The subset of peroxisomal tail-anchored proteins do not reach peroxisomes via ER, instead mitochondria can be involved

Abstract

Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile acid synthesis and biosynthesis of sterols and ether lipids. Defects in peroxisomes result in severe genetic diseases, such as Zellweger syndrome and neonatal adrenoleukodystrophy. 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 the endoplasmic reticulum (ER) in both wild type (WT) and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly 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 ER, peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that some peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes, instead, mitochondria can be involved.

Ribosome-binding protein 1, RRBP1, maintains peroxisomal biogenesis

“…We analyzed RRBP1 KO HEK293T cells expressing GFP-APEX2-ePTS1 and its parental cell line as a control. In addition, as a positive control we used cells lacking essential peroxisomal biogenesis factors, PEX3 or PEX19, generated in our previous study (Somborac et al, 2023). We observed that the steady-state protein levels of peroxisomal matrix protein, catalase, as well as the peroxisomal membrane proteins, PEX3 and PEX19 were decreased in RRBP1 KO cells compared to the parental cell line (Fig.…”

“…PEX3 KO and PEX19 KO HEK293T cells were generated in our previous study (Somborac et al, 2023) using CRISPR/Cas9 genome editing. Cells were co-transfected with two gRNAs and pSpCas9n(BB)-2A-Puro (PX462) V2.0 (Addgene, 62987 (Ran et al, 2013)).…”