Bafilomycin A1 Treatment Retards Transferrin Receptor Recycling More than Bulk Membrane Recycling*
- John F. Presley‡§,
- Satyajit Mayor‡¶,
- Timothy E. McGraw‖,
- Kenneth W. Dunn‡** and
- Frederick R. Maxfield‖‡
- From the ‡Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032 and the ‖Department of Biochemistry, Cornell University Medical College, New York, New York 10021
Abstract
Treatment of Chinese hamster ovary cells with the vacuolar proton pump inhibitor bafilomycin A1 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, 1251–1266). We have used quantitative fluorescence microscopy to determine which 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 normally in bafilomycin A1-treated cells. However, the rate constant for exit of transferrin receptors from recycling endosomes (k e) is reduced from 0.063 min−1 in untreated cells to 0.034 min−1 in the presence of bafilomycin A1. This retardation appears to be dependent on the presence of internalization motifs in the cytoplasmic domain since modified receptors lacking these oligopeptide motifs do not show as large a decrease in recycling rate in the presence of bafilomycin A1. Bulk membrane recycling (measured by efflux of an internalized fluorescent lipid analog, 6-[N-[7-nitrobenzo-2-oxa-1,3-diazol-4-yl]-amino]hexoyl-sphingosylphosphorylcholine) is slowed from an exit rate constant of 0.060 min−1without drug to 0.046 min−1 in the presence of bafilomycin A1. We conclude that bafilomycin A1 slows bulk membrane flow, but it causes additional inhibition of receptor recycling in a manner that is dependent on a peptide motif on the cytoplasmic domain.
Footnotes
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↵* This work was supported by National Institutes of Health Grant DK27083 (to F. R. M.) and American Cancer Society Grant CB-8 (to T. E. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Supported by a fellowship from the American Cancer Society. Present address: Bldg. 18T, CBMB/NICHD, 18 Library Dr., Bethesda, MD 20892.
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↵¶ Received support from the Helen Hay Whitney Foundation. Present address: National Centre for Biological Science, TIFR Centre IISc Campus, Bangalore 560012, India.
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↵** Supported by a National Kidney Foundation Young Investigatorship Award and the American Heart Association, New York City Affiliate. Present address: Dept. of Medicine, Nephrology Section, Indiana University Medical Center, 1120 South Dr., Sesler Hall 115, Indianapolis, IN 46202-5116.
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↵‡ To whom correspondence should be addressed: Dept. of Biochemistry, Cornell University Medical College, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6405; Fax: 212-746-8875; E-mail:frmaxfie{at}mail.med.cornell.edu.
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↵1 The abbreviations used are: Tf, transferrin; Tf-R, transferrin receptor; Tx-Tf, Texas Red-labeled transferrin; BSA, bovine serum albumin; C6-NBD-SM, 6-[N-[7-nitrobenzo-2-oxa-1,3-diazol-4-yl]-amino]hexoyl-sphingosylphosphorylcholine; CCD, charge-coupled device; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; F-R-Tf, fluorescein-rhodamine transferrin; NA, numerical aperture; PBS, phosphate-buffered saline.
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- Received January 28, 1997.
