Fig. 9. TBC1D20 is an ER membrane protein. (A) A schematic showing the proposed topology of TBC1D20. Carbonate extraction experiments were performed as described in the Materials and Methods to investigate the membrane association of TBC1D20. Golgin84 and GM130 were taken as representative integral and peripheral membrane proteins, respectively. The topology of TBC1D20 was investigated using proteinase K (PK) digestion experiments in the presence or absence of Triton X-100 (TX). TGN46 and Golgin84 were taken as controls for proteins with large lumenal or cytosolic domains, respectively. (B) HeLa cells transfected with Myc-tagged TBC1D20 and a series of C-terminal deletions were fixed after 24 hours, and then stained with antibodies to GM130 (green) and the Myc epitope (red). The reticular or cytosolic nature of TBC1D20 constructs is more clearly visible in the enlargements. (C) HeLa cells expressing the GFP-tagged R105A mutant of TBC1D20, or a series of N-terminal deletions were fixed after 24 hours, and then stained with antibodies to GM130 (red). TBC1D20 constructs were visualized by GFP fluorescence (green). DNA was stained with DAPI (blue). Bars, 10 µm in all panels. (D) A summary of the TBC1D20 constructs used and results obtained in B and C. Activity refers to the ability to cause the `loss of Golgi' phenotype; localization of the different constructs is scored as ER, including nuclear envelope, and Golgi. The TBC domain is marked in red, and the putative transmembrane domain in green.