Abstract. Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2). Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7Δ mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7Δ cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata. The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7Δ mutant and were impaired in binding to the PTS2 sequence.
A Mobile PTS2 Receptor for Peroxisomal Protein Import in Pichia pastoris
We are grateful to Dr. A. Koller for providing us the PpFOX3 gene sequence, to Dr. H.F. Tabak for providing us thiolase antibodies and S. cerevisiae strains and plasmids, Dr. Peter van der Sluijs for providing the NH antibody, and Drs. P. Bartel and S. Fields for providing the two-hybrid plasmids. We thank Dr. K.-N. Faber for sharing his preliminary characterization of the pex7 mutant with us.
The Immunocytochemistry Core Facility, University of California, San Diego, was supported by grant CA 58689 to M.G. Farquhar. This work was supported by a National Institutes of Health grant (DK41737) to S. Subramani and by a fellowship from the Netherlands Organization for Scientific Research to Y. Elgersma and by fellowships from the European Molecular Biology Organization to Y. Elgersma and T. Wenzel.
Y. Elgersma and M. Elgersma-Hooisma contributed equally to this work.
Address all correspondence to S. Subramani, Department of Biology, University of California, San Diego, Room 3230, Bonner Hall, 9500 Gilman Drive, La Jolla, CA 92093-0322. Tel.: (619) 534-2327. Fax: (619) 534-0053. E-mail: [email protected]
Y. Elgersma's and M. Elgersma-Hooisma's present addresses are Cold Spring Harbor Laboratory, 1 Bung Town Road, Cold Spring Harbor, NY 11724.
T. Wenzel's present address is Gist Brocades, Food Specialty Division, Delft, The Netherlands.
1. Abbreviations used in this paper: Aco1p, acyl CoA oxidase; AD, activation domain; BLE, bleomycin resistance protein; DB, DNA-binding domain; FOX3, 3-ketoacyl CoA thiolase; GFP, green fluorescent protein; Pex, peroxin; pex, peroxisomal biogenesis/import mutants; Pp, Pichia pastoris; PTS, peroxisomal targeting signal; RCDP, rhizomelic chondrodysplasia punctata; Sc, Saccharomyces cerevisiae; SRP, signal recognition particle; TPR, tetratricopeptide repeat.
Ype Elgersma, Minetta Elgersma-Hooisma, Thibaut Wenzel, J. Michael McCaffery, Marilyn G. Farquhar, Suresh Subramani; A Mobile PTS2 Receptor for Peroxisomal Protein Import in Pichia pastoris . J Cell Biol 23 February 1998; 140 (4): 807–820. doi: https://doi.org/10.1083/jcb.140.4.807
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