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Exp Cell Res. 2001 Feb 1;263(1):98-106.

Quantitative analysis of peroxisomal protein import in vitro.

Author information

1
Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, Michigan 48201, USA. s.r.terlecky@wayne.edu

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.

PMID:
11161709
DOI:
10.1006/excr.2000.5111
[Indexed for MEDLINE]

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