Potential role for Pex19p in assembly of PTS-receptor docking complexes.
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.
Fransen
Marc
M
Katholieke Universiteit Leuven, Faculteit Geneeskunde, Campus Gasthuisberg (O/N), Departement Moleculaire Celbiologie, Afdeling Farmacologie, Herestraat 49, B-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
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002241:Carbohydrates / Q000737:chemistry; D017124:Conserved Sequence; D006224:Cricetinae; D017931:DNA Primers / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D002241:Carbohydrates; D017931:DNA Primers; D008164:Luminescent Proteins; D008565:Membrane Proteins; C101884:PEX13 protein, human; C455539:PEX14 protein, human; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D012097:Repressor Proteins; C085380:peroxisomal targeting sequence receptor; D049452:Green Fluorescent Proteins; C087343:PEX19 protein, human; D005982:Glutathione Transferase