Two Distantly Spaced Basic Patches in the Flexible Domain of Huntingtin-Interacting Protein 1 (HIP1) Are Essential for the Binding of Clathrin Light Chain
Figure 3
Two basic patches in HIP1 are critical for
binding clathrin light chain. Western
blots were performed to analyze GST-pulldown experiments. Samples were run on
the same gel, but after transfer, the membrane was cut apart and blotted with
the appropriate antibody. Bovine
clathrin heavy chain (aa1074-1675, Hub) with an N-terminal histidine-tag was
blotted with a commercial anti-His monoclonal antibody (Anti-His). The neuronal form of clathrin light chain b
(LCb) had an N-terminal histidine-tag, but we used CON.1 to identify LCb. The GST alone and GST-HIP1h constructs were
detected using a commercial monoclonal antibody against GST (Anti-GST). Panel A: lanes 1 and 2: GST alone negative controls
show that there are no nonspecific interactions with 6Hishub alone or
6Hishub + 6HisLCb. Lane 3: wild type
GST-HIP1h pulldown of 6Hishub alone (no bound LCb) indicates that the
interaction needs light chain. Lane 4:
positive control, wild type GST-HIP1h with 6Hishub/6HisLCb. Lanes 5–9: pulldowns of HIP1h single and double mutants
with 6Hishub/6HisLCb. Panel B: lanes 1–3 are controls as
described in panel A. Lane 4: positive
control, wild type GST-HIP1h with 6Hishub/6HisLCb. Lane 5: GST-HIP1h K494A with
6Hishub/6HisLCb. Lane 6: GST-HIP1h K474A
with 6Hishub/6HisLCb.
