The Peroxisomal PTS1-Import Defect of PEX1- Deficient Cells Is Independent of Pexophagy in Saccharomyces cerevisiae

Abstract

The important physiologic role of peroxisomes is shown by the occurrence of peroxisomal biogenesis disorders (PBDs) in humans. This spectrum of autosomal recessive metabolic disorders is characterized by defective peroxisome assembly and impaired peroxisomal functions. PBDs are caused by mutations in the peroxisomal biogenesis factors, which are required for the correct compartmentalization of peroxisomal matrix enzymes. Recent work from patient cells that contain the Pex1(G843D) point mutant suggested that the inhibition of the lysosome, and therefore the block of pexophagy, was beneficial for peroxisomal function. The resulting working model proposed that Pex1 may not be essential for matrix protein import at all, but rather for the prevention of pexophagy. Thus, the observed matrix protein import defect would not be caused by a lack of Pex1 activity, but rather by enhanced removal of peroxisomal membranes via pexophagy. In the present study, we can show that the specific block of PEX1 deletion-induced pexophagy does not restore peroxisomal matrix protein import or the peroxisomal function in beta-oxidation in yeast. Therefore, we conclude that Pex1 is directly and essentially involved in peroxisomal matrix protein import, and that the PEX1 deletion-induced pexophagy is not responsible for the defect in peroxisomal function. In order to point out the conserved mechanism, we discuss our findings in the context of the working models of peroxisomal biogenesis and pexophagy in yeasts and mammals.

Keywords: Atg36; Pex1; peroxisomal protein import; pexophagy.

Figure 1

The matrix protein import defect in PEX1-deficient cells occurs independently of pexophagy. (A) The indicated different Saccharomyces cerevisiae wild-type (WT) strains were transformed with the peroxisomal membrane protein Pex11 genetically fused to green fluorescent protein (GFP). The autophagic degradation of peroxisomes is shown by the occurrence of free *GFP. The pex1Δ strains of the BY4741 and BY4742 background display a constitutive degradation of peroxisomes, while the UTL-7A pex1Δ strain lacks PEX1 deletion-induced pexophagy. The degradation of peroxisomes depended on the presence of Atg36. The mitochondrial protein Por1 was used as loading control. Uncropped versions of the blots can be seen in the Supplemental Figure S1A. (B) The vacuolar membrane of BY4741 cells was stained red with FM4-64, while peroxisomal structures labeled with Pex11-GFP are visible as green dots. In addition, the pex1Δ strain displays a diffuse green staining within the vacuole, demonstrating that a portion of the peroxisome population was degraded via pexophagy. This degradation is fully blocked in the pex1Δatg36Δ double mutant. Bar: 5µm. (C) Cells were transformed with a plasmid encoding the peroxisomal matrix protein marker GFP-PTS1. Cells with punctate pattern displayed a functional import, while cytosolic mislocalization indicated an import defect. Bar: 5µm. (D) The subcellular sedimentation analysis of the prepared post-nuclear supernatant (PNS) showed that the matrix protein GFP-PTS1 can mainly be detected in the organellar pellet (OP) fraction of WT and atg36Δ cells (intact import), while it is mislocalized to the cytosolic supernatant (S) fraction in pex1Δ and pex1Δatg36Δ cells (import defect). The level of the endogenous peroxisomal membrane protein Pex13 was elevated in cells without Atg36. The mitochondrial Por1 and the cytosolic Pgk1 served as controls. Uncropped versions of the blots can be seen in the Supplemental Figure S1B. (E) The indicated strains were spotted as a series of 10-fold dilutions on a glucose medium as well as on a medium with oleate as the sole carbon source. The WT and atg36Δ strains had an intact peroxisome biogenesis and could grow on oleate plates. The utilization of oleate during beta-oxidation was further indicated by the formation of halos around the drop spots. The pex1Δ and pex1Δatg36Δ strains were both unable to grow on oleate medium, indicating a defect in beta-oxidation and peroxisome function. (F) Lower diagram: The densitometry data from the Pex13-positive antibody signals from Western blots of organellar pellet fractions were compared. The block of pexophagy in pex1Δatg36Δ resulted in a rise of the Pex13-level of the pex1Δ strain back to WT level. Upper diagram: The functionality of the indicated strains in beta-oxidation was analyzed by monitoring cell growth in liquid oleate medium (n = 3). The value for PEX1-deficient cells did not improve with the additional deletion of ATG36, suggesting that the block of pexophagy does not improve peroxisomal function in beta-oxidation.