Figures & data
Figure 1. The Rab cycle. (i) GDI extracts inactive Rab proteins from membranes and cycles them between the cytosol and membrane association. (ii) Once membrane bound, the nucleotide is exchanged for GTP with the aid of Guanine nucleotide exchange factors (GEFs) rendering the Rab protein active. GTPase activating proteins (GAPs) increase the generally low intrinsic GTPase activity of Rab proteins to catalyse hydrolysis of GTP to return Rab proteins into their inactive state once their function on the membrane is fulfilled. (iii) In the active state Rab proteins interact with a number of diverse effectors. This Figure is reproduced in color in Molecular Membrane Biology online.
Figure 2. Schematic representation of experimental approach used to investigate Rab membrane targeting. (A) (i) EGFP-Rab fusion proteins were produced by overexpression in HEK293A cells. (ii) The cells were harvested, washed, lysed and the PNS was prepared by centrifugation. (iii) The membrane fraction was then obtained by ultracentrifugation of the PNS. (iv) The resulting membrane pellet was resuspended in buffer by sonication and (v) pelleted again by ultracentrifugation to reduce the amount of excess soluble EGFP-Rab. (vi) The final membrane fraction was then resuspended by brief sonication and the concentration of EGFP-Rab determined based on the EGFP fluorescence in reference to purified Slp1(aa1-116)-EGFP. (B) (i) EGFP-Rab:GDI complexes were formed by extraction of the Rab proteins from the membrane fractions with GDIα. The membrane fraction containing the EGFP-Rab was incubated with GDI, followed by ultracentrifugation to separate the membranes from the solubilized EGFP-Rab. (ii) The resulting membrane pellet was re-solubilized by sonication and (iii) the fluorescence of the membrane fraction and the complex containing supernatant (SN) was compared to determine the yield of the complex. (C) The EGFP-Rab:GDI complex was used in membrane delivery assays. (i) Large vesicles were incubated with the complex and (ii) streptavidin was added to enhance pelleting of the membranes in the following ultracentrifugation step. (iii) The supernatant was then removed and the membrane pellet resuspended in SDS-loading buffer. (iv) The amount of EGFP-Rab in both fractions and the percentage of Rab membrane recruitment was analysed by Western blotting and densitometry. This Figure is reproduced in color in Molecular Membrane Biology online.
Figure 3. Validation of EGFP-Rab:GDI complex formation. (A) Exemplary Western blot of extraction of endogeneous Rab5a and overexpressed EGFP-Rab5a with increasing amounts of GDIα (0–2 μM) from HEK293A membrane fractions showing the supernatant (S) and membrane (P) fractions, probed with αRab5a antibody. (B) Quantification of extraction by densitometry. (C) EGFP-Rab1a:GDIβ co-precipitation. After EGFP-Rab1a extraction from HEK293A membrane fraction with 1 μM GDIβ, his-tagged GDIβ was affinity purified using NiNTA beads. (S, Supernatant of beads; W1/W2, Wash fractions using 250 μl elution buffer; E1/E2, Elution fractions using 250 μl elution buffer containing 200 mM imidazole; B, beads).
Figure 4. Extraction of (A) EGFP-Rab1a and (B) EGFP-Rab5a from HEK293A membrane fractions into the soluble fraction with increasing amount of GDIα. Freshly prepared EGFP-Rab membrane fractions containing 0.6 μM EGFP-Rab were incubated with the indicated concentrations of GDI, followed by separation of the membrane and soluble fractions by ultracentrifugation and quantification of the EGFP fluorescence of both fractions. The percentage of extracted Rab is given by the ratio of the EGFP fluorescence in the supernatant and the total EGFP fluorescence of both fractions.
Figure 5. Membrane delivery of EGFP-Rab1a and EGFP-Rab5a using increasing concentration of lipids (0–2 mM). 0.25 μM EGFP-Rab:GDI complex was incubated with the indicated concentration of lipid vesicles composed of DOPC:DOPE 8:2, containing 1% Biotinyl-Cap-PE. 0.4 µM Streptavidin was added to the mixtures to enhance pelleting of the vesicles by ultracentrifugation. (A) Exemplary Western blots of EGFP-Rab1a and EGFP-Rab5a partitioning between supernatant (S) and membrane (P) fractions with increasing concentration of lipid. (B) Densitometry analysis of Western blots shows the percentage of membrane associated Rab protein and the corresponding membrane saturation curve.
Figure 6. Membrane binding of (A) EGFP-Rab5a and (B) EGFP-Rab1a to vesicles of different lipid compositions. Large vesicles were produced by extrusion of a 2 mM suspension through a 400 nm pore size membrane. Supernatant and membrane fraction were analysed by Western blotting to determine the percentage of membrane partitioning of each Rab by densitometry. The lipid free control shows the amount of non-specific Rab binding to the tube walls. The data is derived from five (Rab5a) or four (Rab1a) separate sets of measurements, each performed in at least duplicates. Error bars represent mean ± SE (standard error).
