Interactions of Pex7p and Pex18p/Pex21p with the peroxisomal docking machinery: implications for the first steps in PTS2 protein import.
Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13delta mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p- and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event.
Stein
Katharina
K
Freie Universität Berlin, Fachbereich Biologie, Chemie, Pharmazie, D-14195 Berlin, Germany.
Schell-Steven
Annette
A
Erdmann
Ralf
R
Rottensteiner
Hanspeter
H
D000102:Acetyl-CoA C-Acyltransferase / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D000074401:Peroxins; D000074437:Peroxisomal Targeting Signal 2 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C491105:PEX13 protein, S cerevisiae; C499195:PEX14 protein, S cerevisiae; C116845:PEX18 protein, S cerevisiae; C116846:PEX21 protein, S cerevisiae; D000074401:Peroxins; D000074437:Peroxisomal Targeting Signal 2 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000102:Acetyl-CoA C-Acyltransferase