Characterization of the interaction between recombinant human peroxin Pex3p and Pex19p: identification of TRP-104 IN Pex3p as a critical residue for the interaction.

Proteins required for peroxisome biogenesis are termed peroxins. The peroxin Pex3p is a peroxisomal membrane protein (PMP), involved in peroxisomal membrane biogenesis. It acts as a docking receptor for another peroxin Pex19p, which is a specific carrier protein for newly synthesized PMPs. Here we have determined the physicochemical properties and binding manners of Pex3p-Pex19p interaction, in terms of the affinity, the stoichiometry, and the binding site in Pex3p. The cytosolic domain of human Pex3p was overproduced, using an Escherichia coli expression system and was highly purified by two chromatography steps. Gel filtration chromatography analyses and intrinsic tryptophan fluorescence titrations revealed that a one-to-one complex is formed between monomeric Pex3p and monomeric Pex19p. The tryptophan fluorescence spectrum of Pex3p showed a large 18-nm blue shift of the maximum emission wavelength by the binding of Pex19p. This result indicates that either one or two tryptophan residues of Pex3p (Trp-104 and Trp-224) are directly involved in binding to Pex19p. We investigated the binding activities of the wild-type and tryptophan mutants of Pex3p by pull-down assays and surface plasmon resonance analyses. As a result, the wild-type and the W104A and W104F mutants showed K(D) values of 3.4 nm, 1080 nm, and 66.2 nm, respectively. The affinity differences with mutation affected their peroxisome restoring activities in pex3 ZPG208 cells. These findings suggest that the indole ring of Trp-104 directly interacts with Pex19p to facilitate the specific peroxisomal translocation of the Pex19p-PMP complexes.

Import of peroxisomal membrane proteins: the interplay of Pex3p- and Pex19p-mediated interactions.

In contrast to the molecular mechanisms underlying import of peroxisomal matrix proteins, those involving the transport of membrane proteins remain rather elusive. At present, two targeting routes for peroxisomal membrane proteins (PMPs) have been depicted: class I PMPs are targeted from the cytoplasm directly to the peroxisome membrane, and class II PMPs are sorted indirectly to peroxisomes via the endoplasmic reticulum (ER). In addition, three peroxins--Pex3p, Pex16p, and Pex19p - have been identified as essential factors for PMP assembly in several species including humans: Pex19p is a predominantly cytoplasmic protein that shows a broad PMP-binding specificity; Pex3p serves as the membrane-anchoring site for Pex19p; and Pex16p - a protein absent in most yeasts--is thought to provide the initial scaffold for recruiting the protein import machinery required for peroxisome membrane biogenesis. Remarkably, the function of Pex16p does not appear to be conserved between different species. In addition, significant disagreement exists about whether Pex19p has a chaperone-like role in the cytosol or at the peroxisome membrane and/or functions as a cycling import receptor for newly synthesized PMPs. Here we review the recent progress made in our understanding of the role of two key players in PMP biogenesis, Pex3p and Pex19p.

Functional domain mapping of peroxin Pex19p: interaction with Pex3p is essential for function and translocation.

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.

In vitro transport of membrane proteins to peroxisomes by shuttling receptor Pex19p.

The peroxin Pex19p comprising 299 amino acids functions in peroxisomal membrane assembly. We here developed a cell-free system for transport of membrane proteins to peroxisomes. Pex19p interacts with multiple membrane peroxins, including other membrane biogenesis peroxins, Pex16p and Pex26p, involved in matrix protein import. Cell-free synthesized, 35S-labeled Pex19p was targeted to subcellular fractions containing peroxisomes from Chinese hamster ovary-K1 cells as well as peroxisomes isolated from rat liver in an ATP-dependent manner. Such translocation was also reproduced with in vitro synthesized 35S-Pex16p with two transmembrane segments and C-tail anchor-type 35S-Pex26p, upon incubation with 35S-Pex19p in the reaction mixtures containing isolated peroxisomes. The transported 35S-Pex16p and 35S-Pex26p were integrated into membranes as assessed by the sodium carbonate extraction method. Peroxisome-associated and partly Na2CO3-resistant 35S-Pex19p was released to the cytosolic fraction upon incubation in the absence of ATP, whereas 35S-Pex16p and 35S-Pex26p remained in the membranes. Furthermore, not only 35S-Pex19p but also 35S-Pex19p complexes each with 35S-Pex16p and 35S-Pex26p were bound to 35S-Pex3p in vitro. Together, these results strongly suggested that Pex19p translocates the membrane peroxins from the cytosol to peroxisomes in an ATP- and Pex3p-dependent manner and then shuttles back to the cytosol.