Molecular anatomy of the peroxin Pex12p - Ring finger domain is essential for Pex12p function and interacts with the peroxisome-targeting signal type1-receptor Pex5p and a ring peroxin, Pex10p

The three peroxin genes, PEX12, PEX2, and PEX10, encode peroxisomal integra l membrane proteins with RING finger at the C-terminal part and are respons ible for human peroxisome biogenesis disorders. Mutation analysis in PEX12 of Chinese hamster ovary cell mutants revealed a homozygous nonsense mutati on at residue Trp263Ter in ZP104 cells and a pair of heterozygous nonsense mutations, Trp170Ter and Trp114Ter, in ZP109. This result and domain mappin g of Pex12p showed that RING finger is essential for peroxisome-restoring a ctivity of Pex12p but not necessary for targeting to peroxisomes. The N-ter minal region of Pex12p, including amino acid residues at positions 17-76, w as required for localization to peroxisomes, while the sequence 17-76 was n ot sufficient for peroxisomal targeting. Peroxins interacting with RING fin ger of Pex2p, Pex10p, and Pex12p were investigated by yeast two-hybrid as w ell as in vitro binding assays. The RING finger of Pex12p bound to Pex10p a nd the PTS1-receptor Pex5p. Pex10p also interacted with Pex2p and Pex5p in vitro. Moreover, Pex12p was co-immunoprecipitated with Pex10p from CHO-K1 c ells, where Pex5p was not associated with the Pex12p-Pex10p complex. This o bservation suggested that Pex5p does not bind to, or only transiently inter acts with, Pex10p and Pex12p when Pex10p and Pex12p are in the oligomeric c omplex in peroxisome membranes. Hence, the RING finger peroxins are most li kely to be involved in Pex5p-mediated matrix protein import into peroxisome s.