Protein Sorting During Endocytosis: Methods

Abstract

Endocytosis in eukaryotic cells is a regulated array of several complex processes and is involved in many cellular activities. The quantitative biochemical assay described herein integrates the qualitative results obtained with the morphological techniques previously used to study endocytosis. A method based on use of a biotinylated reagent that binds covalently to proteins and is cleavable by reducing agents at 4°C allows study of the molecular mechanisms involved and the structural requirements for protein sorting.

Keywords: endocytosis; internalization; recycling; protein sorting

Figure 1.

A schematic of the assay used to study internalization and recycling/degradation of surface expressed proteins. IP, immunoprecipitation; SDS‐PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; NHS‐SS‐biotin, sulfosuccinimidyl–2 (bioTinamido) ethyl–1,3‐dithiopropionate.

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References

Bretscher MS and Lutter R (1998) A new method for detecting endocytosed proteins. EMBO Journal 7: 4087–4092.

Fabbri M, Fumagalli L, Bossi G et al. (1999) A tyrosine‐based sorting signal in the beta2 integrin cytoplasmic domain mediates its recycling to the plasma membrane and is required for ligand‐supported migration. EMBO Journal 18: 4915–4925.

Smythe E, Redelmeier TE and Schmid SL (1992) Receptor‐mediated endocytosis in semiintact cells. Methods in Enzymology 219: 223–234.

Further Reading

Mellman I (1996) Endocytosis and molecular sorting. Annual Review of Cell and Developmental Biology 12: 575–625.

Trowbridge IS, Collawn JF and Hopkins CR (1993) Signal‐dependent membrane protein trafficking in the endocytic pathway. Annual Review of Cell and Developmental Biology 9: 129–161.

Schmid SL (1997) Clathrin‐coated vesicle formation and protein sorting: an integrated process. Annual Review of Biochemistry 66: 511–548.

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How to Cite close
Fabbri, Monica, and Pardi, Ruggero(Apr 2001) Protein Sorting During Endocytosis: Methods. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002613]