title: Quantitative analysis of peroxisomal protein import in vitro.
abstract: Protein import into the peroxisome matrix is mediated by peroxisome-targeting signals (PTSs). We have developed a novel, quantitative, in vitro assay for measuring peroxisomal import of PTS1-containing proteins. This enzyme-linked immunosorbent assay-based system utilizes semi-intact human A431 cells or fibroblasts and a biotinylated version of the PTS1-containing import substrate, luciferase. We show that biotinylated luciferase accumulated in peroxisomes in a time- and temperature-dependent fashion, in a reaction stimulated by exogenously added ATP, cytosol, and zinc. No import was detected in fibroblasts from a human patient belonging to complementation group 2, who suffered from the fatal peroxisomal disorder Zellweger syndrome and lacked a functional PTS1 receptor, Pex5p. Also, the reaction was significantly inhibited by antibodies to the zinc-finger protein, Pex2p. Several lines of evidence demonstrate that biotinylated luciferase was imported into the lumen of bona fide peroxisomes. (a) Biochemical fractionation of cells after the import reaction showed a time-dependent accumulation of the import substrate within intracellular organelles. (b) Confocal fluorescence microscopy indicated that imported biotinylated luciferase colocalized with the peroxisomal protein PMP70. (c) Visualization of the imported biotinylated luciferase by indirect fluorescence or indirect immunofluorescence required disruption of the peroxisomal membrane, indicating true import rather than binding to the outside of the organelle.
authors.affiliation: Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, Michigan 48201, USA. s.r.terlecky@wayne.edu
authors.forename: S R
authors.initials: SR
authors.lastname: Terlecky
authors.forename: J E
authors.initials: JE
authors.lastname: Legakis
authors.forename: S E
authors.initials: SE
authors.lastname: Hueni
authors.forename: S
authors.initials: S
authors.lastname: Subramani
mesh_terms: D020816:Amino Acid Motifs; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D002458:Cell Fractionation; D002478:Cells, Cultured; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005347:Fibroblasts; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D020675:Peroxisomes / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*; D015211:Zellweger Syndrome / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D001360:Avidin; D001710:Biotin; D008156:Luciferases