Peroxisome biogenesis and human peroxisome-deficiency disorders

Abstract

Peroxisome is a single-membrane-bounded ubiquitous organelle containing a hundred different enzymes that catalyze various metabolic pathways such as β-oxidation of very long-chain fatty acids and synthesis of plasmalogens. To investigate peroxisome biogenesis and human peroxisome biogenesis disorders (PBDs) including Zellweger syndrome, more than a dozen different complementation groups of Chinese hamster ovary (CHO) cell mutants impaired in peroxisome biogenesis are isolated as a model experimental system. By taking advantage of rapid functional complementation assay of the CHO cell mutants, successful cloning of PEX genes encoding peroxins required for peroxisome assembly invaluably contributed to the accomplishment of cloning of pathogenic genes responsible for PBDs. Peroxins are divided into three groups: 1) peroxins including Pex3p, Pex16p and Pex19p, are responsible for peroxisome membrane biogenesis via Pex19p- and Pex3p-dependent class I and Pex19p- and Pex16p-dependent class II pathways; 2) peroxins that function in matrix protein import; 3) those such as Pex11pβ are involved in peroxisome division where DLP1, Mff, and Fis1 coordinately function.

Figure 1.

Morphology of peroxisomes in CHO cell mutants defective in peroxisome biogenesis and pathogenic gene cloning of PBDs. (A) CHO cells are stained with antibodies to PMP70 (a-c) and PTS1 (d-f). Cells are as indicated at the top. Scale bar, 20 µm. pex2 Z65 contains PMP70-positive peroxisomal remnants, whereas pex19 ZP119 is absent from such peroxisome ghosts, indicative of the defect of membrane protein import. PTS1 proteins are discernible in the cytosol in pex2 Z65 and pex19 ZP119 cells, in contrast to the wild-type CHO-K1 cells where PTS1 proteins are in peroxisomes. (B) Cloning of pathogenic gene responsible for PBD. Peroxisome-restoring PEX genes were isolated by functional phenotype-complementation assay using CHO mutants. Restoration of peroxisomes was searched by transfection of rat liver cDNA library (a) in Z65 (b). Transformed cells positive in catalase import contained PEX2 (formerly PAF-1). In fibroblasts from a patient with ZS of CG10 (d), expression of PEX2 restored the impaired import of catalase (c). Scale bar, 20 µm (a and b); 30 µm (c and d).

Figure 2.

A schematic view of peroxisome biogenesis in mammalian cells. The subcellular localization and molecular characteristics of peroxins are shown. Peroxins are classified into three groups: 1) peroxins including Pex3p, Pex16p and Pex19p that are responsible for peroxisome membrane assembly via classes I and II pathways required for matrix protein import; 2) those required for matrix protein import; 3) those such as three forms of Pex11p, Pex11pα, Pex11pβ, and Pex11pγ, apparently involved in peroxisome division where DLP1, Mff, and Fis1 coordinately function. PTS1 and PTS2 matrix proteins are recognized by Pex5p and Pex7p, respectively, in the cytoplasm. Two isoforms, Pex5pS and Pex5pL with an internal 37-amino-acid insertion, of Pex5p are identified in mammals. PTS1 proteins are transported by homo- and hetero-oligomers of Pex5pS and Pex5pL to peroxisomes, where Pex14p functions as a convergent, initial docking site of the ‘protein import machinery’ translocon. Pex5pL directly interacts with the PTS2 receptor, Pex7p, carrying its cargo PTS2 protein in the cytosol and translocates the Pex7p-PTS2 protein complex to Pex14p. PTS1 and PTS2 proteins are then released at the inner surface and/or inside of peroxisomes, downstream Pex14p and upstream Pex13p. Pex5p and Pex7p subsequently translocate to other translocon constituents, named translocation complex consisting of the RING peroxins, Pex2p, Pex10p, and Pex12p. Both Pex5p and Pex7p finally shuttle back to the cytosol. At the terminal step of the matrix protein import reaction, Pex1p and Pex6p of the AAA family catalyze the export of Pex5p, where Cys-monoubiquitination of Pex5p is a prerequisite to the Pex5p exit. Moreover, a cytosolic factor, AWP1/ZFAND6 (p40), is involved in the export of Ub-Pex5p in mammals.