Figure 5.

Wild-type Cog4, but not the E53/71A double mutant, restores the Golgi-targeting of SNAREs in Cog4-depleted cells. (A) HeLa cells were transiently cotransfected with shRNA construct encoding siRNA of the human Cog4 together with expression vectors encoding either Myc-tagged Cog3 or Cog4. The specificity of this siRNA was determined by immunoblotting with anti-Myc antibody. Stable HeLa cell lines depleted of Cog4 (Cog4-KD) were established and assessed for Cog4 expression by immunoblotting (B) and immunofluorescence (C) using anti-Cog4 antibody. As shown (C), depletion of Cog4 impairs the compact organization of the Golgi complex. Scale bar, 10 μm. (D) Localization of the SNARE proteins GS15, GS28 and Syntaxin 5 was determined in control and Cog4-knock-down cells by immunofluorescence analysis using the corresponding antibodies. Shown are representative confocal images. Scale bar, 10 μm. (E) Expression levels of GS15, GS28, Syntaxin 5 and Sly1 in control and Cog4-depleted HeLa cells were determined by immunoblotting using the indicated antibodies. The two bands of Syntaxin 5 represent its two isoforms. (F) Silent mutations within the RNAi targeting sequence of Cog4 protect its expression from its RNAi. HeLa cells were transiently cotransfected with the shRNA construct along with Myc-tagged of either the wild-type or the silent Cog4 mutant. Expression of Cog4 was determined by western blotting with anti-Myc antibody. (G) The wild-type Cog4, but not the E53/71A double mutant, restores the targeting of GS15 and GS28 to the Golgi in Cog4-depleted cells and consequently, SNARE pairing. Cog4-depleted HeLa cells were transiently transfected with either the wild-type or the E53/71A double mutant of Myc-tagged Cog4 containing the silent mutations within the RNAi targeting sequence. Two days later, the cells were fixed and double-immunostained with anti-Myc and either anti-GS15 or anti-GS28 antibodies. Transfected cells are indicated by arrowheads. Scale bar, 10 μm.