It has been shown that the Parkinson's disease (PD)-causing mutation in VPS35 (D620N) causes a reduction in association of the retromer CSC with the WASH complex protein Fam21 (Zavodszky et al., 2014; McGough et al., 2014). Therefore, we wondered if the elevated membrane association of the retromer CSC following TBC1D5 knockdown could enhance the interaction of the PD-causing VPS35 mutant with Fam21. Cells expressing either wild-type or D620N GFP-tagged VPS35 were labelled with heavy and light amino acids, treated with crosslinker and the lysates were immunoprecipitated with anti-GFP. In Fig. 3C, the graph shows that for VPS26, the heavy:light ratio is close to 1 as expected. Knockdown of TBC1D5 results in an increased ratio and, for Fam21, the ratio is decreased, indicating that the loss of TBC1D5 can enhance the association of both wild-type and D620N VPS35 with Fam21. A blot of lysates from TBC1D5-knockdown cells expressing GFP-VPS35 wild type or D620N is shown in Fig. 3D and indicates that enhanced association of Fam21 with both wild-type VPS35 and the D620N mutant is not caused by increased levels of Fam21 after TBC1D5 knockdown.
