Analysis of Human Pex19p’s Domain Structure by Pentapeptide Scanning Mutagenesis Marc Fransen*, Ilse Vastiau, Chantal Brees, Vanessa Brys Guy P. Mannaerts and Paul P. Van Veldhoven Katholieke Universiteit Leuven Faculteit Geneeskunde, Campus Gasthuisberg (O/N 6, box 601) Departement Moleculaire Celbiologie, Afdeling Farmacologie, Herestraat 49 3000 Leuven, Belgium Pex19p, a primarily cytosolic protein, is essential for the biogenesis of numerous peroxisomal membrane proteins (PMPs); however, its precise function is unclear. Pex19p might function as a PMP-specific chaperone, a cycling PMP-receptor protein, a PMP membrane insertion factor, or an association/dissociation factor of membrane-associated protein com- plexes. Alternatively, Pex19p might act as a multifunctional peroxin and participate in a number of these activities. Here, we have employed transposon mutagenesis to generate a library of human pex19 alleles coding for Pex19p variants containing random in-frame pentapeptide insertions. A total of 87 different variants were characterized to identify functionally important regions. These studies revealed that Pex19p has a tripartite domain structure consisting of: (i) an amino-terminal domain that binds to Pex3p and is essential for docking at the peroxisome membrane; (ii) a central domain that competes with Pex5p and Pex13p for binding to Pex14p and may play a role in the assembly of PTS-receptor docking complexes; and (iii) a carboxy-terminal domain that interacts with multiple PMPs including Pex3p, Pex11pb, Pex12p, Pex13p, Pex16p, and Pex26p. Whether the latter interactions constitute the chaperone or transport functions (or both), remains to be determined. Finally, our observation that Pex19p contains two distinct binding sites for Pex3p suggests that the peroxin may bind PMPs in multiple places and for multiple purposes. q 2005 Elsevier Ltd. All rights reserved. Keywords: peroxisomes; biogenesis; protein import; Pex19p; transposon mutagenesis *Corresponding author Introduction Peroxisomes play several vital roles in human metabolism. 1 Mutations disrupting peroxisome formation or function result in severe disease phenotypes. 2 The prototype of these is Zellweger syndrome, an often fatal inherited disease caused by a peroxisome biogenesis deficiency. 3 Peroxisome biogenesis is a complex multi-step process that depends on the concerted action of a select group of proteins, called peroxins (abbreviated Pexp and including a number corresponding to the order of discovery; gene acronym: PEX). 4 To date, 16 human PEX-genes have been identified, 5–9 and the corresponding gene products have been implicated in the synthesis and assembly of peroxisome membranes (e.g. Pex3p, Pex16p and Pex19p), the import of peroxisomal matrix proteins (e.g. Pex1p, Pex2p, Pex5p, Pex6p, Pex7p, Pex10p, Pex12p, Pex13p, Pex14p and Pex26p), or the growth in size and number of the organelles (e.g. Pex11pa, Pex11pb and Pex11pg). 6–9 However, the precise function of most of these peroxins from a mechan- istic standpoint remains largely unclear. An important aim of our recent studies was to elucidate the role of human Pex19p 10 in the peroxisome biogenesis process. Like its counterpart in Pichia pastoris, 11 HsPex19p exhibits a broad binding specificity for peroxisomal membrane proteins (PMPs). 12–17 It has been found in the cytosol and, to a minor extent, on the outer surface of the peroxisome membrane. 12,18 Human fibro- blasts, Chinese hamster ovary cells and Saccharomyces cerevisiae cells deficient in Pex19p 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: PMP, peroxisomal membrane protein; b-2HS, bacterial two-hybrid system; y-2HS, yeast two-hybrid system; GFP, green fluorescent protein. E-mail address of the corresponding author: marc.fransen@med.kuleuven.ac.be doi:10.1016/j.jmb.2005.01.013 J. Mol. Biol. (2005) 346, 1275–1286