Quantitative analysis of endocytosis with cytoplasmic pHluorin chimeras.

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Derek C ProsserBeverly Wendland

Abstract

The pH-sensitive green fluorescent protein (GFP) variant pHluorin is typically fused to the extracellular domain of transmembrane proteins to monitor endocytosis. Here, we have turned pHluorin inside-out, and show that cytoplasmic fusions of pHluorin are effective quantitative reporters for endocytosis and multivesicular body (MVB) sorting. In yeast in particular, fusion of GFP and its variants on the extracellular side of transmembrane proteins can result in perturbed trafficking. In contrast, cytoplasmic fusions are well tolerated, allowing for the quantitative assessment of trafficking of virtually any transmembrane protein. Quenching of degradation-resistant pHluorin in the acidic vacuole permits quantification of extravacuolar cargo proteins at steady-state levels and is compatible with kinetic analysis of endocytosis in live cells.

References

Aug 31, 1981·Biochemical and Biophysical Research Communications·S H Bokman, W W Ward
Mar 1, 1995·The Journal of Cell Biology·T A Vida, S D Emr
Jun 3, 1998·Applied and Environmental Microbiology·J B AndersenS Molin
Dec 22, 1999·The Journal of Biological Chemistry·P J PlantN Demaurex
Oct 12, 2000·Biophysical Journal·S SankaranarayananT A Ryan
Jan 7, 2005·Molecular Biology of the Cell·Christopher L BrettRajini Rao
Mar 16, 2006·Proceedings of the National Academy of Sciences of the United States of America·Rubén C AguilarBeverly Wendland
May 13, 2006·Annual Review of Biophysics and Biomolecular Structure·James H Hurley, Scott D Emr
Nov 23, 2007·Molecular Biology of the Cell·Christian DimaanoMarkus Babst
Nov 23, 2007·Molecular Biology of the Cell·Sarah M RueScott D Emr
May 2, 2008·Molecular Biology of the Cell·Lymarie Maldonado-BáezBeverly Wendland
Jul 25, 2009·Cellular and Molecular Life Sciences : CMLS·Irit GottfriedUri Ashery
Oct 7, 2009·Proceedings of the National Academy of Sciences of the United States of America·John H TranRandy Schekman

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Citations

Aug 1, 2013·Bioinformatics·Alberto Giovanni BusettoJoachim M Buhmann
Nov 9, 2011·The Journal of Cell Biology·Derek C ProsserBeverly Wendland
Aug 19, 2015·The Journal of Cell Biology·W Mike HenneScott D Emr
Oct 15, 2013·Traffic·Karen WhitworthBeverly Wendland
Dec 15, 2012·Small GTPases·Derek C Prosser, Beverly Wendland
Jan 30, 2015·Molecular Biology of the Cell·Mary Katherine BradfordBeverly Wendland
May 24, 2018·Molecular Biology of the Cell·Zachary N WilsonGreg Odorizzi
Jun 22, 2018·Molecular Biology of the Cell·Akshay MoharirMarkus Babst
Aug 26, 2017·The Journal of Cell Biology·Masahide OkuYasuyoshi Sakai
Mar 16, 2018·G3 : Genes - Genomes - Genetics·Kristie WrasmanBeverly Wendland
Mar 20, 2020·PLoS Genetics·Jessica M TumoloJason A MacGurn
Dec 19, 2018·Nature Communications·Erin Kate McNally, Christopher Leonard Brett
Jan 6, 2019·The Journal of Cell Biology·Sora LeeJason A MacGurn
Mar 22, 2021·Aging·Nathaniel L HepowitRobert C Dickson
Oct 16, 2015·Journal of Cell Science·Derek C ProsserAllyson F O'Donnell

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