In 2000, the second mammalian nSMase, nSMase2, was identified based on similarity to bacterial SMase (Hofmann et al., 2000). Human nSMase2 is a 655 amino acid protein with a molecular weight of 71 kD and has a C-terminal catalytic domain, two N-terminal hydrophobic segments, with a 200-residue collagen-like triple helices between the two (Fig. 1) (Hofmann et al., 2000). In addition, nSMase2 was found to be palmitoylated in two cysteine clusters (Tani and Hannun, 2007b) with one cluster located between the two hydrophobic segments and the second cluster located in the catalytic region. Mutagenesis studies revealed that palmitoylation of nSMase2 was important for both the protein stability and the plasma membrane (PM) localization of nSMase2. Although the N-terminal hydrophobic segments of nSMase2 were originally proposed to be transmembrane domains, subsequent studies of the enzyme topology suggested that each hydrophobic segment is integrated into the membranes, but does not span the entire membrane (Tani and Hannun, 2007a). The location of the N-terminal palmitoylation cluster between the two hydrophobic segments further supports this as palmitoylation only occurs on the cytosolic side of the PM (Tani and Hannun, 2007b).
