(E) Representative images of the pilot validation screen for the negative control (no siRNA) and for five sets of siRNA-treated cells with reduced TGN retrieval ratio. Different phenotypes that give rise to reduced TGN retrieval ratios are discussed in the text. Scale bar, 100 μm (top two rows) and 38 μm (bottom row).(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and SFT2D2 KD cells (bottom).(B) Cells stably expressing SFT2D2-Myc show localization of SFT2D2 to perinuclear membranes and endosomes, including VPS35-positive endosomes (arrowheads in inset). Treatment with nocodazole disperses the endosomes and even more clearly shows colocalization of SFT2D2-Myc and VPS35 (arrowheads in inset).(C) Cells stably expressing SFT2D2-Myc were costained with antibodies against various post-Golgi SNARE proteins. Colocalization was quantified by Pearson's correlation coefficient (right-hand graph) and indicates very extensive colocalization with STX6, STX7, and VAMP8. The image panels illustrate colocalization of SFT2D2-Myc and STX5 at the Golgi (top, arrowhead) but much more extensive colocalization of SFT2D2-Myc and STX6 (bottom, arrowheads). In (B) and (C), the white dashed box delineates the area magnified in the insets.(D) SNARE protein staining was compared for control and SFT2D2 KD HeLa cells and quantified. The graph shows the change in cellular intensity measured for each post-Golgi SNARE investigated. Images illustrate the increased cellular intensity of VAMP3 in SFT2D2 KD cells.

(E) Control and SFT2D2 KD HeLa cell lysates were separated by LDS-PAGE and blotted for the indicated SNARE proteins or actin.

(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and ZDHHC5 KD cells (bottom).(B) Cells stably expressing ZDHHC5-Myc show localization of ZDHHC5 to the plasma membrane and to intracellular tubules and vesicles. Some colocalization between ZDHHC5 and retromer VPS35 is observed (arrowheads in inset). Following nocodazole treatment endosomes are dispersed and some are labeled with ZDHHC5 and VPS35 (arrowheads in inset).(C) Control and ZDHHC5 siRNA-treated SFT2D2-Myc cells were mixed and stained for ZDHHC5, Myc, and VPS35. KD cells are marked by an asterisk.(D) Control and ZDHHC5 siRNA-treated HeLa cells were mixed and stained for ZDHHC5, α5-integrin, and VPS35. KD cells are indicated with an asterisk.(E) Control (top) and ZDHHC5 KD (bottom) HeLa cells were fixed and stained for TGN46 and β1-integrin.(A) Primary screen anti-CD8 antibody uptake images for control cells (top) and GRINA KD cells (bottom).(B) Control (top) and GRINA KD (bottom) HeLa cells were stained for Golgi marker GM130 and Golgi glycoprotein-1 (GLG1).(C) Cells stably expressing GFP-Rab6 were treated with GRINA siRNA (bottom) and compared to control cells (top) upon staining for TGN46 and VPS35.(D) Quantitation by western blotting of lysates from control and GRINA-silenced HeLa cells and GFP-Rab6 cells.

(E-G) HeLa cells were transiently transfected with GRINA-Myc for 24 hr before fixing and staining. (E and F) GRINA-Myc colocalizes with both TGN46 and VPS35. In this example, GRINA-Myc expression (in the cell marked by an asterisk in E) reduced the cell's TGN46 expression compared to surrounding untransfected cells and caused enlargement of VPS35-positive endosomes. The area in (E) magnified in (F) is indicated by a dashed line. (G) GRINA-Myc expression also perturbs CIMPR localization. In this example, GRINA-Myc transfection (in the cell marked by an asterisk) caused CIMPR to localize to round vesicular structures positive for GRINA-Myc, some of which appeared larger than regular endosomes, whereas TGN46 staining was almost absent in the transfected cell.

(A) Control HeLa cells or cells transfected with the indicated siRNAs were treated for 3 hr with cycloheximide, lysed, and incubated with agarose-bound wheat germ agglutinin to capture glycosylated membrane proteins. Total cell lysates (left) and lectin pull-down samples (right) were assayed by western blotting. The experiment was repeated three times, and representative data are shown.

(B-E) Quantitative analysis of SFT2D2, ZDHHC5, and GRINA KD cells immunofluorescence using automated microscopy (see Experimental Procedures). (B) Representative images showing VPS35, TGN46, and CIMPR staining. Scale bar, 50 μm. (C) Quantitative analysis of CIMPR intensity at the Golgi indicates a significant increase in SFT2D2, ZDHHC5, or GRINA KD cells. (D) SFT2D2, ZDHHC5, and GRINA KD increase the Pearson's correlation coefficient for colocalization between CIMPR and retromer proteins VPS35 or SNX1 while decreasing the correlation between CIMPR and Golgi matrix protein GM130. In some cases, identical correlation coefficients were measured in the replicate experiments. (E) Quantitation of the TGN46 and GLG1 intensity in the three types of KD cells.