1 Introduction

Rho-family small GTPases play key roles in many physiological processes by regulating cytoskeletal organization. The subcellular localization of Rho GTPases is important for their interaction with regulators and effectors and proper signal transduction [1, 2]. Posttranslational lipid modifications of the C-terminal CaaX motif control the subcellular localization of Rho GTPases [3, 4]. In canonical CaaX processing, the cysteine residue of the CaaX motif is modified by a farnesyl or geranylgeranyl isoprenoid, followed by proteolytic cleavage of the -aaX tripeptide and carboxyl methylation of the prenylated cysteine [5]. Prenylation with either a farnesyl or geranylgeranyl group provides Rho GTPases with a membrane anchor.

Rho guanine nucleotide dissociation inhibitors (RhoGDIs) play a regulatory role in the activity and subcellular localization of Rho GTPases [6]. These regulatory proteins were initially identified as cytosolic proteins that bind to the GDP-bound form of Rho GTPases and inhibit their GDP dissociation [7]. However, RhoGDIs also dissociate Rho GTPases from membranes by binding and sequestering the prenyl group within a hydrophobic pocket, forming a stable cytosolic complex [8]. This ability of RhoGDI to extract Rho GTPases from membranes can be demonstrated using a reconstituted system with purified components in which proteoliposomes containing lipid-modified GTPases are incubated with RhoGDI, then fractionated by centrifugation [9, 10]. Rho proteins that are associated with RhoGDI are detected in the supernatant fraction, whereas those that remain associated with liposomes are detected in the pellet (Fig. 1).

Fig. 1
figure 1

Schematic outline of RhoGDIα extraction of proteoliposomes

Full size image

Metabolic labeling with radioactive lipid is a classical assay to detect protein lipidation. Because of potential environmental and health hazards in the handling and disposal of radioisotopes, nonisotopic assays to detect protein lipidation have been developed. Click chemistry involves the copper-catalyzed azide and alkyne cycloaddition (CuAAC) reaction forming a covalent bond between azide and alkyne groups [11, 12]. We previously analyzed the lipid modification of the brain-specific splice variant of Cdc42 (bCdc42) terminating in the CCaX motif using click chemistry-based lipid probes and identified that the CCaX motif of bCdc42 undergoes either canonical CaaX processing or a tandem prenyl–palmitoyl modification [13] (Fig. 1). We found that whereas canonically processed bCdc42 can be extracted from membranes with RhoGDI, the tandem prenyl–palmitoyl form of bCdc42 does not associate with RhoGDI and remains in the membrane. This chapter describes the analysis of RhoGDI extraction of Rho GTPases from membranes using an adaptation of the liposome-based reconstitution assay [9]. The palmitoylated form of the RhoGTPase is labeled with a palmitate analog, 17-octadecynoic acid (17-ODYA) and detected by click chemistry using a fluorescent azide reporter.

2 Materials

2.1 Purification of bCdc42 Labeled with a Clickable Fatty Acid Analogue

  1. 1.

    TriEX Sf9 insect cells (Novagen).

  2. 2.

    TriEX serum-free medium (Novagen).

  3. 3.

    Dialyzed fetal bovine serum (FBS).

  4. 4.

    Baculovirus expressing His-bCdc42 or protein of interest (see Note 1 ).

  5. 5.

    25 mM 17-octadecynoic acid (17-ODYA) in DMSO.

  6. 6.

    Lysis Buffer: 40 mM Hepes–NaOH, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 10 μM GDP.

  7. 7.

    Protease inhibitors: 100 mM PMSF in ethanol (100×), 10 mg/mL Leupeptin in DMSO (1000×).

  8. 8.

    20% sodium cholate.

  9. 9.

    High Salt Buffer A: 20 mM Hepes–NaOH, pH 7.4, 500 mM NaCl, 5 mM MgCl2, 10 μM GDP, 20 mM imidazole, 1% sodium cholate.

  10. 10.

    Elution Buffer A: 20 mM Hepes–NaOH, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 10 μM GDP, 200 mM imidazole, 11 mM CHAPS.

  11. 11.

    Storage Buffer: 20 mM Hepes–NaOH, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 10 μM GDP, 11 mM CHAPS.

  12. 12.

    Cell disruption vessel for nitrogen cavitation (Parr Instrument Co.).

  13. 13.

    Ni-NTA agarose (Qiagen).

  14. 14.

    Amicon Ultra-15, molecular weight cutoff - 10,000.

2.2 Purification of GST-Tagged RhoGDIα from E. coli

  1. 1.

    Bacterial strain E. coli BL21 (DE3) (Novagen).

  2. 2.

    Bacterial expression plasmid for GST-RhoGDIα.

  3. 3.

    1 M isopropyl 1-thio-β-d-galactopyranoside (IPTG).

  4. 4.

    Extraction Buffer: 40 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT.

  5. 5.

    High Salt Buffer B: 20 mM Tris–HCl, pH 7.4, 500 mM NaCl, 1 mM EDTA, 1 mM DTT.

  6. 6.

    Elution Buffer B: 100 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 20 mM glutathione.

  7. 7.

    Dialysis Buffer: 20 mM Hepes–NaOH, pH 7.4, 1 mM EDTA.

  8. 8.

    10% NP-40,

  9. 9.

    1 M MgCl2

  10. 10.

    10 mg/mL lysozyme from chicken egg white in distilled water.

  11. 11.

    1 mg/mL DNase I in distilled water.

  12. 12.

    Glutathione Sepharose 4B.

  13. 13.

    Dialysis tubing, molecular weight cutoff—10,000.

2.3 Liposome Reconstitution of bCdc42

  1. 1.

    10 mM 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in chloroform.

  2. 2.

    10 mM 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) in chloroform.

  3. 3.

    10 mM l-α-phosphatidylinositol (PI) (bovine liver) in chloroform.

  4. 4.

    25 mM cholesterol (ovine) in chloroform.

  5. 5.

    Reconstitution Buffer: 50 mM Hepes–NaOH, pH 7.4, 150 mM NaCl, 5 mM MgCl2.

  6. 6.

    Mini-Extruder equipped with 0.2 μm pore size filter.

  7. 7.

    Vacuum freeze dryer.

2.4 RhoGDIα Extraction and Click Chemistry

  1. 1.

    4 mM Alexa Fluor 488 Azide (Invitrogen) in DMSO.

  2. 2.

    10 mM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) in DMSO.

  3. 3.

    50 mM copper(ii) sulfate in water, prepared just before use.

  4. 4.

    50 mM TCEP HCl in water, prepared just before use.

3 Methods

3.1 Purification of bCdc42 Labeled with Clickable Fatty Acid

  1. 1.

    Culture TriEX Sf9 cells at 27 °C at a density of 0.8 × 106 cells/mL in 100 mL TriEX medium using a 250 mL sterile Erlenmeyer flask (see Note 2 ).

  2. 2.

    The next day, infect cells with 2.5 mL baculovirus stock encoding His-bCdc42 and culture for an additional 48 h (see Note 3 ).

  3. 3.

    Before cells are harvested, label cells with 17-ODYA. Mix 400 μL of 25 mM 17-ODYA with 5 mL of dialyzed FBS. Add this mixture to the cell culture (final 100 μM 17-ODYA), and culture cells for an additional 6 h (see Note 4 ).

  4. 4.

    Harvest cells by centrifugation (5000 × g for 10 min); store cell pellets at −80 °C if purification will be performed later.

  5. 5.

    Resuspend the cell pellet in 40 mL Lysis Buffer with protease inhibitors (see Note 5 ).

  6. 6.

    Disrupt cells by nitrogen cavitation at 500 psi for 30 min (see Note 6 ).

