hRME-6 acts as a rab5GEF through interactions with clathrin-coated pit components. (A, top) Immobilized GST–α-adaptin ear was incubated with HEK293T cytosol and increasing amounts of HEK293T cytosol prepared from cells overexpressing HA–hRME-6^wt as indicated with the total amount of cytosol being maintained constant (120 μg). Bound proteins were detected by Western blotting using anti-HA and anti-AAK1 antibodies. (bottom) Quantitation of HA–hRME-6 versus AAK1 bound proteins. Results are the mean ± the range of three experiments where the values are expressed as a percentage of the maximum HA–hRME-6 and AAK1 bound proteins. (B) Immobilized GST–α-adaptin ear was incubated with cytosol prepared from HEK293T cells overexpressing HA-tagged hRME-6^wt or hRME-6^F1487A. The specific binding of both proteins to GST–α-adaptin ear was quantified from Western blots using anti-HA antibodies. Results are the mean and standard deviation of four experiments where the values are expressed as a percentage of the maximum binding of HA–hRME-6^wt. Asterisk indicates that values are significant at P < 0.05. (C) Immobilized GST–α-adaptin ear was incubated with brain-purified AAK1 (0.75 μg), HEK293T cytosol, or HEK293T cytosol prepared from cells overexpressing HA–hRME-6^wt, preincubated for 15 min at 30°C in the presence (phospho conditions) or absence (dephospho conditions) of 2 mM MgATP, 1 μM microcystin, and 1 mM sodium vanadate. Bound proteins were detected using anti-AAK1 and anti-HA antibodies. (D) SDS gel stained with Sypro Ruby of HA–hRME-6^wt and HA–hRME-6^F1487A affinity purified from HEK293T cells. (E) GEF assays were performed in the presence of 1 mM ATP (regenerating system), 2 μM GST–α-adaptin ear, 8.8 nM HA–hRME-6^wt or HA–RME-6^F1487A, and 120 nM GAPex5 VPS9 domain as indicated. Results are the means ± SD of at least two experiments performed in duplicate.