleotides. Reprinted from Molecular Biology of the Cell (Pagano et al., 2004), with permission of The American Society for Cell Biology.
presence of cytosol (Fig. 2A). This is most likely due to residual membrane‐associated proteins that had not been removed by the high‐salt washes. In a time‐course experiment, formation of H1‐containing vesicles was already detectable within 5 min of incubation and reached a maximum after 20 min (Pagano et al., 2004). Up to 30 min, no decrease of the signal was observed, as might be expected if the vesicles would have fused efficiently with their target compartment. The integrity of the membrane vesicles containing the protein of interest can be tested by protease sensitivity, which in the case of H1 showed its cytoplasmic N‐terminus to be exposed, but its transmembrane and exoplasmic domains to be protected, unless detergent was added (Pagano et al., 2004).
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Efficient formation of H1‐containing vesicles required the presence of both ATP and GTP, suggesting the involvement of ATPase(s) and GTPase (s). The nonhydrolyzable analogues AMP‐PNP and GMP‐PNP did not substitute for ATP and GTP, respectively (Fig. 2B), indicating a requirement for nucleotide hydrolysis for the generation of recycling vesicles. Vesicle Formation Using Modified Cytosol
The in vitro assay to reconstitute vesicle formation opens the possibility of testing the role of candidate proteins by modifying the cytosol. The most basic manipulations are the removal of a component by immunodepletion and the supplementation (or readdition) of a purified component to the cytosol. Here we present examples for these procedures. Immunodepletion To immunodeplete a protein from cytosol, a specific antibody may first be incubated with the cytosol before the antibody–antigen complexes are collected with protein A‐ or G‐Sepharose, as appropriate. Alternatively, antibody may first be immobilized on protein A/G‐Sepharose before incubation with cytosol. This is particularly useful when the antibody is dilute. Protein A‐ and G‐Sepharose (Zymed) are saturated with 5 mg/ml bovine serum albumin (fatty acid free; Sigma) overnight at 4 , washed three times, and resuspended in transport buffer as a 50% slurry. To deplete Rab4, 200 l diluted cytosol (750 g protein at 3.75 mg/ml) is first incubated with 15 l of a rabbit anti‐Rab4 antiserum (from Bruno Goud, Institut Curie, Paris), 35 l transport buffer, and protease inhibitor cocktail overnight at 4 , and then mixed with 30 l each of protein A‐ and G‐ Sepharose for 3 h at 4 . After centrifugation, the supernatant is collected and the beads are washed three times with 1 ml transport buffer and boiled with 200 l SDS‐gel loading buffer. Thirty microliters of the immunodepleted cytosol and of the initial diluted cytosol are boiled in SDS‐ gel loading buffer and analyzed, together with 30 l of the material released from the beads, by SDS‐gel electrophoresis and immunoblotting to determine the extent of depletion. To deplete rabaptin‐5 or Rab5, 30 l each of protein A‐ and G‐Sepharose are incubated overnight at 4 either with 500 l transport buffer and 20 l monoclonal mouse anti‐rabaptin‐5 antibody (Transduction Laboratories), or with 1 ml supernatant of a hybridoma‐producing anti‐Rab5 antibody (CL621.3; from Reinhard Jahn, Max Planck Institute, Go¨ ttingen), in the presence of protease inhibitor cocktail. The beads are then washed three
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times with transport buffer, mixed with 200 l diluted cytosol (750 g protein at 3.75 mg/ml), and incubated at 4 for 6 h to overnight. The depleted cytosol is collected and analyzed as above. Mock depletion of cytosol is performed identically with nonimmune antibody or without antibody. To test the effect on vesicle formation, 85 l depleted or mock‐depleted cytosol is incubated with permeabilized cells (100 g protein), energy regenerating system, and transport buffer to a total volume of 250 l, and the released material is analyzed as described above. Protein Purification Rabaptin‐5 is found in the cell in association with rabex‐5, a Rab5 guanine nucleotide exchange factor (Horiuchi et al., 1997). Purification of His6‐tagged rabaptin‐5 in complex with rabex‐5 produced in Sf9 insect cells infected with recombinant baculovirus vectors has been described in detail by Lippe et al. (2001). To test the effect of the purified protein, different amounts are added to standard reconstitution assays. If the buffer of the protein differs from the transport buffer, a buffer‐only control needs to be included. Experimental Results As is shown in Fig. 3A and B, the formation of H1‐containing endosomal vesicles was strongly inhibited by the removal of Rab4 from the cytosol, whereas it was not affected by depletion of more than 90% of Rab5. This confirms previous in vivo observations pointing to an involvement of Rab4 in receptor recycling (Deneka et al., 2003; Mohrmann et al., 2002; van der Sluijs et al., 1992). Immunodepletion of clathrin‐associated adaptor proteins similarly identified AP‐1 as an essential component to generate H1‐containing vesicles (Pagano et al., 2004). Rabaptin‐5/rabex‐5 is an interesting candidate to connect Rab4 function with the AP‐1 vesicle coat, because it was shown to bind to both Rab4 and the ‐ear domain of AP‐1 (in addition to Rab5; de Renzis et al., 2002; Deneka et al., 2003; Vitale et al., 1998). Upon immunodepletion of rabaptin‐5/rabex‐5, formation of endosomal vesicles was reproducibly stimulated (Fig. 3A and C), suggesting an inhibitory role of the complex. This was confirmed upon addition of purified, recombinantly expressed rabaptin‐5/rabex‐5 in amounts corresponding approximately to once or twice the amount already present in the cytosol, which clearly inhibited the production of H1‐containing vesicles (Fig. 3D). The rabaptin‐5/rabex‐5 complex thus negatively regulates generation of recycling vesicles.
