Membrane association of the cycling peroxisome import receptor Pex5p.
Peroxisomal proteins carrying a peroxisome targeting signal type 1 (PTS1) are recognized in the cytosol by the cycling import receptor Pex5p. The receptor-cargo complex docks at the peroxisomal membrane where it associates with multimeric protein complexes, referred to as the docking and RING finger complexes. Here we have identified regions within the Saccharomyces cerevisiae Pex5p sequence that interconnect the receptor-cargo complex with the docking complex. Site-directed mutagenesis of the conserved tryptophan residue within a reverse WXXXF motif abolished two-hybrid binding with the N-terminal half of Pex14p. In combination with an additional mutation introduced into the Pex13p-binding site, we generated a Pex5p mutant defective in a stable association not only with the docking complex but also with the RING finger peroxins at the membrane. Surprisingly, PTS1 proteins are still imported into peroxisomes in these mutant cells. Because these mutations had no significant effect on the membrane binding properties of Pex5p, we examined yeast and human Pex5p for intrinsic lipid binding activity. In vitro analyses demonstrated that both proteins have the potential to insert spontaneously into phospholipid membranes. Altogether, these data strongly suggest that a translocation-competent state of the PTS1 receptor enters the membrane via protein-lipid interactions before it tightly associates with other peroxins.
Kerssen
Daniela
D
Institut für Physiologische Chemie, Abt. Systembiochemie, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Hambruch
Eva
E
Klaas
Wibke
W
Platta
Harald W
HW
de Kruijff
Ben
B
Erdmann
Ralf
R
Kunau
Wolf-H
WH
Schliebs
Wolfgang
W
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000074401:Peroxins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C491105:PEX13 protein, S cerevisiae; C499195:PEX14 protein, S cerevisiae; C499509:PEX5 protein, S cerevisiae; D000074401:Peroxins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins