Biochem. J. (2002) 365
(41–50) (Printed in Great Britain)
Functional studies on human Pex7p: subcellular localization and interaction with proteins containing a peroxisome-targeting signal type 2 and other peroxins
Karen GHYS , Marc FRANSEN , Guy P. MANNAERTS and Paul P. VAN VELDHOVEN1
Katholieke Universiteit Leuven, Fakulteit Geneeskunde, Campus Gasthuisberg (O/N), Afdeling Farmakologie, Herestraat 49, B-3000 Leuven, Belgium
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.
Key words: biogenesis, peroxin, protein import, thiolase, WD40.
Abbreviations used: PTS, peroxisome-targeting signal; GFP, green fluorescent protein; RCDP, rhizomelic chondrodysplasia punctata; GST, glutathione S-tranferase; HMG-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA synthase; CHO, Chinese hamster ovary; HsPex7p, Homo sapiens Pex7p; Ni-NTA, Ni2+-nitrilotriacetate; TBS, Tris-buffered saline; TNT, transcription/translation.
1To whom correspondence should be addressed (e-mail paul.vanveldhoven@med.kuleuven.ac.be).
Received 5 October 2001/25 March 2002; accepted 3 April 2002
Published as BJ Immediate Publication 3 April 2002, DOI 10.1042/BJ20011432
The Biochemical Society, London ©
2002
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