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FIG. 3. Formation of recycling vesicles requires Rab4 and is inhibited by rabaptin‐5/rabex‐ 5. Bovine brain cytosol was immunodepleted of Rab4, Rab5, or rabaptin‐5 (A). Total cytosol (T) and corresponding aliquots of the depleted cytosol (unbound fraction, U) and the bound material (B) were analyzed by immunoblotting with the respective antibodies. For (B) and (C), biotinylated permeabilized cells were incubated without cytosol (no cyt), with untreated cytosol (cyt), with cytosol depleted of Rab4, Rab5, or rabaptin‐5 (cyt–Rab4; cyt–Rab5; cyt– Rbpt5), or with mock‐depleted cytosol. For (D), the effect of supplementing 12 or 24 g/ml purified rabaptin‐5/rabex‐5 or of the corresponding buffer was tested. Immunoblot analysis of biotinylated H1 in the supernatant after cell pelleting is shown for a representative experiment. Quantitation of three independent experiments (average with standard deviation) is presented below. Reprinted from Molecular Biology of the Cell (Pagano et al., 2004), with permission of The American Society for Cell Biology.
Conclusions Besides immunodepletion and supplementation of purified proteins, there are additional ways to manipulate the cytosol in the reconstitution reaction. Inhibitory antibodies may simply be added to the cytosol to inactivate the antigen, as was done to block clathrin function using the anti‐heavy chain antibody X22 (Pagano et al., 2004). It should also be possible to use cytosol extracted from cultured cells overexpressing specific wild‐type or mutant proteins or depleted of a gene product by RNA interference. Since the assay focuses on a single process, the risk of indirect effects is likely to be very small. Finally, drugs may be applied to the in vitro reaction. For instance, we found the formation of H1‐containing
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endosomal vesicles to be sensitive to brefeldin A (although at higher concentrations than required in vivo to block Arf1 nucleotide exchange factors) and insensitive to LY294002, an inhibitor of phosphatidylinositol 3‐kinase involved in the fast recycling pathway from sorting endosomes (van Dam et al., 2002). The results thus suggest that our procedure reconstitutes the slow recycling pathway from recycling endosomes. In contrast, in a similar in vitro assay, endosome‐derived vesicles containing transferrin receptor and GLUT4 were generated by a brefeldin A‐insensitive but neomycin‐sensitive mechanism (Lim et al., 2001). Since endocytic proteins had been internalized at 15 , a temperature at which transport from sorting to recycling endosomes is blocked, the starting compartment was predominantly sorting endosomes and the vesicles generated are likely to have represented the fast recycling pathway. Acknowledgments This work was supported by Grant 31‐061579.00 from the Swiss National Science Foundation. We thank Drs. Bruno Goud, Reinhard Jahn, and Marino Zerial for reagents, and Dr. Pascal Crottet for helpful discussions.
References Campbell, C., Squicciarini, J., Shia, M., Pilch, P. F., and Fine, R. E. (1984). Identification of a protein kinase as an intrinsic component of rat liver coated vesicles. Biochemistry 23, 4420–4426. de Renzis, S., So¨ nnichsen, B., and Zerial, M. (2002). Divalent Rab effectors regulate the sub‐ compartmental organization and sorting of early endosomes. Nat. Cell Biol. 4, 124–133. de Wit, H., Lichtenstein, Y., Kelly, R. B., Geuze, H. J., Klumperman, J., and van der Sluijs, P. (2001). Rab4 regulates formation of synaptic‐like microvesicles from early endosomes in PC12 cells. Mol. Biol. Cell 12, 3703–3715. Deneka, M., Neeft, M., Popa, I., van Oort, M., Sprong, H., Oorschot, V., Klumperman, J., Schu, P., and van der Sluijs, P. (2003). Rabaptin‐5alpha/rabaptin‐4 serves as a linker between rab4 and gamma(1)‐adaptin in membrane recycling from endosomes. EMBO J. 22, 2645–2657. Hao, M., and Maxfield, F. R. (2000). Characterization of rapid membrane internalization and recycling. J. Biol. Chem. 275, 15279–15286. Horiuchi, H., Lippe, R., McBride, H. M., Rubino, M., Woodman, P., Stenmark, H., Rybin, V., Wilm, M., Ashman, K., Mann, M., and Zerial, M. (1997). A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin‐5 links nucleotide exchange to effector recruitment and function. Cell 90, 1149–1159. Leitinger, B., Hille‐Rehfeld, A., and Spiess, M. (1995). Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes. Proc. Natl. Acad. Sci. USA 92, 10109–10113. Lim, S. N., Bonzelius, F., Low, S. H., Wille, H., Weimbs, T., and Herman, G. A. (2001). Identification of discrete classes of endosome‐derived small vesicles as a major cellular pool for recycling membrane proteins. Mol. Biol. Cell 12, 981–995.
