Biochemical measurement of Rab5 activity

Abstract 

Receptor endocytosis and recycling of the transferrin receptor (TfR) are crucial processes for cellular iron homeostasis.  and involve a well-coordinated series of events. The small GTPase Rab5 controls the early endocytic pathway of transferrin (Tfn), and its deactivation is necessary for Tfn recycling. This deactivation is facilitated by RabGAP5, a GTPase-activating protein, on the endosomes. We report that the recruitment of RabGAP5 alone is insufficient to deactivate Rab5. Instead, developmentally regulated GTP-binding protein 2 (DRG2) is necessary for Rab5 deactivation and transferrin (Tfn) recycling. The spatio-temporal dynamics of Rab5 activity are crucial for TfR recycling. We measured Rab5 activity using a Rab5-GTP-specific antibody and assessed the catalytic activity of DRG2 on Rab5 using a GTPase activity assay.

Materials

Cell lines 

MCF7: (ATCC, HTB-22D) were maintained in media composed of RPMI 1640 Medium (Gibco # 11875135) with 10 % FBS (Gibco # 1600-044) at 37 °C with 5% CO₂. Cells were maintained in an antibiotic’s free environment. For subculture cells were dissociated with TrypLE Express (Thermo Fisher Scientific #12605-010). All cell lines used in this study were not used beyond passage 30 from original derivation.  Cell lines were routinely tested for mycoplasma contamination.

Reagents

Plasmids 

mRFP-Rab5, addgene, (Plasmid #14437), Rab5GAP-EGFP ¹

Purified recombinant proteins

Purified recombinant proteins glutathione S-transferase (GST)–Rab5 and GST-DRG2 were purchased from Bioclone (San Diego, CA).

Antibodies

Anti-Rab5GTP Monoclonal Antibody (used at a 1:500 (vol/vol) ratio, (26911; NewEast Biosciences). 

anti-GFP (G10362; Invitrogen)

anti-DsRed polyclonal antibody (632496; Clontech). 

Dynabeads (11041; thermofischer scientific)

Other 

GTP, [α-32P]- 3000Ci/mmol 10mCi/ml , 250 µCi (part no. BLU006H250UC, Revvity)

PEI Cellulose F plates (VWR Canlab, Catalog #CAM05725‐01)

100 μM GTP-γ-S stock (NewEast Biosciences, 30302).

complete, EDTA free Protease inhibitor Cocktail (Roche; 118735800)

Phosphatase Inhibitor Cocktail (100X) ( Cell signaling technology: 5870)

IP lysis buffer 

50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, 1% Triton X-100, with protease and phosphatase inhibitors.

GTPase activity assay 

To measure the GAP activity DRG2 on Rab5 with [α‐³²P]. 

In brief, this assay measures the hydrolysis of GTP by Rab proteins by quantifying the amount of GDP produced. The assay consists of three steps. First, the Rab5 protein is loaded with [α-³²P] GTP. Second, the hydrolysis reaction converts the bound [α-³²P] GTP to [α-³²P] GDP. Finally, the [α-³²P] GTP and [α-³²P] GDP are separated using thin-layer chromatography (TLC).

 Purified recombinant proteins glutathione s-transferase tagged Rab5 and DRG2. 

1. 200 nM of Rab5 protein was loaded with 0.25 µCi of [α-³²P] for 5 min at room temperature in 30 µl of of the loading buffer (0.002 M Tris-HCl, pH 8, 0.001 M EDTA, and 0.001 M DTT). 

2. The reaction began with the addition of MgCl2 to reach a final concentration of 0.005 M. It was performed at 37 °C for 20 minutes, either with or without 20 nM DRG2.

3. The reaction was stopped by adding a 5× stop solution containing 0.2% SDS, 2 mM EDTA, 2 mM DTT, 0.5 mM GTP, and 0.5 mM GDP.

4. Samples were separated using thin-layer chromatography on polyethyleneimine-cellulose sheets (J. T. Baker) with 0.75 M KH2PO4 (pH 3.5) as the developing solvent. Autoradiography and PhosphorImager analysis (Molecular Dynamics) were then used to quantify the radioactive counts of the GTP and GDP spots, enabling the calculation of GDP production. The phosphorus content of GDP was adjusted to be two-thirds that of GTP. ²

    

Rab5-GTP pull-down assay

1. Day 0: MCF7 (scRNA or shDRG2) stable cells were seeded at a density of 2.2 × 10⁶ in a 10 cm dish.

2.  Day 1: After 16 hours, the cells were transfected with 8 µg of mRFP-Rab5 and 5 µg of EGFP, or 8 µg of mRFP-Rab5 and 8 µg of EGFP-Rab5GAP, using Lipofectamine 2000. The media was replaced 4 hours later.

3. Day 2: After 16 hours, the cells were washed three times with PBS and lysed in 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 1% Triton X-100, with protease and phosphatase inhibitors.

4. The lysate was transferred to a new tube, incubated on ice for 15 minutes, and resuspended 10 times using a pipette.

5. The lysate was centrifuged for 30 minutes at 14,000 rpm at 4°C.

6. An aliquot was saved as the initial input (~10% vol/vol), and approximately 0.3 mg of proteins in 300 µl of IP buffer were incubated with anti-Rab5-GTP or Normal Mouse IgG on Dynabeads for 30 minutes (wash the Dynabeads in the IP lysis buffer three times before antibody incubation).

7. The beads were then washed with 1 ml of IP buffer and incubated with the lysate for 3 hours at 4°C with rotation. 

   Note: Conduct a separate reaction in the presence of 10 μM GTP-γ-S by incubating it with the cell lysate overnight at 4°C before incubating the sample with the respective beads to control for specific binding (positive control).

8. The beads were washed three times with IP lysis buffer.

9. The samples were eluted using 2× sample buffer by boiling at 100°C for 10 minutes with a reducing agent.

10. The amount of Rab5-GTP was determined by SDS-PAGE followed by blotting with an anti-RFP antibody. Amount of GFP/GFP Rab5GAP was measure by blotting with an anti-GFP antibody. 

     

References

1. Mani, M., Lee, U.H., Yoon, N.A., Kim, H.J., Ko, M.S., Seol, W., Joe, Y., Chung, H.T., Lee, B.J., Moon, C.H., et al. (2016). Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling. Mol Biol Cell 27, 334-348. 10.1091/mbc.E15-08-0558.

2. Kebin Liu and Guangpu Li (1998) Catalytic Domain of the p120 Ras GAP Binds to Rab5 and Stimulates Its GTPase Activity* J Biol Chem.273(17),10087-90. doi: 10.1074/jbc.273.17.10087.