Newly Identified Chinese Hamster Ovary Cell Mutants Are Defective in Biogenesis of Peroxisomal Membrane Vesicles (Peroxisomal Ghosts), Representing a Novel Complementation Group in Mammals

Abstract

We isolated peroxisome biogenesis-defective mutants from Chinese hamster ovary cells by the 9-(1-pyrene)nonanol/ultraviolet (P9OH/UV) method. Seven cell mutants, ZP116, ZP119, ZP160, ZP161, ZP162, ZP164, and ZP165, of 11 P9OH/UV-resistant cell clones showed cytosolic localization of catalase, a peroxisomal matrix enzyme, apparently indicating a defect of peroxisome biogenesis. By transfection of PEX cDNAs and cell fusion analysis, mutants ZP119 and ZP165 were found to belong to a novel complementation group (CG), distinct from earlier mutants. CG analysis by cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome indicated that ZP119 and ZP165 were in the same CG as the most recently identified human CG-J. The peroxisomal matrix proteins examined, including PTS1 proteins as well as a PTS2 protein, 3-ketoacyl-CoA thiolase, were also found in the cytosol in ZP119 and ZP165. Furthermore, these mutants showed typical peroxisome assembly-defective phenotype such as severe loss of resistance to 12-(1-pyrene)dodecanoic acid/UV treatment. Most strikingly, peroxisomal reminiscent vesicular structures, so-called peroxisomal ghosts noted in all CGs of earlier Chinese hamster ovary cell mutants as well as in eight CGs of patients' fibroblasts, were not discernible in ZP119 and ZP165, despite normal synthesis of peroxisomal membrane proteins. Accordingly, ZP119 and ZP165 are the first cell mutants defective in import of both soluble and membrane proteins, representing the 14th peroxisomedeficient CG in mammals, including humans.

Functional domain mapping of peroxin Pex19p: interaction with Pex3p is essential for function and translocation

“…We verified various truncated mutants of tandem double hemagglutinin A (HA)-tagged Pex19p, termed HA 2 -Pex19p ( Fig. 1), for the activity in restoring the impaired peroxisomal membrane biogenesis in CHO pex19 ZP119 cells (Kinoshita et al, 1998;Matsuzono et al, 1999), deficient in endogenous Pex19p (supplementary material Fig. S1C, lane 1).…”

“…The molecular mechanisms of peroxisomal import of matrix proteins are well understood, whereas those involving membrane protein transport and membrane vesicle assembly remain elusive (Fujiki, 2000;Lazarow, 2003;. In peroxisome-deficient mutant cell lines, including pex3, pex16 and pex19 Chinese hamster ovary (CHO) cell mutants and fibroblasts from patients with peroxisome biogenesis disorders (PBD) of complementation groups (CGs) 12, 9, and 14, respectively, peroxisome membrane assembly is severely impaired, hence membrane structures such as the so-called 'peroxisomal ghosts' or membrane remnants are morphologically and biochemically undetectable (Ghaedi et al, 2000b;Honsho et al, 2002;Honsho et al, 1998;Jones et al, 2001;Kinoshita et al, 1998;Matsuzono et al, 1999;Muntau et al, 2000;South and Gould, 1999). It is of interest to note that membrane particles are observed in pex19 mutant cells of Pichia pastoris (Snyder et al, 1999), Yarrowia lipolytica (Lambkin and Rachubinski, 2001) and Y. lipolytica pex16 cells (Eitzen et al, 1997).…”

“…Peroxisome membrane assembly is initiated by at least three peroxins, Pex3p, Pex16p and Pex19p, followed by several distinct steps, including the import of membrane and matrix proteins as well as growth and division of peroxisomes. We earlier cloned human PEX19 cDNA encoding the 299 amino acid, hydrophilic peroxin Pex19p with the farnesylation motif CAAx at the C-terminus, by functional complementation strategy using a mutant CHO cell line, ZP119, defective in the import of both matrix and membrane proteins (Kinoshita et al, 1998;Matsuzono et al, 1999). Pex19p is localized, mostly in the cytosol and partly associated with peroxisomes (Goette et al, 1998;James et al, 1994;Matsuzono et al, 1999;Snyder et al, 1999).…”

Disruption of the Interaction of the Longer Isoform of Pex5p, Pex5pL, with Pex7p Abolishes Peroxisome Targeting Signal Type 2 Protein Import in Mammals

“…Three pex1, four pex2, and one pex14 mutant clones were identified; mutant ZPG217 was not in any of these mutant CGs (Table I). TKaG2 cells were used to avoid possible isolation of pex2 mutants, but several PEX2-defective mutants were isolated as in our earlier studies (6,22,27). The integrated rat PEX2 cDNA appears to be occasionally disintegrated.…”

Defective lipid remodeling of GPI anchors in peroxisomal disorders, Zellweger syndrome, and rhizomelic chondrodysplasia punctata

“…It is also likely that cells from patients with ZS are defective in generating 1-alkyl-2-acyl GPI. In this study, we investigated these hypotheses using CHO cells defective in PEX7, PEX5, or PEX19 and fi broblasts from patients with RCDP types 1, 2, or 3, or ZS (21)(22)(23) …”