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Lippe, R., Horiuchi, H., Runge, A., and Zerial, M. (2001). Expression, purification, and characterization of Rab5 effector complex, rabaptin‐5/rabex‐5. Methods Enzymol. 329, 132–145. Maxfield, F. R., and McGraw, T. E. (2004). Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121–132. Mohrmann, K., Leijendekker, R., Gerez, L., and van Der Sluijs, P. (2002). Rab4 regulates transport to the apical plasma membrane in Madin‐Darby canine kidney cells. J. Biol. Chem. 277, 10474–10481. Pagano, A., Crottet, P., Prescianotto‐Baschong, C., and Spiess, M. (2004). In vitro formation of recycling vesicles from endosomes requires adaptor protein‐1/clathrin and is regulated by rab4 and the connector rabaptin‐5. Mol. Biol. Cell 15, 4990–5000. Sheff, D. R., Daro, E. A., Hull, M., and Mellman, I. (1999). The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions. J. Cell Biol. 145, 123–139. Spiess, M. (1990). The asialoglycoprotein receptor—A model for endocytic transport receptors. Biochemistry 29, 10009–10018. Trowbridge, I. S., Collawn, J. F., and Hopkins, C. R. (1993). Signal‐dependent membrane protein trafficking in the endocytic pathway. Annu. Rev. Cell Biol. 9, 129–161. van Dam, E. M., Ten Broeke, T., Jansen, K., Spijkers, P., and Stoorvogel, W. (2002). Endocytosed transferrin receptors recycle via distinct dynamin and phosphatidylinositol 3‐ kinase‐dependent pathways. J. Biol. Chem. 277, 48876–48883. van der Sluijs, P., Hull, M., Webster, P., Male, P., Goud, B., and Mellman, I. (1992). The small GTP‐binding protein rab4 controls an early sorting event on the endocytic pathway. Cell 70, 729–740. Vitale, G., Rybin, V., Christoforidis, S., Thornqvist, P., McCaffrey, M., Stenmark, H., and Zerial, M. (1998). Distinct Rab‐binding domains mediate the interaction of Rabaptin‐5 with GTP‐bound Rab4 and Rab5. EMBO J. 17, 1941–1951.
[9] Assay of Rab4‐Dependent Trafficking on Microtubules By JOHN W. MURRAY and ALLAN W. WOLKOFF Abstract
We present an in vitro method to measure how Rab4 and other regulatory proteins affect microtubule‐based organelle motility. The protocols utilize small‐volume, disposable ‘‘microchambers’’ designed for epifluorescence, confocal, or other microscope platforms and into which microtubules, organelles, and primary and fluorescent secondary antibodies are added. Our work has focused on the isolation and use of endocytic vesicles from rat liver, and we present these protocols. However, the techniques can be adapted for other organelles or cell types. Multiple fluorescent probes, rapid image capture, and immunofluorescence under nonfixation conditions METHODS IN ENZYMOLOGY, VOL. 403 Copyright 2005, Elsevier Inc. All rights reserved.
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Feeley, J. C., Gibson, R. J., Gorman, G. W., Langford, N. C., Rasheed, J. K., Mackel, D. C., and Blaine, W. B. (1979). Charcoal‐yeast extract agar: Primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10, 437–441. Horwitz, M. A. (1983a). Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J. Exp. Med. 158, 1319–1331. Horwitz, M. A. (1983b). The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome lysosome fusion in human monocytes. J. Exp. Med. 158, 2108–2126. Joiner, K. A., Fuhrman, S. A., Miettinen, H. M., Kasper, L. H., and Mellman, I. (1990). Toxoplasma gondii: Fusion competence of parasitophorous vacuoles in Fc receptor‐ transfected fibroblasts. Science 249, 641–646. Kagan, J. C., and Roy, C. R. (2002). Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat. Cell Biol. 4, 945–954. Kagan, J. C., Stein, M. P., Pypaert, M., and Roy, C. R. (2004). Legionella subvert the functions of Rab1 and Sec22b to create a replicative organelle. J. Exp. Med. 199, 1201–1211. Luo, Z. Q., and Isberg, R. R. (2004). Multiple substrates of the Legionella pneumophila Dot/ Icm system identified by interbacterial protein transfer. Proc. Natl. Acad. Sci. USA 101, 841–846. Nagai, H., Kagan, J. C., Zhu, X., Kahn, R. A., and Roy, C. R. (2002). A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295, 679–682. Roy, C. R., and Tilney, L. G. (2002). The road less traveled: Transport of Legionella to the endoplasmic reticulum. J. Cell Biol. 158, 415–419. Roy, C. R., Berger, K., and Isberg, R. R. (1998). Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol. Microbiol. 28, 663–674. Segal, G., Purcell, M., and Shuman, H. A. (1998). Host cell killing and bacterial conjugation require overlapping sets of genes within a 22‐kb region of the Legionella pneumophila genome. Proc. Natl. Acad. Sci. USA 95, 1669–1674. Vogel, J. P., Andrews, H. L., Wong, S. K., and Isberg, R. R. (1998). Conjugative transfer by the virulence system of Legionella pneumophila. Science 279, 873–876. Yamamoto, Y., Klein, T. W., Newton, C. A., Widen, R., and Friedman, H. (1988). Growth of Legionella pneumophila in thioglycolate‐elicited peritoneal macrophages from A/J mice. Infect. Immun. 56, 370–375.
