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A chelator-sensitive protease in the mitochondrial matrix of the yeast, Saccharomyces cerevisiae (Biochem. Biophys. Res. Commun. 144, 277, 1987), was purified and characterized. The purified enzyme, termed protease M, specifically hydrolyzes peptide substrates on the N-side of the paired basic residues. When mastoparan was used as substrate, it cleaved Ala8-Leu9 and Lys11-Lys12 bonds as well as the N-side of Lys11-Lys12 residues. Nucleotide triphosphates stimulated the activity 3-fold at 2.5 mM. The genomic DNA sequence showed that protease M was a gene product of CYM1 known as mitochondrial presequence protease homologue in S. cerevisiae , encoding a 989-amino acid-long precursor protein. The N-terminal sequence of the purified enzyme indicated that protease M has 16-residue signal sequence and the 'mature' protein consists of 973 amino acids with a molecular mass of 110 kDa. Protease M contained consensus sequence motifs of ATP-binding site very near the carboxyl terminus. The alignment of the two ATP-binding motifs is an inverted version of the common alignment. Gene disruption of the enzyme generates mixed subunits in tetrameric MnSOD formed with 23-kDa mature and 24-kDa partial presequence-containing subunits. This report describes newly identified enzyme properties of the CYM1 gene product, protease M and abnormal MnSOD complex formation of the disruption mutant. [ABSTRACT FROM AUTHOR]

Ether glycerolipids, plasmalogens are found in various mammalian cells and tissues. However, physiological role of plasmalogens in epithelial cells remains unknown. We herein show that synthesis of ethanolamine-containing plasmalogens, plasmenylethanolamine (PlsEtn), is deficient in MCF7 cells, an epithelial cell line, with severely reduced expression of alkyl-dihydroxyacetonephosphate synthase (ADAPS), the second enzyme in the PlsEtn biosynthesis. Moreover, expression of ADAPS or supplementation of PlsEtn containing C18-alkenyl residue delays the migration of MCF7 cells as compared to that mock-treated MCF7 and C16-alkenyl-PlsEtn-supplemented MCF7 cells. Localization of E-cadherin to cell–cell junctions is highly augmented in cells containing C18-alkenyl-PlsEtn. Together, these results suggest that PlsEtn containing C18-alkenyl residue plays a distinct role in the integrity of E-cadherin-mediated adherens junction. [ABSTRACT FROM AUTHOR]

Synthesis of ethanolamine plasmalogen (PlsEtn) is regulated by modulating the stability of fatty acyl-CoA reductase 1 (Far1) on peroxisomal membrane, a rate-limiting enzyme in plasmalogen synthesis. Dysregulation of plasmalogen homeostasis impairs cholesterol biosynthesis in cultured cells by altering the stability of squalene epoxidase (SQLE). However, regulation of PlsEtn synthesis and physiological consequences of plasmalogen homeostasis in tissues remain unknown. In the present study, we found that the protein but not the transcription level of Far1 in the cerebellum of the Pex14 mutant mouse expressing Pex14p lacking its C-terminal region (Pex14ΔC/ΔC) is higher than that from wild-type mouse, suggesting that Far1 is stabilized by the lowered level of PlsEtn. The protein level of SQLE was increased, whereas the transcriptional activity of the liver X receptors (LXRs), ligand-activated transcription factors of the nuclear receptor superfamily, is lowered in the cerebellum of Pex14ΔC/ΔC and the mice deficient in dihydroxyacetonephosphate acyltransferase, the initial enzyme for the synthesis of PlsEtn. These results suggest that the reduction of plasmalogens in the cerebellum more likely compromises the cholesterol homeostasis, thereby reducing the transcriptional activities of LXRs, master regulators of cholesterol homeostasis. [ABSTRACT FROM AUTHOR]

A newly isolated binding protein of peroxisomal targeting signal type 2 (PTS2) receptor Pex7, termed P7BP2, is transported into peroxisomes by binding to the longer isoform of Pex5p, Pex5pL, via Pex7p. The binding to Pex7p and peroxisomal localization of P7BP2 depends on the cleavable PTS2 in the N-terminal region, suggesting that P7BP2 is a new PTS2 protein. By search on human database, three AAA+ domains are found in the N-terminal half of P7BP2. Protein sequence alignment and motif search reveal that in the C-terminal region P7BP2 contains additional structural domains featuring weak but sufficient homology to AAA+ domain. P7BP2 behaves as a monomer in gel-filtration chromatography and the single molecule observed under atomic force microscope shapes a disc-like ring. Collectively, these results suggest that P7BP2 is a novel dynein-type AAA+ family protein, of which domains are arranged into a pseudo-hexameric ring structure. [ABSTRACT FROM AUTHOR]

