Functional studies on human Pex7p: subcellular localization and interaction with proteins containing a peroxisome-targeting signal type 2 and other peroxins.
Pex7p is a WD40-containing protein involved in peroxisomal import of proteins containing an N-terminal peroxisome-targeting signal (PTS2). The interaction of human recombinant Pex7p expressed in different hosts/systems with its PTS2 ligand and other peroxins was analysed using various experimental approaches. Specific binding of human Pex7p to PTS2 could be demonstrated only when Pex7p was formed in vitro by a coupled transcription/translation system or synthesized in vivo in Chinese hamster ovary K1 cells transfected with a construct coding for a Pex7p-green fluorescent protein (GFP) fusion protein. Apparently, no cofactors are required and only monomeric Pex7p binds to PTS2. The interaction is reduced upon cysteine alkylation and is impaired upon truncation of the N-terminus of Pex7p. Interaction of Pex7p with other peroxins could not be demonstrated in bacterial or yeast two-hybrid screens, or in pull-down binding assays. The GFP fusion proteins, tagged at either the N- or C-terminus, were able to restore PTS2 import in rhizomelic chondrodysplasia punctata fibroblasts, and Pex7p-GFP was located both in the lumen of peroxisomes and in the cytosol.
Ghys
Karen
K
Katholieke Universiteit Leuven, Fakulteit Geneeskunde, Campus Gasthuisberg (O/N), Afdeling Farmakologie, Herestraat 49, B-3000 Leuven, Belgium.
Fransen
Marc
M
Mannaerts
Guy P
GP
Van Veldhoven
Paul P
PP
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D018902:Chondrodysplasia Punctata, Rhizomelic / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D005816:Genetic Complementation Test; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D000074428:Peroxisomal Biogenesis Factor 2; D000074437:Peroxisomal Targeting Signal 2 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D008565:Membrane Proteins; D000074437:Peroxisomal Targeting Signal 2 Receptor; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000074428:Peroxisomal Biogenesis Factor 2