[8] Reconstitution of Rab4‐Dependent Vesicle Formation In Vitro By ADRIANA PAGANO and MARTIN SPIESS Abstract
We have developed an in vitro assay to reconstitute the formation of endosomal recycling vesicles. To achieve specificity for endosomes as the donor organelle, cells are surface‐biotinylated and allowed to endocytose for 10 min, after which the remaining surface‐biotin is stripped off. The cells are then permeabilized and the cytosol washed away. Upon addition METHODS IN ENZYMOLOGY, VOL. 403 Copyright 2005, Elsevier Inc. All rights reserved.
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of exogenous cytosol and energy, sealed vesicles containing biotinylated recycling receptors are produced. Modification of the cytosol, for example, by immunodepletion or addition of purified proteins, allows the identification of proteins involved in vesicle formation. The results show that recycling is mediated by AP‐1/clathrin‐coated vesicles, requires Rab4, and is negatively regulated by rabaptin‐5/rabex‐5. Introduction
Transport receptors like the transferrin receptor, the low‐density lipoprotein (LDL) receptor, and the asialoglycoprotein (ASGP) receptor cycle continuously between the plasma membrane and early endosomes (Spiess, 1990; Trowbridge et al., 1993). Upon internalization, endocytic vesicles fuse to sorting endosomes. The receptors exit into tubular membranes that form recycling endosomes (or endocytic recycling compartment, ERC), whereas released ligands with the main fluid volume form endosomal carrier vesicles/multivesicular bodies (ECVs/MVBs) to late endosomes (Maxfield and McGraw, 2004). There appear to be two main recycling pathways from early endosomes to the plasma membrane, directly from sorting endosomes or via recycling endosomes (Hao and Maxfield, 2000; Sheff et al., 1999; van Dam et al., 2002). Rab4 and its interactor rabaptin‐5 were implicated in regulating recycling to the cell surface in nonpolarized and to the apical surface in polarized cells based on overexpression and depletion studies in vivo (de Wit et al., 2001; Deneka et al., 2003; Mohrmann et al., 2002; van der Sluijs et al., 1992). We have reconstituted the formation of recycling vesicles in vitro using permeabilized cells (Pagano et al., 2004). The role of candidate proteins can be tested using immunodepleted cytosol or cytosol supplemented with purified proteins or inhibitors. Since the formation of a single cohort of vesicles is analyzed, indirect effects on the organization of endosomes are less likely to affect the results than in in vivo studies. In vitro formation of endosomal vesicles containing the recycling ASGP receptor H1 was found to require Rab4 and to be inhibited by its effector rabaptin‐5/rabex‐5. In Vitro Reconstitution of Endosomal Recycling Vesicles
Overview The basic procedure of the assay is summarized schematically in Fig. 1. Since permeabilized cells, rather than purified endosomes, are used for reconstitution, the specificity for the donor organelle has to be introduced
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FIG. 1. Overview of the procedure to reconstitute the formation of recycling vesicles. For a description see the text. Black membranes contain biotinylated proteins. Recycling receptors are indicated by short lines spanning the membrane (I). G, Golgi; LE, late endosome; Ly lysosome; PM, plasma membrane; RE, recycling endosome; SE, sorting endosome. Other organelles are omitted for simplicity.
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biochemically. (1) The plasma membrane of the cell is biotinylated using a membrane‐impermeant reagent at 4 . (2) Labeled proteins are then allowed to internalize at 37 for 10 min, which limits access to early endosomes. (3) Back at 4 , surface biotin is released with reduced glutathione. Only endocytosed proteins in early endosomes remain labeled. (4) Cells are permeabilized by hypotonic swelling and scraping, and cytosol and small vesicles are washed away. To remove peripherally attached proteins, the membranes are washed with high‐salt buffer. (5) To reconstitute vesicle formation, the permeabilized cells are incubated with exogenous cytosol and energy at 37 . (6) Newly formed vesicles are recovered in the supernatant after pelleting the broken cells and organelles. Upon membrane solubilization, biotinylated proteins, which must have originated from early endosomes, are isolated by avidin precipitation. Among this material, the protein of interest is detected by immunoblot analysis. In the case of a recycling protein like the ASGP receptor, the vesicles that contained them were destined to recycle to the plasma membrane. Protocols Buffer Solutions Buffer A: 0.1 M MES/NaOH, pH 6.6, 0.5 mM MgCl2, 1 mM ethyleneglycoltetraacetic acid (EGTA), 0.2 mM dithiothreitol (DTT). PBSþþ: phosphate‐buffered saline (PBS) with 0.7 mM CaCl2, 0.25 mM MgCl2. Sulfo‐NHS‐SS‐biotin solution: 1 mg/ml sulfosuccinimidyl‐2‐ (biotinamido)‐ethyl‐1,3‐dithiopropionate (Pierce) in PBSþþ, to be prepared immediately before use. Glycine solution: 50 mM glycine in PBSþþ. MEM/HEPES: serum‐free minimal essential medium containing 20 mM HEPES/NaOH, pH 7.2. Glutathione solution: 50 mM reduced glutathione (Sigma), 75 mM NaCl, 75 mM NaOH, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1% bovine serum albumin (Sigma). Dissolve glutathione immediately before use. Iodoacetamide solution: 5 mg/ml iodoacetamide in PBSþþ. Swelling buffer: 15 mM HEPES/KOH, pH 7.2, 15 mM KCl. Transport buffer, 10‐fold concentrated stock solution: 200 mM HEPES/KOH, pH 7.2, 900 mM KOAc, 20 mM Mg(OAc)2. Stripping buffer: 20 mM HEPES/KOH, pH 7.2, 500 mM KOAc, 2 mM Mg(OAc)2.
