FIGURE 4. The protonation state of His⁶⁹ affects the subcellular localization of furin.

A, immunofluorescence microscopy of BSC-40 cells expressing fur/f/ha, H69L:fur/f/ha, or H69K:fur/ f/ha. Cells were fixed and stained with anti-furin PA1-062 and anti-TGN46 and then visualized with species-specific fluorescently labeled secondary antibodies. B, BSC-40 cells expressing fur/f/ha or H69L:fur/f/ha were pulse-labeled for 30 min with 100 μCi each of [³H]Arg and [³H]Leu and then chased with excess unlabeled Arg and Leu at 37 °C for the indicated times. Cell extracts were prepared, and mature furin molecules were immunoprecipitated with mAb M1, separated by 15% Tris-Tricine SDS-PAGE, and co-precipitating propeptide molecules (open arrowheads) were detected by fluorography and quantified by densitometry. C, immunofluorescence microscopy of BSC-40 cells expressing fur/f/ha or H69L:fur/f/ha. Cells were incubated with mAb M1 in the culture medium (30 μg/ml) for 1 h, and internalized mAb M1 was detected with a fluorescently labeled secondary antibody.