CD147 cytoplasmic sequence is sufficient for sorting directly into the recycling route. (A) Sequence alignment of the cytoplasmic tail (carboxyl terminal) of CD147, showing location of the truncated constructs at positions 235, 246, and 261. Identical sequences are highlighted in red, strongly similar in blue, and weakly similar in green. (B) Tac-CD147 carboxyl-terminal truncations were expressed in HeLa cells, and their locations were assessed after internalization for 1 h at 37°C using an anti-Tac antibody. Cells were fixed and processed for immunofluorescence as described in Materials and methods. Insets show enlarged views of the boxed regions. (C) The percentage of colocalization of internal Tac-CD147 truncated chimeras with EEA1 was quantified using MetaMorph software (see Materials and methods). The data presented is the mean of three independent experiments ± SD (error bars). (D) Cells expressing the different Tac-CD147 chimeras were incubated with anti-Tac for 1 h at 37°C. Then the cells were washed with media and chased in media containing 15 mM NH4Cl for 22 h. The arrow in the top row points to the cell surface. Other arrows point to tubular endosomes. The chimeric proteins were visualized with Alexa Fluor 488–conjugated goat anti–mouse, and late endosomes were visualized with antibodies to Lamp1. Bars: (B and D) 10 µm; (B, insets) 5 µm.