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Protease inhibitor cocktail, 500‐fold concentrated stock solution: 5 mg/ml benzamidine, 1 mg/ml antipain (both from Sigma), 1 mg/ml pepstatin A, 1 mg/ml leupeptin, 1 mg/ml chymostatin (all from Applichem) in 40% dimethyl sulfoxide (DMSO) and 60% ethanol. Energy regenerating system, four separate stock solutions: 0.1 M ATP; 0.2 M GTP; 0.6 M creatine phosphate; 8 mg/ml creatine kinase (all from Roche Diagnostics). Cytosol Preparation. Cytosol was obtained from calf brain as the high‐ speed supernatant after homogenization as a side product in the purification of clathrin‐coated vesicles (Campbell et al., 1984). The entire procedure is performed at 4 . Calf brains fresh from the slaughterhouse are cleaned of meninges using paper towels and rinsed with PBS. One volume of brain is homogenized with an equal volume of buffer A in a Waring blender (three times 8 s at medium speed) and centrifuged in a Sorvall GS3 rotor at 7000 rpm for 30 min. The supernatant is centrifuged in a Kontron TFT45.94 rotor at 40,000 rpm for 80 min. The clear supernatant is stored frozen in aliquots at 80 to be used as cytosol. Cell Culture. Madin–Darby canine kidney (MDCK) cell line (strain II) stably expressing the ASGP receptor subunit H1 with a C‐terminal myc‐tag (Leitinger et al., 1995) is grown in minimal essential medium supplemented with 2 mM L‐glutamine, 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin, and 0.5 mg/ml G418 sulfate (all from Life Technologies) at 37 with 7.5% CO2. The assay was also used successfully with A431, NIH/3T3 cells, and baby hamster kidney (BHK) cells. For the experiment, 15‐cm cell culture plates are coated by incubation for 30 min at 37 with 10 mg/ml poly‐L‐lysine (10,000–20,000; Fluka) in PBS followed by rinsing twice with PBS. Cells are seeded to be semiconfluent the next day. Surface Biotinylation, Endocytosis, and Surface Stripping. Four 15‐cm plates of semiconfluent cells are washed three times with ice‐cold PBSþþ and biotinylated at 4 for 30 min with 7 ml/plate of freshly prepared sulfo‐ NHS‐SS‐biotin solution. The reaction is quenched by rinsing the cells with cold PBSþþ, by a 5‐min incubation with glycine solution, and by another two rinses with PBSþþ. The cells are then incubated in prewarmed MEM/HEPES for 10 min at 37 to allow endocytosis. The cells are rinsed twice with ice‐cold PBSþþ, incubated twice for 20 min with 7 ml/plate of freshly prepared glutathione solution to release surface biotin, rinsed twice with PBSþþ, incubated for 5 min at 4 with 5 mg/ml iodoacetamide solution to quench any residual glutathione, followed by another two rinses with PBSþþ. Permeabilization and Cytosol Removal. The cells are permeabilized by addition of 10 ml/plate of swelling buffer at 4 for 15 min, scraped with a rubber policeman into 10 ml/plate of transport buffer, pooled, and
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sedimented at 800g for 5 min. At this point essentially all cells are broken as judged by Trypan blue permeability. The pellet is resuspended twice for 10 min in 50 ml cold stripping buffer and centrifuged as before, and resuspended in transport buffer. The permeabilized cells are resuspended in 400 l transport buffer, which should result in a protein concentration of approximately 1 g/l. Vesicle Formation and Analysis. In siliconized Eppendorf tubes, 100 l of permeabilized cells (100 g protein) is incubated with cytosol (final protein concentration of 1.2 mg/ml) and energy‐regenerating system (1.6 mM ATP, 3.2 mM GTP, 9.6 mM creatine phosphate, and 256 g/ml creatine kinase) in a total reaction volume of 250 l with transport buffer at 37 for 30 min. In control reactions, cytosol, energy, or both are omitted. To test the requirement for nucleotide hydrolysis, the nonhydrolyzable nucleotide analogs adenylyl imidodiphosphate (AMP‐PNP; 0.16 mM) and guanylyl imidodiphosphate (GMP‐PNP; 0.32 mM) are added instead of ATP and GTP. Reactions are stopped on ice and the cells sedimented at 800g for 5 min. The supernatants are carefully aspirated and solubilized with lysis buffer (1% Triton X‐100, 0.5% deoxycholate in PBS) containing protease inhibitor cocktail for 1 h at 4 . Insoluble material is removed by centrifugation in a microcentrifuge at 14,000 rpm for 10 min. Supernatants are recovered and rotated end‐over‐end for 1 h at 4 with 40 l avidin‐ Sepharose (Pierce). The beads are washed three times with lysis buffer and boiled in SDS‐gel sample buffer containing 100 mM dithiothreitol. Proteins are separated by SDS‐gel electrophoresis and transferred to a polyvinylidene fluoride membrane that is then processed for immunoblot analysis using anti‐myc antibody (9E10; ATCC). Antibody is detected using horseradish peroxidase‐conjugated anti‐mouse secondary antibody (Sigma) and the ECL kit (Amersham Biosciences). Experimental Results Figure 2A shows the result of a typical reconstitution experiment. No H1‐containing vesicles were generated upon incubation of the permeabilized cells at 37 without additions or when incubated with nucleotides and cytosol at 4 . In the presence of ATP, GTP, and 1.2 mg/ml cytosol at 37 , typically 10% of biotinylated H1 in the starting material was recovered in the supernatant. With increased concentrations of up to 10 mg/ml cytosol, vesicle formation could be further stimulated 2‐fold (Pagano et al., 2004). Using a limiting amount of cytosol provides higher sensitivity to detect effects of depletion or addition of individual components as described below. In the absence of added cytosol, nucleotides supported a basal release of H1 into the supernatant of typically 20% of that in the
