title: System to quantify the import of peroxisomal matrix proteins by fluorescence intensity.
abstract: 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.
authors.affiliation: Graduate School of Systems Life Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Noguchi
authors.forename: Kanji
authors.initials: K
authors.lastname: Okumoto
authors.forename: Yukio
authors.initials: Y
authors.lastname: Fujiki
mesh_terms: D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008163:Luminescent Measurements; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex