PEX12, the Pathogenic Gene of Group III Zellweger Syndrome: cDNA Cloning by Functional Complementation on a CHO Cell Mutant, Patient Analysis, and Characterization of Pex12p

ABSTRACT

Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11–20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for ²⁹¹Asn and²⁹²Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.

ACKNOWLEDGMENTS

We thank T. Hashimoto for anti-human catalase antiserum and T. Harano, K. Mizuno, and M. Ohara for comments.

This work was supported in part by a CREST grant (to Y.F.) from the Science and Technology Corporation of Japan; Grants-in-Aid for Scientific Research (07408016, 08249232, and 08557011 to Y.F.) from the Ministry of Education, Science, Sports and Culture; and grants (to Y.F.) from the Mitsubishi Foundation, the Uehara Foundation, and the Nagase Science and Technology Foundation.