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FIG. 2. Temperature‐, energy‐, and cytosol‐dependent in vitro formation of endosomal vesicles containing H1. (A) Surface‐biotinylated cells were incubated at 37 for 10 min to allow internalization, chilled in ice, and surface biotin was stripped with reduced glutathione. The cells were then broken and incubated for 30 min at 4 or 37 , with or without cytosol, or nucleotides and energy‐regenerating system as indicated. Cells were pelleted and the supernatant was lysed and analyzed by avidin precipitation of biotinylated proteins, SDS‐gel electrophoresis, and immunoblotting using anti‐myc antibody. Values were normalized to the amount of total labeled H1 by analyzing 10% of a sample before centrifugation as a standard (10% std). Average and standard deviation of at least four independent determinations are shown. (B) Vesicle formation was assayed in the presence of cytosol (12.5 mg/ml) with GTP or GMP‐PNP (GN), and ATP or AMP‐PNP (AN), or without nucleotides. Reprinted from Molecular Biology of the Cell (Pagano et al., 2004), with permission of The American Society for Cell Biology.
presence of cytosol (Fig. 2A). This is most likely due to residual membrane‐associated proteins that had not been removed by the high‐salt washes. In a time‐course experiment, formation of H1‐containing vesicles was already detectable within 5 min of incubation and reached a maximum after 20 min (Pagano et al., 2004). Up to 30 min, no decrease of the signal was observed, as might be expected if the vesicles would have fused efficiently with their target compartment. The integrity of the membrane vesicles containing the protein of interest can be tested by protease sensitivity, which in the case of H1 showed its cytoplasmic N‐terminus to be exposed, but its transmembrane and exoplasmic domains to be protected, unless detergent was added (Pagano et al., 2004).
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Efficient formation of H1‐containing vesicles required the presence of both ATP and GTP, suggesting the involvement of ATPase(s) and GTPase (s). The nonhydrolyzable analogues AMP‐PNP and GMP‐PNP did not substitute for ATP and GTP, respectively (Fig. 2B), indicating a requirement for nucleotide hydrolysis for the generation of recycling vesicles. Vesicle Formation Using Modified Cytosol
The in vitro assay to reconstitute vesicle formation opens the possibility of testing the role of candidate proteins by modifying the cytosol. The most basic manipulations are the removal of a component by immunodepletion and the supplementation (or readdition) of a purified component to the cytosol. Here we present examples for these procedures. Immunodepletion To immunodeplete a protein from cytosol, a specific antibody may first be incubated with the cytosol before the antibody–antigen complexes are collected with protein A‐ or G‐Sepharose, as appropriate. Alternatively, antibody may first be immobilized on protein A/G‐Sepharose before incubation with cytosol. This is particularly useful when the antibody is dilute. Protein A‐ and G‐Sepharose (Zymed) are saturated with 5 mg/ml bovine serum albumin (fatty acid free; Sigma) overnight at 4 , washed three times, and resuspended in transport buffer as a 50% slurry. To deplete Rab4, 200 l diluted cytosol (750 g protein at 3.75 mg/ml) is first incubated with 15 l of a rabbit anti‐Rab4 antiserum (from Bruno Goud, Institut Curie, Paris), 35 l transport buffer, and protease inhibitor cocktail overnight at 4 , and then mixed with 30 l each of protein A‐ and G‐ Sepharose for 3 h at 4 . After centrifugation, the supernatant is collected and the beads are washed three times with 1 ml transport buffer and boiled with 200 l SDS‐gel loading buffer. Thirty microliters of the immunodepleted cytosol and of the initial diluted cytosol are boiled in SDS‐ gel loading buffer and analyzed, together with 30 l of the material released from the beads, by SDS‐gel electrophoresis and immunoblotting to determine the extent of depletion. To deplete rabaptin‐5 or Rab5, 30 l each of protein A‐ and G‐Sepharose are incubated overnight at 4 either with 500 l transport buffer and 20 l monoclonal mouse anti‐rabaptin‐5 antibody (Transduction Laboratories), or with 1 ml supernatant of a hybridoma‐producing anti‐Rab5 antibody (CL621.3; from Reinhard Jahn, Max Planck Institute, Go¨ ttingen), in the presence of protease inhibitor cocktail. The beads are then washed three
