Bafilomycin A1 treatment retards transferrin receptor recycling more than bulk membrane recycling

John F. Presley, Satyajit Mayor, Timothy E. McGraw, Kenneth Dunn, Frederick R. Maxfield

Research output: Contribution to journalArticle

126 Citations (Scopus)

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 A-1. 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-1 without 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.

Original languageEnglish
Pages (from-to)13929-13936
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number21
DOIs
StatePublished - May 23 1997

Fingerprint

Transferrin Receptors
Recycling
Membranes
Endosomes
Oligopeptides
Rate constants
Proton Pump Inhibitors
Cells
Transferrin
Cricetulus
Fluorescence Microscopy
Ovary
Fluorescence microscopy
bafilomycin A1
Sorting
Lipids
Peptides
Assays
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bafilomycin A1 treatment retards transferrin receptor recycling more than bulk membrane recycling. / Presley, John F.; Mayor, Satyajit; McGraw, Timothy E.; Dunn, Kenneth; Maxfield, Frederick R.

In: Journal of Biological Chemistry, Vol. 272, No. 21, 23.05.1997, p. 13929-13936.

Research output: Contribution to journalArticle

Presley, John F. ; Mayor, Satyajit ; McGraw, Timothy E. ; Dunn, Kenneth ; Maxfield, Frederick R. / Bafilomycin A1 treatment retards transferrin receptor recycling more than bulk membrane recycling. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 21. pp. 13929-13936.
@article{91f92a039bb94ee4836b526b8de83ae2,
title = "Bafilomycin A1 treatment retards transferrin receptor recycling more than bulk membrane recycling",
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 A-1. 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-1 without 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.",
author = "Presley, {John F.} and Satyajit Mayor and McGraw, {Timothy E.} and Kenneth Dunn and Maxfield, {Frederick R.}",
year = "1997",
month = "5",
day = "23",
doi = "10.1074/jbc.272.21.13929",
language = "English",
volume = "272",
pages = "13929--13936",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "21",

}

TY - JOUR

T1 - Bafilomycin A1 treatment retards transferrin receptor recycling more than bulk membrane recycling

AU - Presley, John F.

AU - Mayor, Satyajit

AU - McGraw, Timothy E.

AU - Dunn, Kenneth

AU - Maxfield, Frederick R.

PY - 1997/5/23

Y1 - 1997/5/23

N2 - 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 A-1. 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-1 without 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.

AB - 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 A-1. 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-1 without 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.

UR - http://www.scopus.com/inward/record.url?scp=0030610528&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030610528&partnerID=8YFLogxK

U2 - 10.1074/jbc.272.21.13929

DO - 10.1074/jbc.272.21.13929

M3 - Article

VL - 272

SP - 13929

EP - 13936

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 21

ER -