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J. Biol. Chem., Vol. 279, Issue 28, 29351-29358, July 9, 2004

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Conserved Amino Acid Residues in the COOH-terminal Tail Are Indispensable for the Correct Folding and Localization and Enzyme Activity of Neutral Ceramidase^*

Motohiro Tani, Nozomu Okino, Noriyuki Sueyoshi, and Makoto Ito

From the Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Several lines of evidence suggest that neutral ceramidase is involved in the regulation of ceramide-mediated signaling. Recently, the enzymes from mouse and rat were found to be localized at plasma membranes as a type II integral membrane protein, occasionally being detached from the cells after proteolytic processing of the NH₂-terminal anchoring region (Tani, M., Iida, H., and Ito, M. (2003) J. Biol. Chem. 278, 10523–10530). We report here that conserved hydrophobic amino acid residues in the COOH-terminal tail are indispensable for the correct folding and localization, and enzyme activity of neutral ceramidase. Truncation of four, but not three, amino acid residues from the COOH terminus of rat neutral ceramidase resulted in a complete loss of enzyme activity as well as cell surface expression in HEK293 cells. Point mutation analysis revealed that Ile⁷⁵⁸, the 4^th amino acid residue from the COOH terminus, and Phe⁷⁵⁶ are essential for the enzyme to function. The truncated and mutated enzymes were found to be retained in the endoplasmic reticulum (ER) and rapidly degraded without transportation to the Golgi apparatus. Treatment of the cells expressing the aberrant COOH-terminal enzyme with MG-132, a specific inhibitor for the proteasome, increased the accumulation of the enzyme in the ER, indicating that the misfolded enzyme was degraded by the proteasome. It was also found that the COOH-terminal tail was indispensable for the enzyme activity and correct folding of the prokaryote ceramidase from Pseudomonas aeruginosa, indicating that the importance of the COOH-terminal tail of the enzyme has been preserved through evolution.

Received for publication, April 12, 2004 , and in revised form, April 22, 2004.

^* This work was supported in part by a Grant-in-aid for Scientific Research on Priority Area (12140204) and Research B (15380073) from the Ministry of Education, Culture, Sport, Science and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Fax: 81-92-642-2907; E-mail: makotoi{at}agr.kyushu-u.ac.jp.

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