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Peroxisome assembly factor 1: nonsense mutation in a peroxisome-deficient Chinese hamster ovary cell mutant and
T Tsukamoto1, N Shimozawa, Y Fujiki
1Meiji Institute of Health Science, Odawara, Kanagawa, Japan.
Abstract:
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.
Insights
Researchers cloned and sequenced Chinese hamster ovary (CHO) cell peroxisome assembly factor 1 (PAF-1). Truncations in PAF-1 maintained its peroxisome assembly-restoring activity, indicating specific regions are crucial for function.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Peroxisomes are essential organelles involved in various metabolic processes.
- Peroxisome assembly requires specific proteins, including peroxisome assembly factor 1 (PAF-1).
- Understanding PAF-1's structure-function relationship is key to comprehending peroxisome biogenesis.
Purpose of the Study:
- To clone and sequence the cDNA encoding Chinese hamster ovary (CHO) cell PAF-1.
- To investigate the functional domains of PAF-1 essential for peroxisome assembly.
- To analyze the impact of mutations and truncations on PAF-1's activity.
Main Methods:
- cDNA cloning and sequencing of CHO cell PAF-1.
- Site-directed mutagenesis to create truncated PAF-1 variants.
- Functional assays using peroxisome-deficient CHO cell mutants (Z65) and Zellweger patient fibroblasts.
Main Results:
- CHO PAF-1 cDNA was successfully cloned and sequenced, revealing high homology to rat and human PAF-1.
- A conserved cysteine-rich RING finger motif was identified in the C-terminal region.
- Specific N-terminal and C-terminal truncations (19 and 92 amino acids, respectively) retained peroxisome assembly-restoring activity, while larger deletions abolished it.
Conclusions:
- PAF-1 possesses distinct functional domains critical for peroxisome assembly.
- The cysteine-rich C-terminal region and specific N-terminal sequences are important for PAF-1 function.
- PAF-1 is synthesized on free polysomes, suggesting post-translational import into the peroxisomal membrane.