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 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 291Asn and 292Ser, 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.
Okumoto
K
K
Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
Shimozawa
N
N
Kawai
A
A
Tamura
S
S
Tsukamoto
T
T
Osumi
T
T
Moser
H
H
Wanders
R J
RJ
Suzuki
Y
Y
Kondo
N
N
Fujiki
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D006224:Cricetinae; D003600:Cytosol; D018076:DNA, Complementary; D005347:Fibroblasts; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D008830:Microbodies / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D018901:Peroxisomal Disorders / Q000662:veterinary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015211:Zellweger Syndrome / Q000235:genetics*; D016335:Zinc Fingers
D018076:DNA, Complementary; D008565:Membrane Proteins; C105251:PEX12 protein, human; C491336:Pex12 protein, rat