Identification and Characterization of the Putative Human Peroxisomal C-terminal Targeting Signal Import Receptor

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.

TTC4, a novel candidate tumor suppressor gene at 1p31 is often mutated in malignant melanoma of the skin

“…A variety of other genes belong to this family and di erent functions have been assigned to genes with TPR motifs (Blatch and LaÈ ssle, 1999). The members of the human TPR motif gene family include cochaperones like IEFSSP3521 (Honore et al, 1992) or FKBPRr38 (Lam et al, 1995), genes with protein transport functions like PXR1 (Fransen et al, 1995), genes with importance in phosphate turnover like PP5 (Chen et al, 1994), and a gene with cell cycle control functions (Schrick et al, 1995). Very recently, additional genes with TPR motifs have been found which demonstrate interactions with heat shock proteins or are necessary for the formation of the phagocyte NADPH oxidase (Ballinger et al, 1999;Koga et al, 1999;Zhang and Grishin, 1999).…”

Tetratricopeptide repeat domain of Yarrowia lipolytica Pex5p is essential for recognition of the type 1 peroxisomal targeting signal but does not confer full biological activity on Pex5p

“…lipolytica and other yeast species have proved to be invaluable tools for the identification of components required for peroxisome assembly. The PEX5 gene of Y. lipolytica was identified in a genetic screen for mutants of peroxisome biogenesis [12] and shows high amino acid sequence similarity to genes from other organisms that encode PTS1 receptors [4,6,[9][10][11]14,20]. Although the original mutant strain pex5-1 synthesizes a YlPex5p of normal size, unlike the wild-type YlPex5p, it is not found in association with anti-SKL-reactive proteins in i o [12].…”

“…PTS1 receptors are encoded by the PEX5 gene family [4][5][6][7][8][9][10][11][12][13][14][15][16][17]. These peroxins (proteins involved in peroxisome assembly) are characterized by a tetratricopeptide repeat (TPR) domain in the C-terminal half of the protein.…”

“…Interestingly, import of proteins by the PTS1 and PTS2 pathways is not entirely independent in mammalian cells. The human [9][10][11]42], mouse [42] and Chinese hamster [15] PEX5 genes each encode two forms of the protein that differ by the presence or absence of a 37-residue insertion N-terminal to the TPR domain. The short form of Pex5p can functionally complement the PTS1 import defect in pex5 mutant cells but only the long form of the protein can also rescue the PTS2 import defect that is an additional feature of some pex5 cells [15,42].…”

“…Pex5 proteins have been localized to different subcellular compartments, i.e. primarily cytosolic [5,10,11,17,19], associated with the outer side of the peroxisomal membrane [4,9,20], in the peroxisomal matrix [12] and in both the cytosol and peroxisomes [8]. The PEX7 gene family encodes the PTS2 receptor [21][22][23][24][25][26][27].…”