Peroxisomal tail-anchored proteins do not reach peroxisomes via ER, instead mitochondria can be involved.

Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, glycolysis, and biosynthesis of sterols and ether lipids. Defects in peroxisome biogenesis result in severe neurological diseases, such as Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, and myelopathies. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter ER in both WT and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. However, in PEX3 KO cells, which lack peroxisomes as well, the endogenous TA protein, ACBD5, does not target mitochondria, suggesting that PEX3 plays an important role in targeting of peroxisomal TA proteins to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the endoplasmic reticulum (ER), peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes. Instead, mitochondria can play a role in the targeting of TA proteins to peroxisomes.