Human Pex19p binds a broad spectrum of peroxisomal membrane proteins (PMPs). It has been proposed that this peroxin may: (i) act as a cycling PMP receptor protein, (ii) facilitate the insertion of newly synthesized PMPs into the peroxisomal membrane, or (iii) function as a chaperone to associate and/or dissociate complexes comprising integral PMPs already in the peroxisomal membrane. We previously demonstrated that human Pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences. Here we demonstrate that a mutant of Pex13p that fails to bind to Pex19p nevertheless targets to and integrates into the peroxisomal membrane. In addition, through in vitro biochemical analysis, we show that Pex19p competes with Pex5p and Pex13p for binding to Pex14p, supporting a role for this peroxin in regulating assembly/disassembly of membrane-associated protein complexes. To further examine the molecular mechanism underlying this competition, six evolutionarily conserved amino acids in the Pex5p/Pex13p/Pex19p binding domain of Pex14p were subjected to site-directed mutagenesis and the corresponding mutants functionally analyzed. Our results indicate that the physically overlapping binding sites of Pex14p for Pex5p, Pex13p, and Pex19p are functionally distinct, suggesting that competition occurs through induction of structural changes, rather than through direct competition. Importantly, we also found that amino acid substitutions resulting in a strongly reduced binding affinity for Pex13p affect the peroxisomal localization of Pex14p.To date, peroxisome biogenesis studies in a number of evolutionarily diverse organisms have identified 29 gene products (1) called peroxins (abbreviated Pexp 1 and including a number corresponding to the order of discovery), which are essential for formation of the organelle. In man, peroxisome biogenesis requires the concerted action of at least 16 of these peroxins (2). Despite the fact that substantial progress has been made in peroxin identification, the function of most of these proteins in the biogenesis process is still only partially understood. Significant interest has developed in recent years regarding the essentially open question of how PMPs find their way to the organelle (3). In a select few examples, peroxisomal targeting signals (so-called mPTSs) have been identified (4). However, no firm conclusions may yet be drawn about the presence or absence of consensus sequences or common structural properties with respect to these mPTSs (5). The observation that Pex19p interacts with the mPTSs of PpPex2p, HsPex11p␤, HsPex13p, HsPex14p, HsPex16p, HsPMP22, HsPMP34, HsPMP70, HsALDP, and HsALDPR (6 -11), and that a portion of cellular Pex19p is found associated with the outer surface of peroxisomes (6, 12), makes this peroxin a reasonable candidate for a cycling PMP receptor protein. However, as shown by multiple independent point mutations, the physically overlapping Pex19p binding domain and the peroxisomal sorting signals of HsPex13p can be functionally...

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Pex19p exhibits a broad binding specificity for peroxisomal membrane proteins (PMPs), and is essential for the formation of functional peroxisomal membranes. Pex19p orthologues contain a C-terminal CAAX motif common to prenylated proteins. In addition, Saccharomyces cerevisiae and Chinese hamster Pex19p are at least partially farnesylated in vivo. Whether farnesylation of Pex19p plays an essential or merely ancillary role in peroxisome biogenesis is currently not clear. Here, we show that (i) nonfarnesylated and farnesylated human Pex19p display a similar affinity towards a select set of PMPs, (ii) a variant of Pex19p lacking a functional farnesylation motif is able to restore peroxisome biogenesis in Pex19p-deficient cells, and (iii) peroxisome protein import is not affected in yeast and mammalian cells defective in one of the enzymes involved in the farnesylation pathway. Summarized, these observations indicate that the CAAX box-mediated processing steps of Pex19p are dispensable for peroxisome biogenesis in yeast and mammalian cells.