Abstract
To identify proteins interacting with the C-terminal peroxisomal targeting signal (PTS1), we screened a human liver cDNA library by means of a Saccharomyces cerevisiae genetic system, known as the two-hybrid system. We isolated a cDNA encoding a protein that specifically bound the PTS1 topogenic signal in the intact yeast cell but also in vitro after bacterial expression and purification. Sequence analysis of the full-length cDNA revealed the presence of an open reading frame encoding a 70-kDa polypeptide that belongs to the tetratricopeptide repeat family and that is homologous to the PAS8 and PAS10 gene products, which are required for the formation of normal peroxisomes in yeast. Subcellular fractionation of human liver and immunofluorescence studies on HepG2 cells demonstrated that this PTS1-binding protein is present exclusively in peroxisomes and that the PTS1-binding domain is located to the cytosolic side of the peroxisomal membrane. All available evidence indicates that the PTS1-binding protein is part of the peroxisomal protein import machinery and most probably is the long sought after human PTS1 import receptor.
Highlights
Peroxisomes are single membrane-bound organelles found in almost all eukaryotic cells and involved in a variety of metabolic functions (1)
But not all, matrix proteins contain a C-terminal tripeptide that acts as a peroxisomal targeting signal (PTSl)l (3, 4)
In contrast to the well defined characterization of the PTSI topogenic signal, little is known about the mechanism of import including the recognition of the targeting signal
Summary
Vol 270, No 13, Issue of March 31, pp. 7731-7736, 1995 Printed in U.S.A. Identification and Characterization of the Putative Human Peroxisomal C-terminal Targeting Signal Import Receptor*. Complementation studies on cell lines from patients with generalized peroxisomal disorders (e.g. the Zellweger syndrome), believed to be peroxisomal protein translocation defects, have indicated that in the human at least nine gene products are required for peroxisome biogenesis (7). Two of these proteins have been identified: a 35-kDa peroxisomal membrane protein, which was revealed by functional complementation analysis (8), and a previously identified 70-kDa peroxisomal membrane protein, belonging to the ATP-binding cassette transporter family, which proved to be mutated in several Zellweger patients (9). Tel.: 32-16-345802; Fax: 32-16-345699. 1 The abbreviations used are: PTSl, C-terminal peroxisomal targeting signal; TPR, tetratricopeptide repeat; Gal4ad, Gal activation domain; Gal~d' Gal binding domain; TBST, Tria-buffered saline containing 0.05% (v/v) Tween 20; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; RITC, rhodamine isothiocyanate; kb, kilobase pairis)
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