Interaction of Arl1 GTPase with the GRIP domain of Golgin-245 as assessed by GST (glutathione-S-transferase) pull-down experiments.
Arl1 is a member of the Arf/Arl family of Ras-like GTPase superfamily. Arl1 is enriched in the trans-Golgi network (TGN). We have recently shown that Arl1 regulates TGN recruitment of GRIP domain-containing Golgin-97 and Golgin-245 by interacting with the conserved GRIP domain present in their carboxyl (C)-termini. We describe here methods for the analysis of the interaction between Arl1(GTP) and the GRIP domain of Golgin-245 using in vitro GST pull-down experiments. GST-Arl1(GTP) can recover endogenous Golgin-245 from HeLa cell cytosol. Furthermore, GST-GRIP domain of Golgin-245 can efficiently retain endogenous active Arl1. A pull-down assay is developed to quantify the relative level of active Arl1.
Lu
Lei
L
Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore.
Tai
Guihua
G
Hong
Wanjin
W
D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D001324:Autoantigens; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004800:Enzymes, Immobilized; D005982:Glutathione Transferase; D006056:Golgi Apparatus / Q000502:physiology; D000077614:Golgi Matrix Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins
D001324:Autoantigens; D004800:Enzymes, Immobilized; C472298:GCC1 protein, human; C098772:GOLGA4 protein, human; D000077614:Golgi Matrix Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D020126:Brefeldin A; D005982:Glutathione Transferase; C097313:ADP-ribosylation factor related proteins; D020727:ADP-Ribosylation Factors