FIGURE 6.

Cysteine-specific palmitoylation within ISTID2 anchors NMNAT2 at the Golgi complex. A, the tertiary structure prediction for NMNAT2 suggests that Cys¹⁶⁴ and Cys¹⁶⁵ are located at the very tip of ISTID2. B, FLAG-tagged wild-type (wt) NMNAT2, the ISTID2 deletion mutant (NMNAT2_ΔISTID2), or the FLAG-ISTID2-eGFP fusion protein was overexpressed in HeLa S3 cells in the presence of 25 μm 2-BP to inhibit protein palmitoylation. The endogenous 2-BP-resistant Golgi marker GM130 was immunostained for comparison. C, substitution of Cys¹⁶⁴ and Cys¹⁶⁵ with alanine caused relocalization of NMNAT2 to the cytoplasm. After overexpression in HeLa S3 cells, the double mutant NMNAT2_Cys164/165Ala was visualized by FLAG immunocytochemistry. The endogenous Golgi marker GM130 or coexpressed cytosolic eGFP (cyto-eGFP) was used for comparison. D, shown is the in vivo palmitoylation of NMNAT2. HeLa S3 cells overexpressing FLAG-tagged wild-type NMNAT2 or mutant NMNAT2_Cys164/165Ala were incubated with [¹⁴C]palmitic acid. Cell extracts were separated by SDS-PAGE and subjected to autoradiography (left panel) and FLAG immunoblot analyses (right panel). The arrow indicates a radiolabeled band (∼34 kDa) that corresponds to FLAG-tagged wild-type NMNAT2.