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

Hisanori Horiuchi, Oliver Ullrich, Cecilia Bucci, Marino Zerial

研究成果: Article査読

17 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)9-15
ページ数7
ジャーナルMethods in enzymology
257
C
DOI
出版ステータスPublished - 1995 1月 1
外部発表はい

ASJC Scopus subject areas

  • 生化学
  • 分子生物学

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