Wortmannin and Li+ specifically inhibit clathrin-independent endocytic internalization of bulk fluid.

Abstract

Incubation of a human fibrosarcoma cell line HT-1080 in Li(+)-containing medium inhibited internalization of a fluid marker, horseradish peroxidase (HRP), by more than 80%. The ion inhibited the activity enhanced by Ca2+ or phorbol 12-myristate 13-acetate. We also found that wortmannin (WT), a potent inhibitor of phosphoinositide (PI) 3-kinase (PI 3-k), inhibited the non-stimulated and the two stimulated types of endocytosis to the same extent as Li+. In contrast, neither WT nor Li+ influenced the early internalization of transferrin (Tfn), EGF or platelet-derived growth factor. Neither targeting to early endosomes nor recycling of the once-internalized Tfn was influenced. When the cytoplasmic pH was lowered by chasing cells that had been preincubated with 25 mM NH4Cl in an amiloride-containing Na(+)-free medium, more than 90% of internalization of Tfn in HT-1080 cells was inhibited, while that of HRP was reduced by only 35%. In contrast, WT reduced the uptake of HRP by KB cells by 34%, while 60% of the activity was inhibited by the treatment for cytoplasmic acidification. Comparison of other cells i.e., A-549 and a human diploid cell line Miyajima, indicated that cells showing higher sensitivity to WT were less sensitive to low cytoplasmic pH. These results suggest that, in all the cells studied, bulk fluid is internalized either via a clathrin-independent/PI 3-k-dependent route or via a clathrin-dependent/PI 3-k-independent one, though the ratio varied among them. We also found that internalization of a mAb directed toward the 116 (100)-kDa subunit of vacuolar ATPase [OSW2; Sato and Toyama (1994) J. Cell Biol. 127, 39-53] in the fluid phase was inhibited by WT, but the antibody was still internalized in a surface-bound form. Regardless of the treatment with WT, most of the antibody was transported to endosomes that were associated with Tfn receptor. These results suggest that both internalization routes are targeted to the same early endosomal compartments.