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Fig. 10. Reticulon interacts with and modulates TBC1D20 function at the ER. (A) A schematic of reticulon 1 variant 2 (reticulon 1v2) showing the two hydrophobic regions (HR), which by analogy with other reticulons may form transmembrane or membrane anchoring sequences (Voeltz et al., 2006). The minimal binding fragment identified by yeast two-hybrid (Y2H) screening is shown in blue. Directed Y2H analysis was performed using reticulon 1v2 as the prey and full-length TBC1D20 and the various deletion constructs indicated. All combinations grow on non-selective media (–LW), whereas only a subset can grow on the selective media (QDO). GFP-immunoprecipitations were performed from HeLa S3 cells cotransfected with Myc-tagged reticulon, and the GFP-tagged TBC1D20 constructs indicated, using a published method (Voeltz et al., 2006). The immunoprecipitates were blotted with GFP and Myc antibodies to detect TBC1D20 and reticulon, respectively. (B) HeLa cells transfected with reticulon 1v2 (reticulon; green) were fixed after 24 hours, and then stained with antibodies to calnexin or GM130 (red). (C,D) HeLa cells expressing Myc-tagged wild-type, R105A catalytically inactive mutant TBC1D20, or the minimal TBC domain (amino acids 1-317) in the absence or presence of GFP-tagged reticulon 1v2 (reticulon) were fixed, and then stained for GM130 and antibodies to the Myc epitope. In some panels DNA was stained with DAPI. Bars, 10 µm. (D) Quantification of the cells showing intact Golgi, the `loss of Golgi' phenotype, or Golgi fragmentation in cells expressing TBC1D20 constructs and reticulon as indicated. (E) Western blots were performed to control for the expression levels of TBC1D20 and reticulon, with tubulin as a loading control.
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