Figure S9.
GRAF2 and WDR44 are involved in the specific export of neosynthesized E-cadherin, MMP14, and CFTR ΔF508. (A) Confocal stacks of transfected HeLa cells stained with α–E-cadherin under nonpermeabilizing conditions or with α-MICAL1 under permeabilizing conditions. Upon cotransfection of all the dominant negative constructs, E-cadherin–GFP became trapped in intracellular compartments. E-cadherin–GFP colocalized with untagged MICAL1 (uMICAL1) G3W puncta and RFP–WDR44 ΔC patches. (B) Confocal stacks of transfected HeLa cells stained, in the case of uMICAL1 G3W, with α-MICAL1. Occludin-GFP was transported to the plasma membrane in all cases. Occludin-GFP was found in uMICAL1 G3W puncta, but not in WDR44 ΔC patches. (C) Biotinylation of surface proteins of shRNA-expressing HeLa cells transfected with E-cadherin–GFP and Occludin-GFP. The Transferrin receptor (TfnR) was used as loading control; it was also found as a dimer. The four parts of the blots are from the same membranes and correspond to identical exposure times. (D) Western blot analysis of an equal protein amount of shRNA-expressing HeLa cell lysates. The intensity of the Grp78 protein band was normalized by the Actin signal and expressed as a ratio to the value obtained for shControl-transfected cells. n = 2. Down-regulation of GRAF2 or WDR44 expression did not lead to an increase in Grp78 expression, a marker of ER stress (Lee, 2005). (E) Percentage of transfected HeLa cells, incubated or not with BFA (5 µg/ml, 15 min), with endogenous (endo.) WDR44 tubules. n = 24–31 for GFP-transfected cells and 4–6 for the others. Unlike under resting conditions (Fig. 4 D and Fig. 5 F), Rab10 T23N and MICAL1 G3W did not inhibit BFA-induced WDR44 tubules, suggesting that these may not be functional for protein export. (F) Confocal images of transfected HeLa cells, left untreated or incubated with biotin (40 µM, 1 h) and stained with α-Calnexin. In untreated cells, SBP-GFP–E-cadherin was in the ER and was released to the plasma membrane upon incubation with biotin. (D and E) Data are means ± SEM; **, P < 0.01; and ****, P < 0.0001. (A, B, and F) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

GRAF2 and WDR44 are involved in the specific export of neosynthesized E-cadherin, MMP14, and CFTR ΔF508. (A) Confocal stacks of transfected HeLa cells stained with α–E-cadherin under nonpermeabilizing conditions or with α-MICAL1 under permeabilizing conditions. Upon cotransfection of all the dominant negative constructs, E-cadherin–GFP became trapped in intracellular compartments. E-cadherin–GFP colocalized with untagged MICAL1 (uMICAL1) G3W puncta and RFP–WDR44 ΔC patches. (B) Confocal stacks of transfected HeLa cells stained, in the case of uMICAL1 G3W, with α-MICAL1. Occludin-GFP was transported to the plasma membrane in all cases. Occludin-GFP was found in uMICAL1 G3W puncta, but not in WDR44 ΔC patches. (C) Biotinylation of surface proteins of shRNA-expressing HeLa cells transfected with E-cadherin–GFP and Occludin-GFP. The Transferrin receptor (TfnR) was used as loading control; it was also found as a dimer. The four parts of the blots are from the same membranes and correspond to identical exposure times. (D) Western blot analysis of an equal protein amount of shRNA-expressing HeLa cell lysates. The intensity of the Grp78 protein band was normalized by the Actin signal and expressed as a ratio to the value obtained for shControl-transfected cells. n = 2. Down-regulation of GRAF2 or WDR44 expression did not lead to an increase in Grp78 expression, a marker of ER stress (Lee, 2005). (E) Percentage of transfected HeLa cells, incubated or not with BFA (5 µg/ml, 15 min), with endogenous (endo.) WDR44 tubules. n = 24–31 for GFP-transfected cells and 4–6 for the others. Unlike under resting conditions (Fig. 4 D and Fig. 5 F), Rab10 T23N and MICAL1 G3W did not inhibit BFA-induced WDR44 tubules, suggesting that these may not be functional for protein export. (F) Confocal images of transfected HeLa cells, left untreated or incubated with biotin (40 µM, 1 h) and stained with α-Calnexin. In untreated cells, SBP-GFP–E-cadherin was in the ER and was released to the plasma membrane upon incubation with biotin. (D and E) Data are means ± SEM; **, P < 0.01; and ****, P < 0.0001. (A, B, and F) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

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