Figure 3.

Overexpression of the N-terminal region of RINT-1 perturbs membrane trafficking between the ER and Golgi. (A) HeLa cells were transfected with the plasmids encoding GST or the indicated FLAG-tagged constructs. After 24 h, the cells were fixed and double-stained. Staining for p115 (top two panels), Man II (middle two panels), and ZW10 (bottom two panels). Asterisks indicate cells overexpressing FLAG-RINT-1 full-length (Full) or FLAG-RINT-1N (N). Bar, 5 μm. The quantitative results are shown on the right. Error bars, SE of the mean for three experiments. (B) The plasmid for VSVG-GFP was cotransfected with the plasmid for GST, as a control, FLAG-RINT-1 full-length, or FLAG-RINT-1N into HeLa cells, and the protein transport assay was performed. To identity cells expressing GST or FLAG-tagged constructs, the fixed cells were stained with an anti-GST or anti-FLAG antibody followed by a Texas Red–conjugated secondary antibody. Only GFP fluorescence is shown. Bar, 5 μm. The quantitative results are shown at the bottom. “Peripheral dots” denotes the dispersed dotlike distribution pattern of VSVG-GFP. When VSVG-GFP was found to be concentrated in the perinuclear area, this localization pattern was categorized into “Golgi.” When VSVG-GFP was detected on the plasma membrane, this pattern was counted as “Plasma membrane.” In many of the cells categorized into “Plasma membrane,” VSVG-GFP was detected not only on the plasma membrane but also in the perinuclear area.