| Research Abstract |
With the support of this Giant-in Aid for Scientific Research 09044094, we obtained two major types of findings :
1) Isolation, characterization, and complementation group analysis of novel CHO cell mutants defective in peroxisome biogenesis.
In addition to the previously isolated, three complementation groups (CGs) of peroxisome-deficient CHO cell mutants, ZP24, Z65, and ZP92, we isolated and classified in CGs novel mutant cell lines, including ZP107 of CG1 (the same group as Z24), CG2, ZP105/ZP139, CG3, ZP109, ZP110, ZP114, CG-J, ZP119, CG8, ZP124, ZP126, CG-H, ZP128, CG11, ZPG207, and ZPG208 by the P9OH/UV method. ZP110, ZP114, ZP126, and ZPG208 were found to be distinct from human 13 CDs of fibroblasts from patients with peroxisome biogenesis disorder (PBDs) such as Zellweger syndrome, indicating that totally 17 genotypes are present in mammals. Thus, it is evident that peroxisome assembly requires at least 17 gene-products.
2) Cloning of novel peroxin genes
By genetic functional complementation assay of newly isolated CHO cell mutants, we cloned several peroxin cDNAs (PEXs), including PEX1, PEX12, PEX13, PEX14, and PEX19 for ZP107, ZP109, ZP128, ZP110, and ZP119 respectively. We then delineated that gene mutants in PEX1, PEX12, PEX13, and PEX19 are responsible for PBDs of CG1(E), CG3, CG-H, and CG-J, respectively. Moreover, we isolated PEX10 and PEX16 by expressed sequence tag (EST) DNA search using yeast genes and demonstrated that inactivation of PEX10 and PEX16 is the genetic cause of CG7 (B) and CG9 (D) PBDs, respectively. We also found two isoforms of the peroxin Pex5p (PTS1 receptor) using CG2 mutants, ZP105 and ZP139, demonstrating the longer Pex5p to be involved in PTS2 import as well. Moreover, we showed the evidence that peroxisomal membrane protein Pex14p plays a key role in import of PTS1 and PTS2 proteins into peroxisomes.
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