The reduced colocalisation of Glut1 with Snx1, GM130 and Lamp1 after silencing TBC1D5 in the GFP-VPS35 D620N cells indicates that loss of TBC1D5 can enhance retromer function. We tested whether increased retromer function could enhance protein sorting at endosomes by using two methods. First, we determined whether TBC1D5 knockdown could enhance the colocalisation of retromer cargo proteins (e.g. CIMPR, Atg9a and CD8-SorL1) with the TGN marker protein TGN46. Fig. 6A shows that knockdown of TBC1D5 enhances the colocalisation of the CIMPR with TGN46 although the degree of enhancement is modest and not statistically significant in all the cell lines tested. The colocalisation of Atg9a, a protein that cycles through the endocytic system to the TGN, is also enhanced after TBC1D5 knockdown (see Fig. 6B), a gain that is not due to changes in levels of Atg9a (see Fig. S1D,E). It is worth noting, however, that increased Atg9a and TGN46 colocalisation is not statistically significant in cells expressing the VPS35 D620N mutant that causes impaired Atg9a trafficking (Zavodszky et al., 2014). Not all retromer cargo proteins respond to TBC1D5 knockdown equally
