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.