Abstract
The peroxin Pex19p functions in peroxisomal membrane assembly. Here we mapped functional domains of human Pex19p comprising 299 amino acids. Pex19p mutants deleted in the C-terminal CAAx farnesylation motif, the C-terminal 38 amino acid residues and the N-terminal 11 residues, maintained peroxisome-restoring activity in pex19 cells. The sequence 12-261 was essential for re-establishing peroxisome activity. Pex19p was partly localized to peroxisomes but mostly localized in the cytosol. Pex19p interacted with multiple membrane proteins, including the other two membrane biogenesis peroxins, Pex3p and Pex16p, those involved in matrix protein import such as Pex14p, Pex13p, Pex10p, and Pex26p, peroxisome morphogenesis factor Pex11pbeta, and a PMP70 peroxisome-targeting signal region at residues 1-123. In yeast two-hybrid assays, Pex10p and Pex11pbeta interacted only with full-length Pex19p. Of various truncated Pex19p variants active in translocating to peroxisomes, the mutants with the shortest sequence (residues 12-73 and 40-131) were localized to peroxisomes and competent in binding to Pex3p. Furthermore, membrane peroxins were initially discernible in a cytosolic staining pattern in pex19 cells only when co-expressed with Pex19p and were then localized to peroxisomes in a temporally differentiated manner. Pex19p probably functions as a chaperone for membrane proteins and transports them to peroxisomes by anchoring to Pex3p using residues 12-73 and 40-131.
Highlights
Peroxisomal proteins are encoded by nuclear genes, translated on free polyribosomes in the cytosol, and imported to peroxisomes (Lazarow and Fujiki, 1985)
Functional domain mapping of Pex19p As a step toward understanding the molecular mechanisms involved in peroxisome membrane biogenesis, we first searched for functional regions of Pex19p
Restoration of peroxisome biogenesis was assessed by immunofluorescent cell staining with antibodies recognizing matrix peroxisome-targeting signal type 1 (PTS1) proteins and the membrane peroxin Pex14p as markers for membrane proteins
Summary
Peroxisomal proteins are encoded by nuclear genes, translated on free polyribosomes in the cytosol, and imported to peroxisomes (Lazarow and Fujiki, 1985). We earlier cloned human PEX19 cDNA encoding the 299 amino acid, hydrophilic peroxin Pex19p with the farnesylation motif CAAx at the C-terminus, by functional complementation strategy using a mutant CHO cell line, ZP119, defective in the import of both matrix and membrane proteins (Kinoshita et al, 1998; Matsuzono et al, 1999). Pex19p binds multiple peroxisomal integral membrane proteins (PMPs), including several peroxins such as Pex3p and Pex13p (Fransen et al, 2001; Ghaedi et al, 2000b; Snyder et al, 2000). Pex19p has been proposed to function in recruiting newly synthesized PMPs from their site of synthesis on free polyribosomes to the peroxisomes as a soluble receptor and/or a chaperone in targeting of PMPs (Fransen et al, 2001; Jones et al, 2001; Matsuzono and Fujiki, 2006; Snyder et al, 2000)
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