Peroxisome-proliferator activated receptor α (PPARα) is a ligand-activated transcription factor, playing a key role in several essential pathways including lipid metabolism. Although nuclear localization of PPARα is essential for its transactivation activity, mechanisms underlying intracellular traffics of PPARα remain undefined. We here identify and characterize a nuclear localization signal (NLS) residing in the junction between DNA-binding domain and hinge regions of PPARα. The NLS consists of two basic-amino acid clusters locating in the sequence encompassing amino acid residues at 144–187. We evidently show by mutational analysis that the basic residues in this NLS are essential for the nuclear import. Moreover, the PPARα NLS binds well-known nuclear transporters, importin α and importin β, in a manner independent of DNA-binding activity. [ABSTRACT FROM PUBLISHER]

We earlier isolated peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, ZPEG241, by the 9-(1′-pyrene)nonanol/ultraviolet selection method, from TKaEG2, the wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal type 2 (PTS2)-tagged enhanced green fluorescent protein (EGFP). Peroxisomal localization of PTS2-EGFP was specifically impaired in ZPEG241 due to the failure of Pex5pL expression. Analysis of partial genomic sequence of PEX5 revealed one-point nucleotide-mutation from G to A in the 3′-acceptor splice site located at 1 nt upstream of exon 7 encoding Pex5pL specific 37-amino acid insertion, thereby generating 21-nt deleted mRNA of PEX5L in ZPEG241. When ZPEG241-derived Pex5pL was ectopically expressed in ZPEG241, PTS2 import was not restored because of no interaction with Pex7p. Together, we confirm the pivotal role of Pex5pL in PTS2 import, showing that the N-terminal 7-amino acid residues in the 37-amino acid insertion of Pex5pL are essential for the binding to Pex7p. [ABSTRACT FROM PUBLISHER]

We isolated peroxisome biogenesis mutants ZP128 and ZP150 from rat PEX2-transformed Chinese hamster ovary (CHO) cells, by the 9-(1"-pyrene)nonanol/ultraviolet method. The mutants lacked morphologically recognizable peroxisomes and showed a typical peroxisome assembly-defective phenotype such as a high sensitivity to 12-(1"-pyrene)dodecanoic acid/UV treatment. By means of PEX cDNA transfection and cell fusion, ZP128 and ZP150 were found to belong to a recently identified complementation group H. Expression of human PEX13 cDNA restored peroxisome assembly in ZP128 and ZP150. CHO cell PEX13 was isolated; its deduced sequence comprises 405 amino acids with 93% identity to human Pex13p. Mutation in of PEX13 mutant ZP150 was determined by RT-PCR: G to A transition resulted in one amino acid substitution, Ser319Asn, in one allele and truncation of a 42 amino acid sequence from Asp265 to Lys306 in another allele. Therefore, ZP128 and ZP150 are CHO cell lines with a phenotype of impaired PEX13. [ABSTRACT FROM AUTHOR]

Examines the hypothesis that nonsense and temperature-sensitive mutations in the human cDNA encoding PEX13 are the cause of complementation group H of peroxisome biogenesis disorders. Analysis of fibroblasts from group H patients; Expression of hsPEX13; Mutation analysis and TS mutation of hsPEX13 in group H.

The peroxisome biogenesis factor, peroxin Pex2p, is an integral membrane protein of peroxisomes [Tsukamoto, T., Miura, S., and Fujiki, Y. (1991) Nature 350,77–81]. As a step toward elucidating the structure and biological function of Pex2p, we determined the transmembrane topology of Pex2p by expressing epitope-tagged rat Pex2p in COS-7 cells. Pex2p tagged with myc at the C-terminus was detected as a punctate staining pattern, when the cells were permeabilized with 50 μg/ml of digitonin, under which conditions intra-peroxisomal proteins such as PTSl-proteins are inaccessible to exogenous antibodies. N-terminally flag-tagged Pex2p was likewise detected upon the same treatment. These results strongly suggest that both the N- and C-terminal parts of Pex2p are exposed to the cytosol. The transmembrane orientation of Pex2p was also assessed by using rat liver peroxisomes and Pex2p region-specific antibodies. The two types of antibodies used, raised to the N-(amino acid residues 1-131) and C-terminal part (residues 226 to the C-terminus), respectively, specifically recognized Pex2p and immunoprecipitated intact, whole peroxisomes. Pex2p was not recognized by the antibodies when the peroxisomes were treated with Proteinase K. Furthermore, in situ crosslinking studies involving bifunctional reagents revealed an apparently dimeric form of Pex2p. Therefore, Pex2p is anchored to the peroxisomal membrane by two membrane-spanning segments, with its N- and C-terminal regions exposed to the cytosol. [ABSTRACT FROM AUTHOR]

Demonstrates that mutation in PEX10 is the genetic cause of complementation group B peroxisome biogenesis disorders. Cloning of human PEX10 cDNA; Immunostaining patterns of peroxisome targeting signal type 1 proteins in fibroblasts; Intracellular localization and topology of Pex10p.

Investigates the molecular mechanisms involved in temperature-sensitive mutation in peroxisome biogenesis (PB) and the genetic cause of PB disorders. Functional importance of human peroxisomes; Identification of a missense PEX1 mutant allele; Activity of peroxisome-specific enzymes.