The SH3 Domain of the Saccharomyces cerevisiae Peroxisomal Membrane Protein Pex13p Functions as a Docking Site for Pex5p, a Mobile Receptor for the Import of PTSl-containing Proteins

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pexl3p, that is essential for protein import. A point mutation in the COOHterminal Src homology 3 (SH3) domain of Pexl3p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pexl3p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pexl3p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein. S ELECTIVE protein sorting in eukaryotic cells requires (a) specific signals in the transported proteins, (b) cellular components that recognize and bind these signals, and (c) membrane-associated protein complexes to carry out the actual translocation step. Two types of targeting signals have been described that function in protein sorting to peroxisomes. The peroxisomal targeting signal type 1 (PTS1) t, which is present in the majority of the peroxisomal matrix proteins, consists of a tripeptide at the extreme carboxy terminus of a protein and was first identified in luciferase (Gould et al., 1987; Keller et al., 1987). The second peroxisomal targeting signal (PTS2) resides at the NH2 terminus of a protein, and was first delineated in thiolase (Osumi et al., 1991; Swinkels et al., 1991). Only a limited number of peroxisomal matrix proteins is imported via this signal. To identify components involved in peroxisomal protein import several groups have set out to isolate peroxisome assembly mutants in various yeast species (Erdmann et al., 1989; Cregg et al., 1990; Gould et al., 1992; Liu et al., 1992; Van der Leij et al., 1992; Elgersma et al., 1993; Nuttley et al., 1993). These peroxisome assembly mutants, formerly described as pas, per, peb, or pay have now been collecPlease address all correspondence to B. Distel, Department of Biochemistry, Academic Medical Centre, Meibergdreef 15, 1105 A Z Amsterdam, The Netherlands. Tel.: 31 20 566 5127. Fax: 31 20 691 5519. 1. Abbreviat ions used in this paper: DB, DNA-binding; DHFR, dihydrofolate reductase; NH, hemagglutinin epitope; pex, peroxin; PTS1, peroxisomal targeting signal type 1; SH3, Src homology 3; TA, transcriptional activation. tively renamed pex (see accompanying letter in this issue). Among the 15 complementation groups ofpex mutants in Saccharomyces cerevisiae, the phenotypes of the pex7 (pas7) and pex5 (paslO) mutants are very informative since they display selective protein import defects. The pex7 mutant is specifically disturbed in the import of PTS2-containing proteins, whereas pex5 does not import PTSl-containing proteins (Van der Leij et al., 1992, 1993; Marzioch et al., 1994; Zhang and Lazarow, 1995). The PEX5 gene encodes a 69-kD protein with multiple degenerate repeats of a 34-amino acid motif (tetratricopeptide repeat, TPR), linking it to the family of TPR proteins (Goebl and Yanagida, 1991; Van der Leij et al., 1993). In a two-hybrid assay Pex5p specifically interacts with PTS1containing proteins such as luciferase and peroxisomal malate dehydrogenase (Tabak et al., 1995). A possible homologue of the PEX5 gene has been cloned from Pichia pastoris and more recently from man (McCollum et al., 1993; Dodt et al., 1995; Fransen et al., 1995; Terlecky et al., 1995; Wiemer et al., 1995). Both the human and the P. pastoris proteins exhibit binding of PTSl-containing peptides in vitro. Together these results suggest that the PEX5 gene encodes the receptor for PTSl-containing proteins. Besides pex7 and pex5, 13 additional pex (pas) mutants have been isolated in S. cerevisiae. The phenotype of most of these mutants is absence of morphologically distinguishable peroxisomes and cytosolic localization of all peroxisomal matrix proteins, which does not reveal any details of the function of the gene product in question, other than that it is essential for peroxisome biogenesis. However, several of the PEX proteins, collectively called peroxins, © The Rockefeller University Press, 0021-9525/96/10/97/13 $2.00 The Journal of Cell Biology, Volume 135, Number 1, October 1996 97-109 97 on F ebuary 1, 2013 jcb.rress.org D ow nladed fom Published October 1, 1996