Pex5p stabilizes Pex14p: a study using a newly isolated pex5 CHO cell mutant, ZPEG101

2012 ◽  
Vol 449 (1) ◽  
pp. 195-207 ◽  
Author(s):  
Ryuichi Natsuyama ◽  
Kanji Okumoto ◽  
Yukio Fujiki
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Cell Mutant ◽  
Peroxisomal Membrane ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Signal Type ◽  
Mutant Phenotypes

Pex5p [PTS (peroxisome-targeting signal) type 1 receptor] plays an essential role in peroxisomal matrix protein import. In the present study, we isolated a novel PEX5-deficient CHO (Chinese-hamster ovary) cell mutant, termed ZPEG101, showing typical peroxisomal import defects of both PTS1 and PTS2 proteins. ZPEG101 is distinct from other known pex5 CHO mutants in its Pex5p expression. An undetectable level of Pex5p in ZPEG101 results in unstable Pex14p, which is due to inefficient translocation to the peroxisomal membrane. All of the mutant phenotypes of ZPEG101 are restored by expression of wild-type Pex5pL, a longer form of Pex5p, suggesting a role for Pex5p in sustaining the levels of Pex14p in addition to peroxisomal matrix protein import. Complementation analysis using various Pex5p mutants revealed that in the seven pentapeptide WXXXF/Y motifs in Pex5pL, known as the multiple binding sites for Pex14p, the fifth motif is an auxiliary binding site for Pex14p and is required for Pex14p stability. Furthermore, we found that Pex5p–Pex13p interaction is essential for the import of PTS1 proteins as well as catalase, but not for that of PTS2 proteins. Therefore ZPEG101 with no Pex5p would be a useful tool for investigating Pex5p function and delineating the mechanisms underlying peroxisomal matrix protein import.

scholarly journals Peroxisome assembly factor 1: nonsense mutation in a peroxisome-deficient Chinese hamster ovary cell mutant and deletion analysis.

1994 ◽  
Vol 14 (8) ◽  
pp. 5458-5465 ◽  
Author(s):  
T Tsukamoto ◽  
N Shimozawa ◽  
Y Fujiki
Keyword(s):  
Amino Acids ◽  
Chinese Hamster Ovary ◽  
Nonsense Mutation ◽  
Chinese Hamster ◽  
General Rule ◽  
Ovary Cell ◽  
Cell Mutant ◽  
Peroxisomal Membrane ◽  
C Terminus ◽  
Assembly Factor

A cDNA encoding 35-kDa peroxisome assembly factor 1 (PAF-1), a peroxisomal integral membrane protein, was cloned from Chinese hamster ovary (CHO) cells and sequenced. The CHO PAF-1 comprised 304 amino acids, one residue shorter than rat or human PAF-1, and showed high homology to rat and human PAF-1: 90 and 86% at the nucleotide sequence level and 92 and 90% in amino acid sequence, respectively. PAF-1 from these three species contains a conserved cysteine-rich sequence at the C-terminal region which is exactly the same as that of a novel cysteine-rich RING finger motif family. PAF-1 cDNA from a peroxisome-deficient CHO cell mutant, Z65 (T. Tsukamoto, S. Yokota, and Y. Fujiki, J. Cell Biol. 110:651-660, 1990), contained a nonsense mutation at the codon for Trp-114, resulting in premature termination. Truncation in PAF-1 of either 19 amino acids from the N terminus or 92 residues from the C terminus maintained the peroxisome assembly-restoring activity when tested in both the Z65 mutant and the fibroblasts from a Zellweger patient. In contrast, deletion of 27 or 102 residues from the N or C terminus eliminated the activity. PAF-1 is encoded by free polysomal RNA, consistent with a general rule for biogenesis of peroxisomal proteins, including membrane polypeptides, implying the posttranslational transport and integration of PAF-1 into peroxisomal membrane.

Peroxisome assembly factor 1: nonsense mutation in a peroxisome-deficient Chinese hamster ovary cell mutant and deletion analysis

1994 ◽  
Vol 14 (8) ◽  
pp. 5458-5465
Author(s):  
T Tsukamoto ◽  
N Shimozawa ◽  
Y Fujiki
Keyword(s):  
Amino Acids ◽  
Chinese Hamster Ovary ◽  
Nonsense Mutation ◽  
Chinese Hamster ◽  
General Rule ◽  
Ovary Cell ◽  
Cell Mutant ◽  
Peroxisomal Membrane ◽  
C Terminus ◽  
Assembly Factor

