PD-causing VPS35 D620N mutant. The interaction of Fam21 with the retromer CSC requires the binding of VPS29 to VPS35, and TBC1D5 interacts with VPS29 (Helfer et al., 2013; Harbour et al., 2010). Thus, it is possible that the gain in association of the retromer CSC with Fam21 after loss of TBC1D5 expression could be due to a reduction in steric hindrance imposed by TBC1D5 binding to VPS29. However, we feel that this is unlikely as GFP-tagged versions of TBC1D5 and the Fam21 tail both coimmunoprecipitate not only retromer proteins but Fam21 (and other WASH complex proteins) and TBC1D5, respectively (Freeman et al., 2014). Additionally, proteomic analyses have revealed that expression levels of Fam21 and TBC1D5 are a fraction of that of retromer proteins (i.e. VPS35 or VPS29) (Itzhak et al., 2016) and thus loss of TBC1D5 expression will not markedly increase binding sites for Fam21 on the retromer CSC as the retromer CSC already outnumbers TBC1D5 and Fam21 by approximately 60:1 and 20:1, respectively (see Fig. S3).
