Abstract 1438 Unveiling a novel peroxisomal sorting signal of a human membrane-bound reductase involved in plasmalogen biosynthesis

Plasmalogens are the major ether glycerophospholipid constituents in most mammalian tissues. The synthesis of ether lipids begins in peroxisomes and comprises four enzymatic steps. Each of the four enzymes involved is targeted to peroxisomes through a different import pathway. The first one is catalyzed by an acyltransferase (GNPAT) imported to peroxisomes via a C-end peroxisomal targeting signal (PTS1). A synthase (AGPS) imported through a PTS2 is responsible for the second step. The proposed rate-limiting step of the pathway is catalyzed by a reductase (FAR1/2) which associates with peroxisomes peripherally from their cytosolic side. The last peroxisomal step is catalyzed by a membrane-bound reductase called alkyl/acyl dihydroxyacetone-phosphate reductase (ADHAPR). ADHAPR catalyzes the reduction of alkyl dihydroxyacetone-phosphate into alkyl glycerol-3-phosphate and bridges the movement of ether lipids precursors to the endoplasmic reticulum, where the synthesis of complex plasmalogens is finalized. The gene encoding ADHAPR was identified in mammals as DHRS7B based on homology with Ayr1, a yeast reductase catalyzing a similar reaction. Ether lipids are absent in yeast and Ayr1 is a lipid droplet resident protein that participates in a shunt pathway for biosynthesis of glycerolipids. In contrast, human ADHAPR (hADHAPR) is a peroxisomal membrane protein (PMP) with a single transmembrane domain that belongs to the short-chain dehydrogenases/reductases (SDR) family. We aim to characterize the peroxisomal sorting mechanism of hADHAPR. In this work we have validated the use of budding yeast Saccharomyces cerevisiae to tackle these studies. Using live fluorescence microscopy, we have determined that hADHAPR-GFP localizes to peroxisomes when expressed in yeast. hADHAPR degradation was observed in yeast mutants lacking Pex19, suggesting Pex19 stabilizes the protein. We then evaluated the sorting of hADHAPR during the de novo biogenesis of peroxisomes. Interestingly, hADHAPR was found in Pex14-containing pre-peroxisomal vesicles (PPVs) in yeast mutants lacking Pex3, which is a critical receptor for the biogenesis of peroxisomes and partner for Pex19. In addition, hADHAPR also localized to PPVs budding sites in the ER enriched in Pex30. Altogether these results suggest that hADHAPR can be targeted to peroxisomes in PPVs derived from the ER in a Pex3 independent manner. Albeit peroxisomal membrane proteins do not have a conserved targeting signal like the ones found in peroxisomal matrix proteins (PTS1 and PTS2), hADHAPR was sorted into yeast peroxisomes indicating a generally conserved sorting signal mechanism for PMPs. Notably, overproduction of hADHAPR induced peroxisomal proliferation, an effect not seen when other peroxisomal enzymes of this metabolic pathway like hGNPAT or hAGPS were overproduced at similar levels. Using a toolbox of mutants, we have determined that the sequence including the N-terminal 44 residues of hADHAPR is necessary and sufficient for targeting a GFP reporter to peroxisomes. In summary, we provide evidence for hADHAPR target to peroxisomes via a ER-PPV dependent mechanism which involves recognition of its N-end by peroxins. Our study contributes to better understand signature motifs required for sorting of membrane proteins to peroxisomes and the impact of PMPs in peroxisome biogenesis and dynamics. This work has been financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant to VZ.