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times with transport buffer, mixed with 200 l diluted cytosol (750 g protein at 3.75 mg/ml), and incubated at 4 for 6 h to overnight. The depleted cytosol is collected and analyzed as above. Mock depletion of cytosol is performed identically with nonimmune antibody or without antibody. To test the effect on vesicle formation, 85 l depleted or mock‐depleted cytosol is incubated with permeabilized cells (100 g protein), energy regenerating system, and transport buffer to a total volume of 250 l, and the released material is analyzed as described above. Protein Purification Rabaptin‐5 is found in the cell in association with rabex‐5, a Rab5 guanine nucleotide exchange factor (Horiuchi et al., 1997). Purification of His6‐tagged rabaptin‐5 in complex with rabex‐5 produced in Sf9 insect cells infected with recombinant baculovirus vectors has been described in detail by Lippe et al. (2001). To test the effect of the purified protein, different amounts are added to standard reconstitution assays. If the buffer of the protein differs from the transport buffer, a buffer‐only control needs to be included. Experimental Results As is shown in Fig. 3A and B, the formation of H1‐containing endosomal vesicles was strongly inhibited by the removal of Rab4 from the cytosol, whereas it was not affected by depletion of more than 90% of Rab5. This confirms previous in vivo observations pointing to an involvement of Rab4 in receptor recycling (Deneka et al., 2003; Mohrmann et al., 2002; van der Sluijs et al., 1992). Immunodepletion of clathrin‐associated adaptor proteins similarly identified AP‐1 as an essential component to generate H1‐containing vesicles (Pagano et al., 2004). Rabaptin‐5/rabex‐5 is an interesting candidate to connect Rab4 function with the AP‐1 vesicle coat, because it was shown to bind to both Rab4 and the ‐ear domain of AP‐1 (in addition to Rab5; de Renzis et al., 2002; Deneka et al., 2003; Vitale et al., 1998). Upon immunodepletion of rabaptin‐5/rabex‐5, formation of endosomal vesicles was reproducibly stimulated (Fig. 3A and C), suggesting an inhibitory role of the complex. This was confirmed upon addition of purified, recombinantly expressed rabaptin‐5/rabex‐5 in amounts corresponding approximately to once or twice the amount already present in the cytosol, which clearly inhibited the production of H1‐containing vesicles (Fig. 3D). The rabaptin‐5/rabex‐5 complex thus negatively regulates generation of recycling vesicles.
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FIG. 3. Formation of recycling vesicles requires Rab4 and is inhibited by rabaptin‐5/rabex‐ 5. Bovine brain cytosol was immunodepleted of Rab4, Rab5, or rabaptin‐5 (A). Total cytosol (T) and corresponding aliquots of the depleted cytosol (unbound fraction, U) and the bound material (B) were analyzed by immunoblotting with the respective antibodies. For (B) and (C), biotinylated permeabilized cells were incubated without cytosol (no cyt), with untreated cytosol (cyt), with cytosol depleted of Rab4, Rab5, or rabaptin‐5 (cyt–Rab4; cyt–Rab5; cyt– Rbpt5), or with mock‐depleted cytosol. For (D), the effect of supplementing 12 or 24 g/ml purified rabaptin‐5/rabex‐5 or of the corresponding buffer was tested. Immunoblot analysis of biotinylated H1 in the supernatant after cell pelleting is shown for a representative experiment. Quantitation of three independent experiments (average with standard deviation) is presented below. Reprinted from Molecular Biology of the Cell (Pagano et al., 2004), with permission of The American Society for Cell Biology.
Conclusions Besides immunodepletion and supplementation of purified proteins, there are additional ways to manipulate the cytosol in the reconstitution reaction. Inhibitory antibodies may simply be added to the cytosol to inactivate the antigen, as was done to block clathrin function using the anti‐heavy chain antibody X22 (Pagano et al., 2004). It should also be possible to use cytosol extracted from cultured cells overexpressing specific wild‐type or mutant proteins or depleted of a gene product by RNA interference. Since the assay focuses on a single process, the risk of indirect effects is likely to be very small. Finally, drugs may be applied to the in vitro reaction. For instance, we found the formation of H1‐containing
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endosomal vesicles to be sensitive to brefeldin A (although at higher concentrations than required in vivo to block Arf1 nucleotide exchange factors) and insensitive to LY294002, an inhibitor of phosphatidylinositol 3‐kinase involved in the fast recycling pathway from sorting endosomes (van Dam et al., 2002). The results thus suggest that our procedure reconstitutes the slow recycling pathway from recycling endosomes. In contrast, in a similar in vitro assay, endosome‐derived vesicles containing transferrin receptor and GLUT4 were generated by a brefeldin A‐insensitive but neomycin‐sensitive mechanism (Lim et al., 2001). Since endocytic proteins had been internalized at 15 , a temperature at which transport from sorting to recycling endosomes is blocked, the starting compartment was predominantly sorting endosomes and the vesicles generated are likely to have represented the fast recycling pathway. Acknowledgments This work was supported by Grant 31‐061579.00 from the Swiss National Science Foundation. We thank Drs. Bruno Goud, Reinhard Jahn, and Marino Zerial for reagents, and Dr. Pascal Crottet for helpful discussions.
