BAFILOMYCIN A(1) TREATMENT RETARDS TRANSFERRIN RECEPTOR RECYCLING MORE THAN BULK MEMBRANE RECYCLING

Treatment of Chinese hamster ovary cells with the vacuolar proton pump inhibitor bafilomycin A(1) causes a 2-fold retardation in the rate of recycling of transfected human transferrin receptors back to the cell surface as measured using biochemical assays (Johnson, L. S., Dunn, K . W., Pytowski, B., and McGraw, T. E. (1993) Mol. Biol. Cell 4, 1251-1 266). We have used quantitative fluorescence microscopy to determine w hich step(s) in the endocytic recycling pathway are affected. We show that removal of transferrin from sorting endosomes and accumulation in the peri-centriolar endocytic recycling compartment takes place norma lly in bafilomycin A(1)-treated cells. However, the rate constant for exit of transferrin receptors from recycling endosomes (k(e)) is reduc ed from 0.063 min(-1) in untreated cells to 0.034 min(-1) in the prese nce of bafilomycin A(1). This retardation appears to be dependent on t he presence of internalization motifs in the cytoplasmic domain since modified receptors lacking these oligopeptide motifs do not show as la rge a decrease in recycling rate in the presence of bafilomycin A(1). Bulk membrane recycling (measured by efflux of an internalized fluores cent lipid analog, ol-4-yl]-amino]hexoyl-sphingosylphosphorylcholine) is slowed from an exit rate constant of 0.060 min(-1) without drug to 0.046 min(-1) in the presence of bafilomycin A(1). We conclude that ba filomycin A(1) slows bulk membrane flow, but it causes additional inhi bition of receptor recycling in a manner that is dependent on a peptid e motif on the cytoplasmic domain.