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Determination of Rab5 activity in the cell by effector pull-down assay.

ABSTRACT: Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins.

SUBMITTER: Qi Y

PROVIDER: S-EPMC5901726 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Determination of Rab5 activity in the cell by effector pull-down assay.

Qi Yaoyao Y Liang Zhimin Z Wang Zonghua Z Lu Guodong G Li Guangpu G

Methods in molecular biology (Clifton, N.J.) 20150101

Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we ...[more]

PMID: 25800849

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