Expression of BIG2 and analysis of its function in mammalian cells.
BIG2 is a member of brefeldin A-inhibited guanine nucleotide exchange factors for ADP-ribosylation factors (ARFs). Although BIG2 is associated mainly with the trans-Golgi network, we have recently revealed that some population of BIG2 is associated with the recycling endosome. Moreover, we have found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We have also demonstrated that the exchange activity of BIG2 is specific for class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF enhances the BIG2(E738K)-induced membrane tubulation. Therefore, we have proposed that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs. This article describes methods used for examining the function of BIG2 including basic protocols, which are not conventional because of cytotoxicity of BIG2 in Escherichia coli cells and its low expression efficiency in mammalian cells.
Shin
Hye-Won
HW
Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan.
Shinotsuka
Chisa
C
Nakayama
Kazuhisa
K
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D000818:Animals; D020662:Guanine Nucleotide Exchange Factors / Q000096:biosynthesis* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D014162:Transfection
C488152:Arfgef2 protein, rat; D020662:Guanine Nucleotide Exchange Factors; D020823:ADP-Ribosylation Factor 1