  7. 7.

    Centrifuge the homogenate at 500 × g for 5 min to pellet nuclei, unbroken cells, and large debris. Transfer the supernatant into ultracentrifugation tubes.

  8. 8.

    Resuspend the pellet in 40 mL of Lysis Buffer with protease inhibitors and repeat steps 6 and 7.

  9. 9.

    Collect membranes by ultracentrifugation at 100,000 × g for 45 min (see Note 7 ). Recover the pellets and resuspend in 19 mL of Lysis Buffer using a Dounce homogenizer.

  10. 10.

    Add 1 mL of 20% sodium cholate to the resuspended membranes and rotate for 1 h to extract lipidated bCdc42.

  11. 11.

    Centrifuge at 100,000 × g for 45 min. The supernatant contains the detergent-extracted bCdc42. Check the protein concentration of the supernatant by Bradford assay and adjust to 1 mg/mL.

  12. 12.

    Save an aliquot of the supernatant as Input for later analysis.

  13. 13.

    Mix the supernatant with 250 μL of Ni-NTA agarose (preequilibrated in Lysis Buffer) and 300 μL of 1 M imidazole (final 15 mM), and incubate for 2 h at 4 °C with gentle rotation.

  14. 14.

    Transfer the sample into a column. Collect and save the flow-through for later analysis.

  15. 15.

    Wash the column with 10 mL High-Salt Buffer A.

  16. 16.

    Add 3 mL Elution Buffer A and collect the eluate in 0.5 mL fractions.

  17. 17.

    Analyze the input, flow-through, washes, and elutions by SDS-PAGE. Detect by Coomassie Brilliant Blue (CBB) staining or western blot. Concentrate the peak elution fractions containing His-bCdc42 and buffer exchange with Storage Buffer using an Amicon Ultra-15. Divide the final pool into 20 μL aliquots, snap-freeze in liquid nitrogen, and store at −80 °C.

  18. 18.

    Serially dilute samples and BSA standards and analyze by SDS-PAGE. Stain with CBB and determine sample concentration by densitometric analysis (see Note 8 ).

3.2 Purification of GST-Tagged RhoGDIα from E. coli

  1. 1.

    Culture E. coli BL21 (DE3) transformed with a GST-RhoGDIα expression plasmid in 300 mL LB medium at 30 °C. When the culture reaches an OD600 of 0.5, induce protein expression by adding IPTG to a final concentration of 300 μM. Culture the bacteria for an additional 4 h at 30 °C.

  2. 2.

    Harvest the bacteria by centrifugation (8000 × g for 10 min).

  3. 3.

    Store the pellets at −80 °C until purification.

  4. 4.

    Resuspend the cell pellet in 40 mL of Extraction Buffer with protease inhibitors (see Note 9 ).

  5. 5.

    Mix the homogenate with 2 mL of 10 mg/mL lysozyme and incubate for 10 min with gentle rotation (see Note 10 ).

  6. 6.

    Add 4.2 mL of 10% NP-40 (final 1%), 140 μL of 1 M MgCl2 (final 3 mM), and 400 μL of 1 mg/mL DNase I, and incubate for 10 min with gentle rotation.

  7. 7.

    Disrupt the bacteria by passing the mixture through a 23G needle 15 times.

  8. 8.

    Centrifuge at 100,000 × g for 45 min and save the supernatant.

  9. 9.

    Save an aliquot of the supernatant as Input for later analysis.

  10. 10.

    Mix the supernatant with 1 mL of Glutathione Sepharose 4B (preequilibrated in Extraction Buffer) and incubate for 1 h with gentle rotation.

  11. 11.

    Load the sample into the column; collect and save the flow-through for later analysis.

  12. 12.

    Wash the column resin with 10 mL High Salt Buffer B.

  13. 13.

    Add 6 mL Elution Buffer B and collect the eluate in 1 mL fractions.

  14. 14.

