Purification and analysis of RIN family-novel Rab5 GEFs.
The small GTPase Rab5 plays important roles in membrane budding and trafficking in the early endocytic pathways, and the activation of this GTPase is mediated by several guanine nucleotide exchange factors (GEFs) at each of the transport steps. The RIN family has been identified as GEFs for Rab5 and shown to possess unique biochemical properties. The RIN family preferentially interacts with an activated form of Rab5, although it enhances guanine nucleotide exchange reaction. Moreover, biochemical analysis indicates that the RIN family functions as a tetramer. In this chapter, we describe the isolation of the recombinant RIN family via expression in Spodoptera frugiperda (Sf9) insect cells and in mammalian cells. In addition, functional analysis is also provided to assess the physiological properties of the RIN family.
Kota
K
Saito
Hiroaki
H
Kajiho
Yasuhiro
Y
Araki
Hiroshi
H
Kurosu
Kenji
K
Kontani
Hiroshi
H
Nishina
Toshiaki
T
Katada
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors / Q000302:isolation & purification* / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D020696:rab5 GTP-Binding Proteins / Q000302:isolation & purification* / Q000502:physiology*
D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors; D011994:Recombinant Proteins; D020696:rab5 GTP-Binding Proteins