Pex7p and Pex20p ofNeurospora crassaFunction Together in PTS2-dependent Protein Import into Peroxisomes

Abstract

Recruiting matrix proteins with a peroxisomal targeting signal type 2 (PTS2) to the peroxisomal membrane requires species-specific factors. In Saccharomyces cerevisiae, the PTS2 receptor Pex7p acts in concert with the redundant Pex18p/Pex21p, whereas in Yarrowia lipolytica, Pex20p might unite the function of both S. cerevisiae peroxins. Herein, the genome of the filamentous fungus Neurospora crassa was analyzed for peroxin-encoding genes. We identified a set of 18 peroxins that resembles that of Y. lipolytica rather than that of S. cerevisiae. Interestingly, proteins homologous to both S. cerevisiae Pex7p and Y. lipolytica Pex20p exist in N. crassa. We report on the isolation of these PTS2-specific peroxins and demonstrate that NcPex20p can substitute for S. cerevisiae Pex18p/Pex21p, but not for ScPex7p. Like Pex18p, NcPex20p did not bind PTS2 protein or the docking proteins in the absence of ScPex7p. Rather, NcPex20p was required before docking to form an import-competent complex of cargo-loaded PTS2 receptors. NcPex7p did not functionally replace yeast Pex7p, probably because the N. crassa PTS2 receptor failed to associate with Pex18p/Pex21p. However, once NcPex7p and NcPex20p had been coexpressed, it proved possible to replace yeast Pex7p. Pex20p and Pex18p/Pex21p are therefore true orthologues, both of which are in need of Pex7p for PTS2 protein import.

The N Terminus of the Peroxisomal Cycling Receptor, Pex5p, Is Required for Redirecting the Peroxisome-associated Peroxin Back to the Cytosol

“…In this context, it may be relevant to note that, although the N-terminal half of Pex5p is poorly conserved among different species, the sequence Cys-X n -Asn-Pro-Leu (where X n is any 3-9 amino acid residues) is found at the N termini of most Pex5p proteins (data not shown). Also interesting is the observation that similar motifs (Cys-X n -Asn-(Ala/Gly)-(Leu/Ala)) are found at the N termini of Neurospora crassa and Yarrowia lipolytica Pex20p (19,20). This peroxin is thought to escort the PTS2 receptor to the peroxisomal membrane, a role that in mammals and plants is performed by Pex5p (19 -21).…”

“…This peroxin recognizes a degenerated nona-peptide complying to the consensus sequence (R/K)(L/I/V)X 5 (H/Q)(L/A/F), the socalled peroxisomal targeting signal type 2 (PTS2) (8 -10). In mammals and plants, but not in lower eukaryotes, targeting of these PTS2-containing proteins seems to require Pex5p, suggesting that the two import pathways actually converge at the level of the PTS1 receptor (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

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

“…The auxiliary protein(s) for Pex7p-mediated import varies depending on the species (Figure 3): Pex18p and Pex21p in S. cerevisiae (Purdue et al, 1998); Pex20p in Hansenula polymorpha (Otzen et al, 2005), Neurospora crassa (Sichting et al, 2003), Pichia pastoris (Leon et al, 2006b) and Yarrowia lipolytica (Einwachter et al, 2001); and PEX5 in plants (Hayashi et al, 2005;Woodward and Bartel, 2005) and mammals. In mammals, only the PEX5L isoform can interact with PEX7 , and it is essential for PTS2 import (Matsumura et al, 2000).…”

Pex30p, Pex31p, and Pex32p Form a Family of Peroxisomal Integral Membrane Proteins Regulating Peroxisome Size and Number inSaccharomyces cerevisiae

“…Others have used this methodology to detect interactions between peroxins (for examples, see Girzalsky et al, 1999;Smith and Rachubinski, 2001;Sichting et al, 2003). Chimeric genes were made by ligating the ORFs of YLR324w, YGR004w, YBR168w, PEX11, PEX25, PEX28, PEX29, and VPS1 in-frame and downstream of sequences encoding one of the two functional domains (AD or DB) of the GAL4 transcriptional activator.…”