We have previously reported that overexpression of TBC1D5 leads to reduced association of the retromer CSC with endosomes (Seaman et al., 2009). We repeated our previous observation and show that transient overexpression of wild-type GFP-tagged TBC1D5 results in reduced punctate endosomal staining of both VPS35 and VPS26 (Fig. 1A, asterisks). Previous structural studies revealed that critical residues are required for the activity of TBC proteins (Pan et al., 2006). Expression of the catalytically inactive TBC1D5 RQ mutant does not generate the same dominant-negative effect, however, suggesting that the TBC1D5 protein is exerting its effect through a Rab GTPase. As overexpression of TBC1D5 is deleterious to the endosomal localisation of the retromer CSC, we next tested whether knockdown of TBC1D5 could enhance membrane association. Control cells were mixed with TBC1D5-knockdown cells and then stained with antibodies against TBC1D5 and either VPS35 or VPS26 (Fig. 1B). Loss of TBC1D5 appears to lead to somewhat brighter staining of VPS35 and VPS26 (asterisks). To more quantitatively investigate the effect of loss of TBC1D5 on the membrane association of the retromer CSC, we used automated
