Identification of a novel, intraperoxisomal pex14-binding site in pex13: association of pex13 with the docking complex is essential for peroxisomal matrix protein import.
The peroxisomal docking complex is a key component of the import machinery for matrix proteins. The core protein of this complex, Pex14, is thought to represent the initial docking site for the import receptors Pex5 and Pex7. Associated with this complex is a fraction of Pex13, another essential component of the import machinery. Here we demonstrate that Pex13 directly binds Pex14 not only via its SH3 domain but also via a novel intraperoxisomal site. Furthermore, we demonstrate that Pex5 also contributes to the association of Pex13 with Pex14. Peroxisome function was affected only mildly by mutations within the novel Pex14 interaction site of Pex13 or by the non-Pex13-interacting mutant Pex5(W204A). However, when these constructs were tested in combination, PTS1-dependent import and growth on oleic acid were severely compromised. When the SH3 domain-mediated interaction of Pex13 with Pex14 was blocked on top of that, PTS2-dependent matrix protein import was completely compromised and Pex13 was no longer copurified with the docking complex. We conclude that the association of Pex13 with Pex14 is an essential step in peroxisomal protein import that is enabled by two direct interactions and by one that is mediated by Pex5, a result which indicates a novel, receptor-independent function of Pex5.
Schell-Steven
Annette
A
Institut für Physiologische Chemie, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Stein
Katharina
K
Amoros
Mara
M
Landgraf
Christiane
C
Volkmer-Engert
Rudolf
R
Rottensteiner
Hanspeter
H
Erdmann
Ralf
R
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D005656:Fungal Proteins / Q000378:metabolism*; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019301:Oleic Acid / Q000378:metabolism; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D025941:Protein Interaction Mapping; D021381:Protein Transport / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology
D005656:Fungal Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C491105:PEX13 protein, S cerevisiae; C499509:PEX5 protein, S cerevisiae; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D019301:Oleic Acid