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PMID: 3031085 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

The Journal of cell biology ·Vol. 104 ·No. 4 ·1987-04-00 ·Pages 875-86

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

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 micrograms protein) sufficient for biochemical, immunological, and functional analysis.

MeSH Terms
Animals Cell Fractionation Cell Line Centrifugation, Density Gradient/methods Electrophoresis/methods Endocytosis Golgi Apparatus/ultrastructure Lysosomes/ultrastructure Microscopy, Electron Microsomes/ultrastructure Povidone Silicon Dioxide
Chemicals
Percoll Silicon Dioxide Povidone
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Marsh M
Schmid S
Kern H
Harms E
Male P
Mellman I
Helenius A
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53 references, click to expand
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.104.4.875
Published
1987-04-00
Pages
875-86
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2114435
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.104.4.875 PMC Full Text View journal details
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