Okumoto K, Shimozawa N, Kawai A, Tamura S, Tsukamoto T, Osumi T, Moser H, Wanders R J, Suzuki Y, Kondo N, Fujiki Y
Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
Mol Cell Biol. 1998 Jul;18(7):4324-36. doi: 10.1128/MCB.18.7.4324.
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.
通过对突变型CHO细胞系ZP109（K. Okumoto、A. Bogaki、K. Tateishi、T. Tsukamoto、T. Osumi、N. Shimozawa、Y. Suzuki、T. Orii和Y. Fujiki，《实验细胞研究》233:11 - 20，1997）的过氧化物酶体缺陷进行功能互补，利用瞬时转染分析和一个异位的、易于观察的标记物绿色荧光蛋白，分离出大鼠PEX12 cDNA。该cDNA编码一种359个氨基酸的过氧化物酶体膜蛋白，具有两个跨膜结构域和一个富含半胱氨酸的锌指结构，即RING基序。用PEX12构建的ZP109稳定转化体在形态学和生物化学上恢复了过氧化物酶体生物发生。通过表达真实的以及带有表位标签的Pex12p表明，Pex12p将其N端和C端区域都暴露于细胞质中。来自III型互补组（CG - III）过氧化物酶体缺陷的齐-韦二氏综合征患者的成纤维细胞，也通过PEX12恢复了过氧化物酶体生物发生。该组两名无关的表现出过氧化物酶体缺陷疾病的患者具有纯合的、失活的PEX12突变：一名患者发生一个点突变，导致Arg180Thr，另一名患者在291Asn和292Ser的密码子中缺失两个核苷酸，产生一个明显未改变的Asn密码子和一个292位的终止密码子。这些结果表明，编码过氧化物酶体组装因子Pex12p的基因是CG - III型过氧化物酶体缺陷的致病基因。此外，包括对患者PEX12分析在内的截短和位点突变研究表明，Pex12p面向细胞质的N端和C端部分对其生物学功能至关重要。
