System to quantify the import of peroxisomal matrix proteins by fluorescence intensity

Masafumi Noguchi, Kanji Okumoto, Yukio Fujiki

研究成果: ジャーナルへの寄稿記事

5 引用 (Scopus)

抄録

Fourteen distinct peroxins are essential for peroxisome biogenesis in mammals, of which ten are involved in the import of matrix proteins into peroxisomes. Peroxisomal matrix protein import is regulated by various cellular factors; however, the mechanisms underlying this regulation are poorly understood. This is primarily because no quantitative detection method with high resolution is available to study the import of peroxisomal matrix proteins. Here, we developed a monitoring system that uses a fluorescent reporter that is stabilized in peroxisomes but is degraded in the cytosol. An FK506 binding protein 12 variant, termed destabilization domain (DD), is rapidly and constitutively degraded by proteasomes when expressed in mammalian cells. DD is reversibly protected by the addition of a specific synthetic ligand. In the absence of the ligand, a reporter molecule, enhanced GFP (EGFP) fused with DD and peroxisomal targeting signal 1 (DD-EGFP-PTS1), is largely degraded in the cytosol. By contrast, in the presence of the ligand, the reporter is stabilized and translocates into peroxisomes. Upon withdrawal of the ligand, the reporter in peroxisomes remains intact, whereas that in the cytosol is rapidly degraded. Thus, peroxisomal protein import can be readily quantified by measuring the fluorescence intensity of whole cells.

元の言語英語
ページ(範囲)476-492
ページ数17
ジャーナルGenes to Cells
18
発行部数6
DOI
出版物ステータス出版済み - 6 1 2013

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Peroxisomes
Fluorescence
Cytosol
Ligands
Proteins
Tacrolimus Binding Proteins
Proteasome Endopeptidase Complex
Mammals

All Science Journal Classification (ASJC) codes

  • Genetics
  • Cell Biology

これを引用

System to quantify the import of peroxisomal matrix proteins by fluorescence intensity. / Noguchi, Masafumi; Okumoto, Kanji; Fujiki, Yukio.

:: Genes to Cells, 巻 18, 番号 6, 01.06.2013, p. 476-492.

研究成果: ジャーナルへの寄稿記事

Noguchi, Masafumi ; Okumoto, Kanji ; Fujiki, Yukio. / System to quantify the import of peroxisomal matrix proteins by fluorescence intensity. :: Genes to Cells. 2013 ; 巻 18, 番号 6. pp. 476-492.
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