Analysis of human Pex19p's domain structure by pentapeptide scanning mutagenesis.
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 complexes. 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, Pex11pbeta, 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.
Fransen
Marc
M
Departement Moleculaire Celbiologie, Afdeling Farmacologie, Katholieke Universiteit Leuven, Faculteit Geneeskunde, Campus Gasthuisberg (O/N 6, box 601), Herestraat 49, 3000 Leuven, Belgium. marc.fransen@med.kuleuven.ac.be
Vastiau
Ilse
I
Brees
Chantal
C
Brys
Vanessa
V
Mannaerts
Guy P
GP
Van Veldhoven
Paul P
PP
D000595:Amino Acid Sequence; D001665:Binding Sites; D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000074428:Peroxisomal Biogenesis Factor 2; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism
D004251:DNA Transposable Elements; D008565:Membrane Proteins; D010446:Peptide Fragments; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; C085380:peroxisomal targeting sequence receptor; D000074428:Peroxisomal Biogenesis Factor 2; D049452:Green Fluorescent Proteins; C087343:PEX19 protein, human