FIGURE 4.

ISTID2 mediates targeting to the Golgi complex and reduces catalytic activity. A, wild-type (wt) NMNAT2 bearing a C-terminal eGFP tag (NMNAT2-eGFP) and the FLAG-ISTID2-eGFP fusion protein were overexpressed in HeLa S3 cells to determine their subcellular localization. B, a mutant construct of NMNAT1 (NMNAT1-ISTID2) in which ISTID1 was replaced with ISTID2 from NMNAT2 was overexpressed in HeLa S3 cells. The proteins were visualized by FLAG immunocytochemistry. C, substitution of ISTID1 with ISTID2 in NMNAT1 redirected the protein to the Golgi complex and reduced the activity to the level of wild-type NMNAT2. Note that purified recombinant wild-type NMNAT1 is ∼20 times more active compared with wild-type NMNAT2 (27). The experimental details were identical to those described in the legend to Fig. 2. RLU, relative light units; c, control.