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Results: 5

1.
Figure 2

Figure 2. From: Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex.

Rabaptin-5 enhances Rabex-5 GEF activity. GDP to GTP nucleotide exchange on Rab5 was measured by filter binding assay in the presence of 5 μM [32P]GTPγs for the indicated times. This was achieved with 1.5 μM Rab5 alone (▴) or with 100 nM Rabex-5 (✦), 100 nM Rabex-5 and 100 nM Rabaptin-5 (●), or 100 nM Rabaptin-5/Rabex-5 complex (▪).

Roger Lippé, et al. Mol Biol Cell. 2001 July;12(7):2219-2228.
2.
Figure 5

Figure 5. From: Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex.

Rabaptin-5/Rabex-5 complex is essential for homotypic early endosome fusion. Rescue of cytosol depleted of endogenous Rabaptin-5/Rabex-5 complex was investigated in the homotypic early endosome fusion assay. (A) Rabaptin-5/Rabex-5 complex is needed for fusion. Low physiological concentrations of recombinant Rabaptin-5 (lanes 3–5), Rabex-5 (lanes 6–8), or complex (lanes 9–11) were used for the rescue. (B) Rabaptin-5/Rabex-5 complex must be preformed to be functional. Rabaptin-5 (150 nM) (lane 4), 150 nM Rabaptin-5, and 30 nM Rabex-5 (lane 5) or 150 nM Rabaptin-5/30 nM Rabex-5 complex (lane 6) was added to the test tubes. (C) Excess of Rabex-5 overrides the need for the Rabaptin-5/Rabex-5 complex. Rabaptin-5 (100 nM) (lane 4), 50 or 300 nM Rabex-5 (lanes 5–6), and 100 nM recombinant Rabaptin-5/Rabex-5 complex (lane 7) was, respectively, tested.

Roger Lippé, et al. Mol Biol Cell. 2001 July;12(7):2219-2228.
3.
Figure 3

Figure 3. From: Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex.

Recruitment of Rabaptin-5 and Rabex-5 on early endosomes. (A) Recruitment of the Rabaptin-5/Rabex-5 complex. The recruitment of Rabaptin-5, Rabex-5, and Rabaptin-5/Rabex-5 complex was evaluated by incubation of 35S-labeled in vitro transcribed/translated proteins with purified early endosomes for 30 min at 37°C. This was performed in the presence of 1 mM GTP or 1 μM GDI plus 1 mM GDP to determine the Rab5 dependence of the recruitment. After the recovery of the endosomes by flotation, the endosomes were pelleted and loaded onto a 12% SDS-PAGE gel and analyzed by autoradiography. (B) Western. Essentially, the above-described gels were cut below the 50-kDa molecular mass marker before drying and the lower part of the gels transferred to nitrocellulose. Removal of Rab5 by GDI was confirmed with the use of an α Rab5 antibody. As loading control, the amount of an integral protein present on endosomes was determined by Western with a Syntaxin 6-specific antibody.

Roger Lippé, et al. Mol Biol Cell. 2001 July;12(7):2219-2228.
4.
Figure 4

Figure 4. From: Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex.

Rabaptin-5 recruitment is dependent on its association to Rabex-5. (A) In vitro formation of Rabaptin-5/Rabex-5 complex. To test whether Rabaptin-5 and Rabex-5 can form a complex in the test tube during the recruitment incubations, we pooled Rabaptin-5 and Rabex-5 for 5 or 30 min at 37°C in the conditions used for the recruitment (ATP-regenerating system, 1 mM GTP in the in vitro fusion assay buffer). The samples were then immunoprecipitated with preimmune, α Rabaptin-5 or α Rabex-5 serum (as described in MATERIALS AND METHODS). The controls included the incubation of a sample left at 4°C for 30 min (i.e., 0 min at 37°C) as well as the immunoprecipitation of Rabaptin-5/Rabex-5 complex preformed in vivo. (B) GTPγS-loaded Rab5 fails to recruit uncomplexed Rabaptin-5. To evaluate whether the recruitment of Rabaptin-5 is solely dependent on the activation of Rab5 by Rabex-5, we performed the recruitment experiments as in Figure 3 in the presence of 1 mM GTP or GTPγS.

Roger Lippé, et al. Mol Biol Cell. 2001 July;12(7):2219-2228.
5.
Figure 1

Figure 1. From: Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex.

Purification of functional recombinant Rabaptin-5/Rabex-5 complex. (A) Recombinant protein expression. Histidine-tagged Rabaptin-5 or Rabex-5 was expressed in High Five insect cells with the use of recombinant baculoviruses as described in MATERIALS AND METHODS. In addition, histidine-tagged Rabaptin-5 and untagged Rabex-5 baculoviruses were coexpressed to produce a complex in vivo. All recombinant proteins were purified in a single step via a nickel agarose affinity column. The purified proteins were then loaded onto a 12% SDS-PAGE gel and stained with Coomassie. The molecular mass markers are indicated to the left of the gels, whereas the position of the recombinant proteins (∗, Rabaptin-5; ∗∗, Rabex-5) is shown by arrows. (B) Coexpression of the two proteins results in the formation of a complex in vivo. The recombinant complex purified from insect cells coexpressing the two proteins was immunoprecipitated with preimmune, α Rabex-5 or α Rabaptin-5 serum as detailed in MATERIALS AND METHODS. Both the washed beads and the supernatants were loaded onto a 12% SDS-PAGE gel and Coomassie stained. A minimal immunoprecipitation was observed with the preimmune anti body. (C) Homotypic early endosome fusion assay. Endosomes were purified and incubated in the presence of cytosol and energy, unless otherwise indicated. In lanes 4–7, the endogenous native Rabaptin-5/Rabex-5 complex was immunodepleted from the cytosol. The rescue of the depleted cytosol by 100 nM recombinant Rabaptin-5 (lane 5) or Rabaptin-5/Rabex-5 complex purified from insect cells coexpressing the two proteins (lane 6) was compared with the rescue by 100 nM purified native Rabaptin-5/Rabex-5 complex (lane 7).

Roger Lippé, et al. Mol Biol Cell. 2001 July;12(7):2219-2228.

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