Separation of early steps in endocytic membrane transport

Electrophoresis
F G van der Goot

Abstract

We describe a simple subcellular fractionation scheme aimed at separating early endosomes from the plasma membrane in view of studying the possible arrival of plasma membrane-bound toxins, proteins or other extracellular ligands in endosomes. Plasma membrane proteins were labeled with the impermeable reagent sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC) biotin at 4 degrees C. In a separate set of cells, early endosomes were labeled by internalization of horseradish peroxidase from the medium for 5 min. The first step of the purification, which consists of a step sucrose gradient, led to three fractions, respectively: enriched in biosynthetic membranes (interface 3), in plasma membrane and early endosomes (interface 2), and in late endosomes (interface 1). The second step, in which interface 2 was loaded at the bottom of a 17% Percoll gradient, led to the separation of the plasma membrane, including caveolae and cholesterol-glycolipid rafts, from early endosomes. Western blot analysis of the fractions from the Percoll gradient showed that the transferrin receptor, the small GTPases rab5 and Arf6, as well as annexin II were present both at the plasma membrane and in early endosomes, whereas the caveolar marker caveolin, 1co...Continue Reading

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Citations

Jul 13, 2000·Journal of Biochemical and Biophysical Methods·H Pertoft
Jul 30, 2002·The EMBO Journal·Marc FivazF Gisou van der Goot
May 4, 2012·Biochimica Et Biophysica Acta·Martina MalnarSilva Hecimovic
Mar 6, 2004·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·G AndringaM C Bennett
Nov 22, 2000·The Journal of Biological Chemistry·T YamazakiY Ihara
May 2, 2003·Journal of Cell Science·Erica A PetersonEdward L G Pryzdial

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