ABC transporters required for endocytosis and endosomal pH regulation in Dictyostelium

In Dictyostelium, the RtoA protein links both initial cell-type choice and physiological state to cell-cycle phase. rtoA(-) cells (containing a disrup tion of the rtoA gene) generally do not develop past the mound stage, and h ave an abnormal ratio of prestalk and prespore cells. RtoA is also involved in fusion of endocytic/exocytic vesicles. Cells lacking RtoA, although hav ing a normal endocytosis rate, have a decreased exocytosis rate and endosom es with abnormally low pHs. RtoA levels vary during the cell cycle, causing a cell-cycle-dependent modulation of parameters such as cytosolic pH (Braz ill et al., 2000). To uncover other genes involved in the RtoA-mediated dif ferentiation, we identified genetic suppressors of rtoA. One of these suppr essors disrupted two genes, mdrA1 and mdrA2, a tandem duplication encoding two members of the ATP binding cassette (ABC) transporter superfamily. Disr uption of mdrA1/mdrA2 results in release from the developmental block and s uppression of the defect in initial cell type choice caused by loss of the rtoA gene. However, this is not accomplished by re-establishing the link be tween cell type choice and cell cycle phase. MdrA1 protein is localized to the endosome. mdrA1(-)/mdrA2(-) cells (containing a disruption of these gen es) have an endocytosis rate roughly 70% that of wild-type or rtoA(-) cells , whereas mdrA1(-)/ mdrA2(-)/rtoA(-) cells have an endocytosis rate roughly 20% that of wild-type. The exocytosis rates of mdrA1(-)/mdrA2(-) and mdrA1 (-)/mdrA2(-)/rtoA(-) are roughly that of wild-type. mdrA1(-)/mdrA2(-) endos omes have an unusually high pH, whereas mdrA1(-)/mdrA2(-)/rtoA(-) endosomes have an almost normal pH. The ability of mdrA1/mdrA2 disruption to rescue the cell-type proportion, developmental defects, and endosomal pH defects c aused by rtoA disruption, and the ability of rtoA disruption to exacerbate the endocytosis defects caused by mdrA1/mdrA2 disruption, suggest a genetic interaction between rtoA, mdrA1 and mdrA2.