Previously, we reported two types of neutral ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229 -11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that the N-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside.Over the past decade, sphingolipids and their metabolites have emerged as a new class of lipid biomodulators of various cell functions (1, 2). Ceramide (N-acylsphingosine; Cer), 1 a common lipid backbone of sphingolipids, functions as a second messenger in a variety of cellular events including apoptosis and cell differentiation (3, 4). Sphingosine (Sph) has bifunctional effects on cell growth, i.e. it exerts mitogenic (5) and apoptosis inducing (6) activities, depending on the cell type and cell cycle. Sph-1-phosphate (S1P) was found to function as an intra-and intercellular second messenger to regulate cell growth (7), motility (8), and morphology (9). Interestingly, S1P inhibits the apoptosis induced by Cer and Fas ligand (10), indicating that the balance of Cer/Sph/S1P affects cell phenotype. Ceramidase (CDase, EC 3.5.1.23) is an enzyme that catalyzes hydrolysis of the N-acyl linkage of Cer to produce Sph, which can be phosphorylated to S1P by sphingosine kinase (11). Sph is not produced by de novo synthesis (12), and thus the activity of CDase is crucial not only for switching off the Cer-induced signaling but also for generation of Sph and S1P. CDase is classified into two categories: acid and neutral/alkaline enzymes depending on pH optimum. Acid CDase is thought to be a housekeeping enzyme to catabolize Cer in lysosomes. The enzyme was purified from human urine (13), and cDNA encoding the enzyme was isolated from cDNA libraries of human (14) and mouse (15). A deficiency of the enzyme could cause Farber disease in which Cer is accumulated in lysosomes (16). Neutral/alkaline CDase seems...