title: Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction.
abstract: Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.
authors.affiliation: Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
authors.forename: Márcia E M
authors.initials: ME
authors.lastname: Oliveira
authors.forename: Carlos
authors.initials: C
authors.lastname: Reguenga
authors.forename: Alexandra M M
authors.initials: AM
authors.lastname: Gouveia
authors.forename: Carla P
authors.initials: CP
authors.lastname: Guimarães
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schliebs
authors.forename: Wolf-H
authors.initials: WH
authors.lastname: Kunau
authors.forename: Manuel T
authors.initials: MT
authors.lastname: Silva
authors.forename: Clara
authors.initials: C
authors.lastname: Sá-Miranda
authors.forename: Jorge E
authors.initials: JE
authors.lastname: Azevedo
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D008565:Membrane Proteins; C455539:PEX14 protein, human; C499193:Pex14 protein, mouse; C499194:Pex14 protein, rat; D011994:Recombinant Proteins; D012097:Repressor Proteins