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

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 Kanji Okumoto 1 , Nobuyuki Shimozawa 2 , Atsusi Kawai 1 , Shigehiko Tamura 1 , Toshiro Tsukamoto 3 , Takashi Osumi 3 , Hugo Moser 4 , Ronald J. A. Wanders 5 , Yasuyuki Suzuki 2 , Naomi Kondo 2 , and Yukio Fujiki 1 , 6 , * Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-8581, 1 Department of Pediatrics, Gifu University School of Medicine, Gifu 500-8076, 2 Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo 678-1297, 3 and CREST, Japan Science and Technology Corporation, Tokyo 170-0013, 6 Japan; Kennedy-Krieger Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 4 ; and Department of Pediatrics, Academic Medical Centre, University of Amsterdam, 1100DE Amsterdam, The Netherlands 5 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 PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for 291 Asn and 292 Ser, 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 PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.