Figure 2. DENND1A/1B are GEFs for Rab35. (A) Human Rabex-5 was tested against a representative panel of human Rab proteins using the GDP-releasing assay. In brief, 10 µg of each GST-tagged Rab to be tested was incubated in 50 mM Hepes-NaOH, pH 6.8, 0.1 mg/ml BSA, 125 µM EDTA, 10 µM Mg-GDP, and 5 µCi [3H]-GDP (10 mCi/ml; 5,000 Ci/mmol) in a total volume of 200 µl for 15 min at 30°C to load the Rab with the radioactive GDP probe. For standard GDP-releasing GEF assays, 100 µl of the loading reaction was then mixed with 10 µl of 10 mM Mg-GTP and 10 nM His6-tagged Rabex-5 purified from bacteria or a buffer control, then adjusted to 120 µl final volume with assay buffer. The GEF reaction occurred for 20 min at 30°C. After this, 2.5 µl was taken for a specific activity measurement; the remainder was split into two tubes, then incubated with 500 µl of ice-cold assay buffer containing 1 mM MgCl2 and 20 µl of packed glutathione-sepharose for 60 min at 4°C to separate Rab–GDP complexes from free “released” GDP. After washing three times with 500 µl of ice-cold assay buffer, the sepharose was transferred to a vial containing 4 ml of scintillation fluid and counted. The amount of nucleotide exchange was calculated in pmoles of GDP released. (B and C) A representative panel of human Rab proteins was tested against 10 nM of His6-tagged DENND1B-S in the GDP-releasing (B) or GTP-binding assay (C). For GTP-binding assays, the following modifications were made: only unlabeled GDP was used in the loading reaction; in the GEF reaction, 0.5 µl of 10 mM GTP and 1 µCi [35S]-GTPγS (10 mCi/ml; 5000 Ci/mmol) were used. The amount of nucleotide exchange was calculated in pmoles of GTP bound. (D and E) Human DENND1A (D), DENND1B-L (D), and DENND1C (E) were tested against a subset of Rab35-related Rabs using the GTP-binding assay. For these assays, 10 nM of FLAG-tagged DENND1A or DENND1C purified from HeLa cells, or 10 nM of His6-tagged DENND1B-L purified from bacteria were used. Errors bars show the standard error from the mean. The red line marks double the median value taken as a threshold.
Figure 2.

DENND1A/1B are GEFs for Rab35. (A) Human Rabex-5 was tested against a representative panel of human Rab proteins using the GDP-releasing assay. In brief, 10 µg of each GST-tagged Rab to be tested was incubated in 50 mM Hepes-NaOH, pH 6.8, 0.1 mg/ml BSA, 125 µM EDTA, 10 µM Mg-GDP, and 5 µCi [3H]-GDP (10 mCi/ml; 5,000 Ci/mmol) in a total volume of 200 µl for 15 min at 30°C to load the Rab with the radioactive GDP probe. For standard GDP-releasing GEF assays, 100 µl of the loading reaction was then mixed with 10 µl of 10 mM Mg-GTP and 10 nM His6-tagged Rabex-5 purified from bacteria or a buffer control, then adjusted to 120 µl final volume with assay buffer. The GEF reaction occurred for 20 min at 30°C. After this, 2.5 µl was taken for a specific activity measurement; the remainder was split into two tubes, then incubated with 500 µl of ice-cold assay buffer containing 1 mM MgCl2 and 20 µl of packed glutathione-sepharose for 60 min at 4°C to separate Rab–GDP complexes from free “released” GDP. After washing three times with 500 µl of ice-cold assay buffer, the sepharose was transferred to a vial containing 4 ml of scintillation fluid and counted. The amount of nucleotide exchange was calculated in pmoles of GDP released. (B and C) A representative panel of human Rab proteins was tested against 10 nM of His6-tagged DENND1B-S in the GDP-releasing (B) or GTP-binding assay (C). For GTP-binding assays, the following modifications were made: only unlabeled GDP was used in the loading reaction; in the GEF reaction, 0.5 µl of 10 mM GTP and 1 µCi [35S]-GTPγS (10 mCi/ml; 5000 Ci/mmol) were used. The amount of nucleotide exchange was calculated in pmoles of GTP bound. (D and E) Human DENND1A (D), DENND1B-L (D), and DENND1C (E) were tested against a subset of Rab35-related Rabs using the GTP-binding assay. For these assays, 10 nM of FLAG-tagged DENND1A or DENND1C purified from HeLa cells, or 10 nM of His6-tagged DENND1B-L purified from bacteria were used. Errors bars show the standard error from the mean. The red line marks double the median value taken as a threshold.

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