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

Rat PEX12 cDNA was isolated by functional complementation of peroxisom e 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 tra nsfection assay and an ectopic, readily visible marker, green fluoresc ent protein. This cDNA encodes a 359-amino-acid membrane protein of pe roxisomes with two transmembrane segments and a cysteine rich zinc fin ger, 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. Fibro blasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group LII (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this g roup 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 co don 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-m peroxi some deficiency. Moreover, truncation and site mutation studies, inclu ding patient PEX12 analysis, demonstrated that the cytoplasmically ori ented N- and C-terminal parts of Pex12p are essential for biological f unction.