Purification of posttranslationally modified and unmodified Rab5 protein expressed in Spodoptera frugiperda cells

Hisanori Horiuchi, Oliver Ullrich, Cecilia Bucci, Marino Zerial

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Rab5 is a 25-kDa GTP-binding protein localized to the plasma membrane, clathrin-coated vesicles, and early endosomes, and it functions as a regulatory factor of endocytosis. As for other Rab proteins, Rab5 is geranylgeranylated at its C terminus, and this modification is essential for its function. To obtain Rab5 in the isoprenylated form, the chapter makes use of a baculovirus expression system. This chapter describes a method to purify both posttranslationally modified and unmodified Rab5 from Spodoptera frugiperda (Sf9) insect cells overexpressing the protein. Purified posttranslationally modified and unmodified Rab5 protein efficiently binds GTP and GDP. However, as expected, Rab-GDI is active only on modified Rab5. When modified Rab5 complexed with Rab-GDI is introduced into permeabilized cells, Rab5 is localized to its correct site of function and induces the formation of enlarged early endosomes as previously observed in vivo, indicating that it is functionally active.

Original languageEnglish
Pages (from-to)9-15
Number of pages7
JournalMethods in enzymology
Volume257
Issue numberC
DOIs
Publication statusPublished - 1995 Jan 1
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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