Multiple PEX genes are required for proper subcellular distribution and stability of Pex5p, the PTS1 receptor: evidence that PTS1 protein import is mediated by a cycling receptor.

Abstract

PEX5 encodes the type-1 peroxisomal targeting signal (PTS1) receptor, one of at least 15 peroxins required for peroxisome biogenesis. Pex5p has a bimodal distribution within the cell, mostly cytosolic with a small amount bound to peroxisomes. This distribution indicates that Pex5p may function as a cycling receptor, a mode of action likely to require interaction with additional peroxins. Loss of peroxins required for protein translocation into the peroxisome (PEX2 or PEX12) resulted in accumulation of Pex5p at docking sites on the peroxisome surface. Pex5p also accumulated on peroxisomes in normal cells under conditions which inhibit protein translocation into peroxisomes (low temperature or ATP depletion), returned to the cytoplasm when translocation was restored, and reaccumulated on peroxisomes when translocation was again inhibited. Translocation inhibiting conditions did not result in Pex5p redistribution in cells that lack detectable peroxisomes. Thus, it appears that Pex5p can cycle repeatedly between the cytoplasm and peroxisome. Altered activity of the peroxin defective in CG7 cells leads to accumulation of Pex5p within the peroxisome, indicating that Pex5p may actually enter the peroxisome lumen at one point in its cycle. In addition, we found that the PTS1 receptor was extremely unstable in the peroxin-deficient CG1, CG4, and CG8 cells. Altered distribution or stability of the PTS1 receptor in all cells with a defect in PTS1 protein import implies that the genes mutated in these cell lines encode proteins with a direct role in peroxisomal protein import.

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

“…cDNAs were subjected to in vitro transcription using T7 RNA polymerase (Roche Applied Science). 35 S-Labeled proteins were synthesized using the translation kit Retic Lysate IVT TM (Ambion) in the presence of Redivue TM L-[ 35 S]methionine (specific activity Ͼ1000 Ci/ mmol) following the manufacturer's instructions. In antibody inhibition experiments, 150 g of rat liver PNS or 80 g of purified peroxisomes were preincubated with 30 g of nonimmune or anti-Pex14p immunoglobulins (29) or 10 g of nonimmune or anti-Pex13p (42) Fab fragments in 100 l of import buffer for 20 min on ice, before starting the import reaction by adding the radiolabeled proteins.…”

“…Thus, although the data supporting this general model are now abundant (35)(36)(37), it is clear that we are still far from understanding the mechanistic details of this process. Much of what we know regarding this issue has been inferred from steady-state level analysis of peroxisomal Pex5p in several mutant cell lines (35,36).…”

“…Much of what we know regarding this issue has been inferred from steady-state level analysis of peroxisomal Pex5p in several mutant cell lines (35,36). It is true that this type of studies has been quite valuable in assigning general functions to components involved in protein translocation across the peroxisomal membrane.…”

“…It consists of incubating 35 S-labeled Pex5p with a post-nuclear supernatant from rat liver, and it explores the fact that Pex5p acquires a protease-resistant status when inserted into the peroxisomal membrane (38,39). By using this experimental strategy, it was shown that insertion of Pex5p into the peroxisomal membrane requires the presence of PTS1-containing proteins in the import medium and depends on available Pex14p, an intrinsic membrane component of the peroxisomal docking/translocation machinery (39,40).…”

Quantitative Analysis of Peroxisomal Protein Import in Vitro

“…This topogenic determinant, called PTS1, is recognized in a high-affinity interaction by the receptor molecule Pex5p [8]. This receptor protein appears to shuttle between the cytosol and the peroxisome [9]. Although an understanding of how Pex5p functions mechanistically to facilitate import is not yet worked out, the basis of its association with the peroxisome is.…”