A cDNA encoding 35-kDa peroxisome assembly factor 1 (PAF-1), a peroxisomal integral membrane protein, was cloned from Chinese hamster ovary (CHO) cells and sequenced. The CHO PAF-1 comprised 304 amino acids, one residue shorter than rat or human PAF-1, and showed high homology to rat and human PAF-1: 90 and 86% at the nucleotide sequence level and 92 and 90% in amino acid sequence, respectively. PAF-1 from these three species contains a conserved cysteine-rich sequence at the C-terminal region which is exactly the same as that of a novel cysteine-rich RING finger motif family. PAF-1 cDNA from a peroxisome-deficient CHO cell mutant, Z65 (T. Tsukamoto, S. Yokota, and Y. Fujiki, J. Cell Biol. 110:651-660, 1990), contained a nonsense mutation at the codon for Trp-114, resulting in premature termination. Truncation in PAF-1 of either 19 amino acids from the N terminus or 92 residues from the C terminus maintained the peroxisome assembly-restoring activity when tested in both the Z65 mutant and the fibroblasts from a Zellweger patient. In contrast, deletion of 27 or 102 residues from the N or C terminus eliminated the activity. PAF-1 is encoded by free polysomal RNA, consistent with a general rule for biogenesis of peroxisomal proteins, including membrane polypeptides, implying the posttranslational transport and integration of PAF-1 into peroxisomal membrane.

Metabolic control of peroxisome abundance

1999 ◽  
Vol 112 (10) ◽  
pp. 1579-1590 ◽  
Author(s):  
C.C. Chang ◽  
S. South ◽  
D. Warren ◽  
J. Jones ◽  
A.B. Moser ◽  
...  
Keyword(s):  
Metabolic Control ◽  
Matrix Protein ◽  
Protein Import ◽  
Zellweger Syndrome ◽  
Mutant Gene ◽  
Peroxisomal Membrane ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Complementation Groups ◽  
Peroxisomal Targeting Signal

Zellweger syndrome and related disorders represent a group of lethal, genetically heterogeneous diseases. These peroxisome biogenesis disorders (PBDs) are characterized by defective peroxisomal matrix protein import and comprise at least 10 complementation groups. The genes defective in seven of these groups and more than 90% of PBD patients are now known. Here we examine the distribution of peroxisomal membrane proteins in fibroblasts from PBD patients representing the seven complementation groups for which the mutant gene is known. Peroxisomes were detected in all PBD cells, indicating that the ability to form a minimal peroxisomal structure is not blocked in these mutants. We also observed that peroxisome abundance was reduced fivefold in PBD cells that are defective in the PEX1, PEX5, PEX12, PEX6, PEX10, and PEX2 genes. These cell lines all display a defect in the import of proteins with the type-1 peroxisomal targeting signal (PTS1). In contrast, peroxisome abundance was unaffected in cells that are mutated in PEX7 and are defective only in the import of proteins with the type-2 peroxisomal targeting signal. Interestingly, a fivefold reduction in peroxisome abundance was also observed for cells lacking either of two PTS1-targeted peroxisomal beta-oxidation enzymes, acyl-CoA oxidase and 2-enoyl-CoA hydratase/D-3-hydroxyacyl-CoA dehydrogenase. These results indicate that reduced peroxisome abundance in PBD cells may be caused by their inability to import these PTS1-containing enzymes. Furthermore, the fact that peroxisome abundance is influenced by peroxisomal 105-oxidation activities suggests that there may be metabolic control of peroxisome abundance.

scholarly journals Pex24 and Pex32 are required to tether peroxisomes to the ER for organelle biogenesis, positioning and segregation in yeast

2020 ◽  
Vol 133 (16) ◽  
pp. jcs246983 ◽  
Author(s):  
Fei Wu ◽  
Rinse de Boer ◽  
Arjen M. Krikken ◽  
Arman Akşit ◽  
Nicola Bordin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Matrix Protein ◽  
Protein Import ◽  
Hansenula Polymorpha ◽  
Major Effect ◽  
Organelle Biogenesis ◽  
Peroxisomal Membrane ◽  
Contact Sites ◽  
Membrane Growth

ABSTRACTThe yeast Hansenula polymorpha contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four H. polymorpha Pex23 family proteins localize to the endoplasmic reticulum (ER). Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome–ER contacts. Upon deletion of PEX24 or PEX32 – and to a much lesser extent, of PEX23 or PEX29 – peroxisome–ER contacts are lost, concomitant with defects in peroxisomal matrix protein import, membrane growth, and organelle proliferation, positioning and segregation. These defects are suppressed by the introduction of an artificial peroxisome–ER tether, indicating that Pex24 and Pex32 contribute to tethering of peroxisomes to the ER. Accumulation of Pex32 at these contact sites is lost in cells lacking the peroxisomal membrane protein Pex11, in conjunction with disruption of the contacts. This indicates that Pex11 contributes to Pex32-dependent peroxisome–ER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 deletion cells.

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