    Analyze the input, flow-through, and fractions by SDS-PAGE by CBB staining.

  15. 15.

    Pool peak elution fractions containing GST-RhoGDIα; dialyze three times for a minimum of 4 h against 500 mL of Dialysis Buffer.

  16. 16.

    Divide the sample into 150 μL aliquots, snap-freeze in liquid nitrogen, and store at −80 °C (see Note 11 ).

3.3 Liposome Reconstitution of bCdc42

Liposomes are prepared with a composition of 35% PE, 25% PS, 5% PI, and 35% cholesterol to mimic the plasma membrane [9], then reconstituted with purified bCdc42. The proteoliposomes are recovered by centrifugation.

  1. 1.

    Add 100 μL of chloroform to a clean glass tube.

  2. 2.

    To make 700 μL of 1 mM liposomes, add 24.5 μL of 10 mM DOPE, 17.5 μL of 10 mM DOPS, 3.5 μL of 10 mM PI, and 9.8 μL of 25 mM cholesterol using a glass syringe.

  3. 3.

    Dry the lipid mixture with a gentle stream of argon gas for 10 min. While under the gas stream, rotate the tube slowly to form a thin lipid film on the wall.

  4. 4.

    Freeze-dry the lipid mixture using a vacuum freeze dryer for 1 h.

  5. 5.

    Resuspend the dry lipid film in 700 μL of Reconstitution Buffer by pipetting up and down.

  6. 6.

    Freeze the hydrated liposome solution in liquid nitrogen and thaw it in a 37 °C water bath. Repeat this freeze–thaw cycle five times to disrupt large multilamellar vesicles.

  7. 7.

    Make unilamellar vesicles using a mini-extruder equipped with a 0.2 μm pore size filter. Pass the liposome solution through the filter 21 times, and collect extruded liposomes in the syringe opposite of the sample loading syringe.

  8. 8.

    Mix 1 μM 17ODYA-labeled bCdc42 with 700 μL of liposome solution, and incubate for 30 min at room temperature with gentle agitation.

  9. 9.

    Centrifuge the samples at 16,000 × g for 20 min at room temperature, and discard the supernatant to remove liposome-unbound bCdc42.

  10. 10.

    Resuspend the liposome pellet in 700 μL of Reconstitution buffer.

  11. 11.

    Divide the liposome solution into 6 × 100 μL aliquots.

3.4 RhoGDIα Extraction and Click Chemistry

  1. 1.

    Add 20 μL of increasing amounts of GST-RhoGDIα (0, 30, 60, 90, 120, and 150 pmol) to each tube containing 100 μL of proteoliposomes. Incubate the samples for 30 min at room temperature with gentle agitation.

  2. 2.

    Centrifuge the samples at 16,000 × g for 20 min at room temperature. Transfer 84 μL of supernatants into new tubes, add 10 μL of 10% SDS, and save as “Unbound” samples.

  3. 3.

    Resuspend the pellet in 120 μL of Reconstitution Buffer. Transfer 84 μL into new tubes, add 10 μL of 10% SDS, and save as “Bound” samples (see Note 12 ).

  4. 4.

    Prepare click chemistry premix: for a 6 μL premix/sample, mix 1 μL of 4 mM Alexa 488 azide, 1 μL of 10 mM TBTA, 2 μL of freshly prepared 50 mM TCEP, and 2 μL of freshly prepared 50 mM CuSO4.

  5. 5.

    To perform click chemistry, mix 94 μL of “Unbound” or “Bound” samples with 6 μL of click chemistry premix.

  6. 6.

    Incubate the samples for 1 h at room temperature in the dark with gentle agitation.

  7. 7.

    Perform a methanol–chloroform precipitation to remove the free Alexa probe (see Note 13 ). Add 400 μL of methanol and vortex. Then add 100 μL of chloroform and vortex.

  8. 8.

    Add 300 μL of water and vortex. Centrifuge at 16,000 × g for 5 min at room temperature.

