Figure 6.
NRBF2 is required for CCZ1-MON1A GEF activity. (A) GEF activity assay was used to determine whether NRBF2 enhances GEF activity of CCZ1-MON1A (CM1). Mant-GDP bound RAB7 were diluted to a concentration of 50 nM in exchange buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, and 0.5 mM MgCl2). The dissociation of mant-GDP from RAB7 was determined by measuring the decrease in fluorescence signal that accompanies the release of mant-GDP in the presence of a large excess of GTP. Nucleotide release reactions were initiated by the addition of GTP (1 mM final concentration) in the reaction buffer containing CCZ1 (500 pmol) and MON1A (500 pmol), with or without adding NRBF2 (500 pmol). Samples are excited at 360 nm, and the emission was monitored at 440 nm. (B) GEF activity of CCZ1-MON1A toward RAB7 is increased in the presence of PtdIns3P (PtdIns3P concentration is 20 pmol). (C) GEF activity of the CCZ1-MON1A purified from nrbf2-/- mice was decreased obviously compared with that from WT group. Immunopurified CCZ1-MON1A protein by CCZ1 antibody from WT or nrbf2-/- mice were used to perform the GEF assay. (D) GEF activity of the CCZ1-MON1A purified from nrbf2-/- and SAR405-treated cells were decreased compared with that purified from WT or untreated group. Immunopurified CCZ1-MON1A protein by CCZ1 antibody from WT or nrbf2-/- cells were used to perform the GEF assay. (E) NRBF2 dCCD domain is important for regulating CCZ1-MON1A GEF activity. nrbf2-/- N2a cells were transiently transfected with CFP, Nrbf2-CFP, dMIT-CFP or dCCD-CFP, Immunopurified CCZ1-MON1A protein by CCZ1 antibody were used to perform the GEF assay. (F) NRBF2-associated GEF activity was increased in a starvation-induced autophagy condition. (G) Schematic model for NRBF2 regulating CCZ1-MON1A GEF activity