Peroxisomal Targeting of PTS2 Pre‐Import Complexes in the Yeast Saccharomyces cerevisiae

Abstract

†These authors contributed equally to this work.Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…This localization suggested that Pex7p functions in a similar manner to Pex5p, only instead operating as an import receptor for newly synthesized PTS2-containing proteins. Gel-filtration chromatography has shown mammalian PEX7 to be monomeric (Mukai and Fujiki, 2006), consistent with MS analysis of S. cerevisiae PTS2 pre-import complexes which contain Fox3p (thiolase)/Pex7p/Pex18p in the following proportions 2:1:1 (Grunau et al, 2009). Although the structure of Pex7p is unknown, it is predicted to be a member of the β-transducinrelated (WD-40) protein family, forming a sevenbladed β-propeller, consisting of six WD repeats and a distinct N-terminal region.…”

“…PTS2 proteins appear to bind to monomeric PEX7 (Mukai and Fujiki, 2006;Grunau et al, 2009), and there is some evidence to suggest that mammalian PEX7 only binds PEX5L in the presence of PTS2 cargo (Mukai and Fujiki, 2006). Furthermore, these authors suggest that the PTS2-PEX7 complex can only interact with PEX14 via PEX5L, consistent with the known docking function of the W-X 3 -F/Y motifs of PEX5.…”

“…However, thiolase can be imported as a heterodimer when one subunit possesses a PTS2 and the other does not, which suggests that PTS2 dimerization is not required for import (Glover et al, 1994;Flynn et al, 1998). This is also supported by a recent study suggesting that one thiolase dimer interacts with one PTS2 receptor molecule (Grunau et al, 2009). Therefore the biological relevance of PTS2 dimerization is yet to be determined.…”

“…The number of PTS2-targeted proteins ranges from zero in Caenorhabditis elegans, which has lost the PTS2 import pathway altogether (Motley et al, 2000), to an estimated 60 in A. thaliana (Reumann et al, 2009). Interestingly, S. cerevisiae appears to maintain a PTS2 import pathway solely for the transport of 3-ketoacyl thiolase (Grunau et al, 2009). The biological rationale for these differences remains unknown.…”

Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

“…The solubilization of membrane proteins was followed by centrifugation at 100,000 ϫ g (Sorvall T-647.5) for 1 h at 4°C to remove insolubilized membrane proteins. Soluble and/or membrane-bound Gpd1p-TEVcs or Pex21p-TEVcs-Protein A complexes were purified as described earlier (42,43). The eluate of soluble Gpd1p complexes was subjected to size-exclusion chromatography using a Superose 6 PC3.2/30 column (GE Healthcare).…”