Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis

M. Marsh, S. Schmid, H. Kern, E. Harms, P. Male, I. Mellman, A. Helenius

Research output: Contribution to journalArticle

140 Citations (Scopus)

Abstract

Endosomes are prelysosomal organelles that serve as an intracellular site for the sorting, distribution, and processing of receptors, ligands, fluid phase components, and membrane proteins internalized by endocytosis. Whereas the overall functions of endosomes are increasingly understood, little is known about endosome structure, composition, or biogenesis. In this paper, we describe a rapid procedure that permits analytical and preparative isolation of endosomes from a variety of tissue culture cells. The procedure relies on a combination of density gradient centrifugation and free flow electrophoresis. It yields a fraction of highly purified, functionally intact organelles. As markers for endosomes in Chinese hamster ovary cells, we used endocytosed horseradish peroxidase, FITC-conjugated dextran, and [35S]methionine-labeled Semliki Forest virus. Total postnuclear supernatants, crude microsomal pellets, or partially purified Golgi fractions were subjected to free flow electrophoresis. Endosomes and lysosomes migrated together as a single anodally deflected peak separated from most other organelles (plasma membrane, mitochondria, endoplasmic reticulum, and Golgi). The endosomes and lysosomes were then resolved by centrifugation in Percoll density gradients. Endosomes prepared in this way were enriched up to 70-fold relative to the initial homogenate and were still capable of ATP-dependent acidification. By electron microscopy, the isolated organelles were found to consist of electron lucent vacuoles and tubules, many of which could be shown to contain an endocytic tracer (e.g., horseradish peroxidase). SDS PAGE analysis of integral and peripheral membrane proteins (separated from each other by condensation in Triton X-114) revealed a unique and restricted subset of proteins when compared with lysosomes, the unshifted free flow electrophoresis peak, and total cell protein. Altogether, the purification procedure takes 5-6 h and yields amounts of endosomes (150-200 μg protein) sufficient for biochemical, immunological, and functional analysis.

Original languageEnglish (US)
Pages (from-to)875-886
Number of pages12
JournalJournal of Cell Biology
Volume104
Issue number4
DOIs
StatePublished - 1987

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Endosomes
Electrophoresis
Organelles
Lysosomes
Horseradish Peroxidase
Endocytosis
Membrane Proteins
Semliki forest virus
Proteins
Density Gradient Centrifugation
Vacuoles
Cricetulus
Centrifugation
Endoplasmic Reticulum
Methionine
Polyacrylamide Gel Electrophoresis
Ovary
Electron Microscopy
Mitochondria
Cell Culture Techniques

ASJC Scopus subject areas

  • Cell Biology

Cite this

Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis. / Marsh, M.; Schmid, S.; Kern, H.; Harms, E.; Male, P.; Mellman, I.; Helenius, A.

In: Journal of Cell Biology, Vol. 104, No. 4, 1987, p. 875-886.

Research output: Contribution to journalArticle

Marsh, M. ; Schmid, S. ; Kern, H. ; Harms, E. ; Male, P. ; Mellman, I. ; Helenius, A. / Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis. In: Journal of Cell Biology. 1987 ; Vol. 104, No. 4. pp. 875-886.
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