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Molecular and Cellular Biology, September 2002, p. 6056-6069, Vol. 22, No. 17
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.17.6056-6069.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Interactions of Pex7p and Pex18p/Pex21p with the Peroxisomal Docking Machinery: Implications for the First Steps in PTS2 Protein Import

Katharina Stein, Annette Schell-Steven, Ralf Erdmann,* and Hanspeter Rottensteiner

Freie Universität Berlin, Fachbereich Biologie, Chemie, Pharmazie, D-14195 Berlin, Germany

Received 19 February 2002/ Returned for modification 28 March 2002/ Accepted 23 May 2002

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 pex13{Delta} 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.

* Corresponding author. Mailing address: Freie Universität Berlin, Institut für Chemie/Biochemie, Thielallee 63, D-14195 Berlin, Germany. Phone: 43-30-838 52935. Fax: 43-30-838 52936. E-mail: ralferdm{at}zedat.fu-berlin.de.

Molecular and Cellular Biology, September 2002, p. 6056-6069, Vol. 22, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.17.6056-6069.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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