Fig. 2.

Mutational analysis of Cog4 function in Cog4-silenced HeLa cells. (A) Cells transfected with the indicated siRNA-resistant allele were incubated with GNL-Alexa-647, which labels mis-glycosylated cell surface proteins. All constructs (except “vector”) include Cog4 residues 1–524, indicated by the white bars. Red, green, and blue bars represent the protein regions color-coded as in Fig. 1A. Cyan and magenta boxes indicate sets of mutations as indicated in Fig. S2. A yellow box indicates mutations of R729 or interacting residues as noted on the bar itself (e.g., R729W). (B) Quantification of the percentage of cells [± SD, n = 3 (>100 cells each)] as shown in Fig. 2A with detectable GNL staining of plasma membrane glycoproteins. (C) To assess protein expression level, cells transfected with silencing-resistant Cog4 alleles carrying C-terminal 3× Myc tags were analyzed by western blotting. To assess the integrity of the COG complex, the quantity of tagged Cog4 present in Cog6 immunoprecipitation reactions was evaluated by western blotting. (D) Surface representations of the Cog4-(525–785) monomer. In the left-hand pair of images, the surface is color-coded by sequence conservation (darker blue is more conserved). In the right-hand pair, the residues that were studied by site-directed mutagenesis are color-coded to match the triangles in Fig. S2.