Fig. 5

Multimolecular complex formation including SMCT1-PDZK1-URAT1. GST-fused SMCT1-CTwt was incubated together with in vitro translated full length PDZK1 and MBP-fused URAT1-CTwt in a Glutathione Sephrarose affinity column and were eluted and analyzed by SDS PAGE. The full length PDZK1 PCR product was in vitro-translated in the presence of Transcend biotinylated lysine tRNA (Promega). The in vitro translated products were incubated with MBP-URAT1-CTwt (lane 1), GST alone (lane 2), positive control: GST-URAT1-CTwt alone (lane 5), GST-SMCT1-CTwt (lane 6), GST-SMCT1-CTwt and MBP-URAT1-CTwt (lane 7), negative controls were GST alone and MBP-URAT1-CTwt without PDZK1 (lane 3) and GST-SMCT1-CTwt and MBP-URAT1-CTwt without PDZK1 (lane 4). MBP-fused URAT1 could only be precipitated in the presence of PDZK1 implicating the role of PDZK1 as a tether between SMCT1 and URAT1 in vitro. The input corresponds to the crude in vitro translation reaction and MBP-URAT1-CT lysate. The positions of molecular mass standards are shown on the left