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      Pex18p and Pex21p, a Novel Pair of Related Peroxins Essential for Peroxisomal Targeting by the PTS2 Pathway

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          Abstract

          We have identified ScPex18p and ScPex21p, two novel S. cerevisiae peroxins required for protein targeting via the PTS2 branch of peroxisomal biogenesis. Targeting by this pathway is known to involve the interaction of oligopeptide PTS2 signals with Pex7p, the PTS2 receptor. Pex7p function is conserved between yeasts and humans, with defects in the human protein causing rhizomelic chondrodysplasia punctata (RCDP), a severe, lethal peroxisome biogenesis disorder characterized by aberrant targeting of several PTS2 peroxisomal proteins, but uncertainty remains about the subcellular localization of this receptor. Previously, we have reported that ScPex7p resides predominantly in the peroxisomal matrix, suggesting that it may function as a highly unusual intraorganellar import receptor, and the data presented in this paper identify Pex18p and Pex21p as key components in the targeting of Pex7p to peroxisomes. They each interact specifically with Pex7p both in two-hybrid analyses and in vitro. In cells lacking both Pex18p and Pex21p, Pex7p remains cytosolic and PTS2 targeting is completely abolished. Pex18p and Pex21p are weakly homologous to each other and display partial functional redundancy, indicating that they constitute a two-member peroxin family specifically required for Pex7p and PTS2 targeting.

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          Most cited references42

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          Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.

          The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.
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            A conserved tripeptide sorts proteins to peroxisomes

            The firefly luciferase protein contains a peroxisomal targeting signal at its extreme COOH terminus (Gould et al., 1987). Site-directed mutagenesis of the luciferase gene reveals that this peroxisomal targeting signal consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine. When this tripeptide is appended to the COOH terminus of a cytosolic protein (chloramphenicol acetyltransferase), it is sufficient to direct the fusion protein into peroxisomes. Additional mutagenesis experiments reveal that only a limited number of conservative changes can be made in this tripeptide targeting signal without abolishing its activity. These results indicate that peroxisomal protein import, unlike other types of transmembrane translocation, is dependent upon a conserved amino acid sequence.
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              Biogenesis of peroxisomes.

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                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                28 December 1998
                : 143
                : 7
                : 1859-1869
                Affiliations
                Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, New York 10029-6574
                Author notes

                Address correspondence to Dr. P. Edward Purdue, Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, 1190 Fifth Avenue, Box 1007, New York, NY 10029-6574. Tel.: (212) 241-9740. Fax: (212) 860-1174. E-mail: epurdue@ 123456smtplink.mssm.edu

                Article
                10.1083/jcb.143.7.1859
                2175223
                9864360
                5aaf0ead-c1ce-4620-b36f-8843884a273f
                Copyright @ 1998
                History
                : 2 September 1998
                : 29 October 1998
                Categories
                Regular Articles

                Cell biology
                microbodies,peroxisomes,biogenesis,protein import,saccharomyces cerevisiae
                Cell biology
                microbodies, peroxisomes, biogenesis, protein import, saccharomyces cerevisiae

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