Figure 2.
GEF activity of Rabex-5 and mutants in vitro and in vivo. (A) Purified Rabex-5(1-399), Rabex-5(135-399), Rabex-5(135-480), and Rabex-5(135-480)/Rabaptin-5(572-641) complex were examined for their ability to stimulate the loading of [35S]GTPγS onto Rab5-GDP. The reaction without any of the Rabex-5 constructs (Rab5 alone) served as a negative control. Samples were taken at the indicated times, and the amount of [35S]GTPγS bound to Rab5 in each case was determined by the filter binding assay. The results were reproducible in two independent experiments. (B) FLAG-tagged Rab5 was coexpressed in BHK cells with either the pBI vector control or pBI constructs expressing Rabex-5(1-135), Rabex-5, Rabex-5(135-399), Rabex-5(135-480), Rabex-5(135-480)/Rabaptin-5, Rabex-5(81-399) as indicated. Top, amount of Rab5-GTP in each case as determined by GST-R5BD pull-down assay, followed by immunoblot analysis with the anti-FLAG antibody. Middle, total amount of Rab5 in each cell lysate used for the pull-down assay as determined by immunoblot analysis of the lysate directly (1% of the amount for the pull-down assay) with the anti-FLAG antibody. Bottom, expression of the indicated Rabex-5 constructs (Myc-tagged) in the cell as determined by immunoblot analysis with the anti-Myc antibody. Molecular mass standards (in kilodaltons) are indicated on the left. The results were reproducible in three experiments. (C) Ratio of Rab5-GTP over total Rab5 quantified by densitometry of the immunoblots in B. Error bars indicate SEM in three experiments.