Figure 6.

Attenuated endosome-to-TGN retrograde transport in Cog8-deficient cells. A) Endosome-to-TGN transport of HA-tagged TGN38 is attenuated in Cog8-depleted HeLa cells. Control and Cog8-depleted (KD) stable HeLa cell lines expressing TGN38-HA were incubated at 37°C with the anti-HA monoclonal antibody for various time periods. The colocalization of TGN38-HA (red) with the TGN marker Golgin 97 (green) was determined by immunostaining and confocal microscopy analysis. Shown are representative confocal images of control and Cog8-depleted cells at 3 and 30 min of antibody uptake. Bar: 10 µm. B, C) Deficiency of the Cog8 subunit impairs the Golgi localization of CI-MPR and TGN46. B) HeLa cells were transiently transfected with a Cog8 shRNA construct or cotransfected with the Cog8 shRNA construct together with a vector encoding YFP-Gal-T (5:1 molar ratio). Seventy-two hours later the cells were fixed and immunostained with the indicated antibodies. The Cog8-depleted cells (marked by arrowheads) were identified by immunostaining with anti-Cog8 antibody (upper panels) or by the YFP-Gal-T expression (lower panels). As shown, both CI-MPR and TGN46 were completely dispersed from the Golgi in Cog8-depleted HeLa cells. Bars: 10 µm. C) Control and Cog8-deficient fibroblasts obtained from a human patient (Cog8p) were fixed and co-immunostained for CI-MPR or TGN46 (red) and the Golgi marker p115 (green). As shown, the Golgi localization of CI-MPR was abolished and TGN46 was partially dispersed to the cytosol in the patient’s cells. Bars: 10 µm.