  9. 9.

    Carefully discard the upper aqueous phase (see Note 14 ).

  10. 10.

    Add 300 μL of methanol and vortex. Centrifuge again.

  11. 11.

    Discard supernatant as completely as possible and air-dry the pellet for 10 min.

  12. 12.

    Resuspend the pellet in 50 μL Binding Buffer.

  13. 13.

    Add 12.5 μL of 5× sample buffer, and boil the samples.

  14. 14.

    Apply the samples to duplicate SDS polyacrylamide gels; one for in-gel fluorescence imaging (Fig. 2a) and the second for CBB staining (Fig. 2b).

Fig. 2
figure 2

(a) Total bCdc42 detected by CBB staining is extracted from the liposome-bound fraction to the unbound (Un) fraction in a RhoGDIα concentration-dependent manner. On the other hand, palmitoylated (17-ODYA-labeled) bCdc42 is detected by in-gel fluorescence only in the liposome bound fraction, suggesting that palmitoylation inhibits bCdc42 binding to RhoGDIα. (b) Quantification of liposome-bound bCdc42. (Figure adapted from Ref. [13])

Full size image

The results are shown in Fig. 2. Total bCdc42 that is detected by CBB staining is moved from the “Bound” fraction to “Unbound” fraction in a RhoGDIα concentration-dependent manner. On the other hand, palmitoylated (17-ODYA labeled) bCdc42 is only detected in “Bound” fraction, consistent with palmitoylation inhibiting bCdc42 binding to RhoGDIα.

4 Notes

  1. 1.

    In our study of bCdc42 [13], the mouse cDNA (NM_001243769) gene is inserted into the baculovirus expression vector pFastBac HT B vector (Invitrogen) to generate N-terminal His-tagged bCdc42. Recombinant baculovirus is generated using the Bac-to-Bac baculovirus expression system (Invitrogen) according to the manufacturer’s instructions.

  2. 2.

    Culture medium volume should be less than 40% of flask size to give sufficient aeration.

  3. 3.

    The volume of virus to add is dependent on the titer of the virus stock. A small-scale expression test should be performed in advance to determine the volume of virus stock to be added.

  4. 4.

    The concentration and incubation time for ODYA labeling is dependent on the protein of interest. A small-scale expression test should be performed in advance.

  5. 5.

    All purification steps are performed at 4 °C after the cell harvest.

  6. 6.

    Nitrogen cavitation is a gentle and efficient method to disrupt mammalian cells. Mechanical homogenization methods using Dounce or Potter-Elvehjem homogenizers can also be used.

  7. 7.

    The supernatant contains nonlipidated bCdc42, which can be purified by Ni-NTA affinity chromatography if desired.

  8. 8.

    We purified more than 300 μg His-bCdc42 from a 100-mL culture.

  9. 9.

    All purification steps are performed at 4 °C after cell harvest.

  10. 10.

    In steps 4–6, bacterial cell walls and DNA are digested using lysozyme and DNase I. Sonication can also be used for bacterial lysis.

  11. 11.

    We purified more than 2.5 mg GST-RhoGDIα from a 300-mL culture.

  12. 12.

    Buffer composition may affect the efficiency of click chemistry. Accordingly, we added SDS to a final concentration of 1% to the “Unbound” samples to make them equivalent to the buffer composition of the “Bound” samples.

  13. 13.

    There is a large fluorescence signal from free Alexa 488 azide that is detected at around 15 kDa on the gel. This signal interferes with detection of the 25 kDa GTPase labeled with ODYA. The methanol–chloroform precipitation removes the free Alexa probe to reduce the background. If the protein of interest is well resolved from the free probe, this step may not be necessary and 5× sample buffer can be added directly to samples after the click chemistry reaction.

  14. 14.

    Protein is precipitated at the interface of upper and bottom layer. Be careful not to disturb the interface.