References Campbell, C., Squicciarini, J., Shia, M., Pilch, P. F., and Fine, R. E. (1984). Identification of a protein kinase as an intrinsic component of rat liver coated vesicles. Biochemistry 23, 4420–4426. de Renzis, S., So¨ nnichsen, B., and Zerial, M. (2002). Divalent Rab effectors regulate the sub‐ compartmental organization and sorting of early endosomes. Nat. Cell Biol. 4, 124–133. de Wit, H., Lichtenstein, Y., Kelly, R. B., Geuze, H. J., Klumperman, J., and van der Sluijs, P. (2001). Rab4 regulates formation of synaptic‐like microvesicles from early endosomes in PC12 cells. Mol. Biol. Cell 12, 3703–3715. Deneka, M., Neeft, M., Popa, I., van Oort, M., Sprong, H., Oorschot, V., Klumperman, J., Schu, P., and van der Sluijs, P. (2003). Rabaptin‐5alpha/rabaptin‐4 serves as a linker between rab4 and gamma(1)‐adaptin in membrane recycling from endosomes. EMBO J. 22, 2645–2657. Hao, M., and Maxfield, F. R. (2000). Characterization of rapid membrane internalization and recycling. J. Biol. Chem. 275, 15279–15286. Horiuchi, H., Lippe, R., McBride, H. M., Rubino, M., Woodman, P., Stenmark, H., Rybin, V., Wilm, M., Ashman, K., Mann, M., and Zerial, M. (1997). A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin‐5 links nucleotide exchange to effector recruitment and function. Cell 90, 1149–1159. Leitinger, B., Hille‐Rehfeld, A., and Spiess, M. (1995). Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes. Proc. Natl. Acad. Sci. USA 92, 10109–10113. Lim, S. N., Bonzelius, F., Low, S. H., Wille, H., Weimbs, T., and Herman, G. A. (2001). Identification of discrete classes of endosome‐derived small vesicles as a major cellular pool for recycling membrane proteins. Mol. Biol. Cell 12, 981–995.
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Lippe, R., Horiuchi, H., Runge, A., and Zerial, M. (2001). Expression, purification, and characterization of Rab5 effector complex, rabaptin‐5/rabex‐5. Methods Enzymol. 329, 132–145. Maxfield, F. R., and McGraw, T. E. (2004). Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121–132. Mohrmann, K., Leijendekker, R., Gerez, L., and van Der Sluijs, P. (2002). Rab4 regulates transport to the apical plasma membrane in Madin‐Darby canine kidney cells. J. Biol. Chem. 277, 10474–10481. Pagano, A., Crottet, P., Prescianotto‐Baschong, C., and Spiess, M. (2004). In vitro formation of recycling vesicles from endosomes requires adaptor protein‐1/clathrin and is regulated by rab4 and the connector rabaptin‐5. Mol. Biol. Cell 15, 4990–5000. Sheff, D. R., Daro, E. A., Hull, M., and Mellman, I. (1999). The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions. J. Cell Biol. 145, 123–139. Spiess, M. (1990). The asialoglycoprotein receptor—A model for endocytic transport receptors. Biochemistry 29, 10009–10018. Trowbridge, I. S., Collawn, J. F., and Hopkins, C. R. (1993). Signal‐dependent membrane protein trafficking in the endocytic pathway. Annu. Rev. Cell Biol. 9, 129–161. van Dam, E. M., Ten Broeke, T., Jansen, K., Spijkers, P., and Stoorvogel, W. (2002). Endocytosed transferrin receptors recycle via distinct dynamin and phosphatidylinositol 3‐ kinase‐dependent pathways. J. Biol. Chem. 277, 48876–48883. van der Sluijs, P., Hull, M., Webster, P., Male, P., Goud, B., and Mellman, I. (1992). The small GTP‐binding protein rab4 controls an early sorting event on the endocytic pathway. Cell 70, 729–740. Vitale, G., Rybin, V., Christoforidis, S., Thornqvist, P., McCaffrey, M., Stenmark, H., and Zerial, M. (1998). Distinct Rab‐binding domains mediate the interaction of Rabaptin‐5 with GTP‐bound Rab4 and Rab5. EMBO J. 17, 1941–1951.
[9] Assay of Rab4‐Dependent Trafficking on Microtubules By JOHN W. MURRAY and ALLAN W. WOLKOFF Abstract
We present an in vitro method to measure how Rab4 and other regulatory proteins affect microtubule‐based organelle motility. The protocols utilize small‐volume, disposable ‘‘microchambers’’ designed for epifluorescence, confocal, or other microscope platforms and into which microtubules, organelles, and primary and fluorescent secondary antibodies are added. Our work has focused on the isolation and use of endocytic vesicles from rat liver, and we present these protocols. However, the techniques can be adapted for other organelles or cell types. Multiple fluorescent probes, rapid image capture, and immunofluorescence under nonfixation conditions METHODS IN ENZYMOLOGY, VOL. 403 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)03009-0