Identifying and characterizing a missing peroxin—PEX8—in <i>Arabidopsis thaliana</i>

Peroxisomes are dynamic organelles that contribute to diverse metabolic functions, including β-oxidation, photorespiration, and phytohormone biosynthesis. Peroxisomes import proteins from the cytosol through the action of peroxins (PEX proteins), many of which are conserved among fungi, plants, and animals. An apparent exception is Pex8, which is essential for lumenal protein import in several yeast species but has not been reported outside of fungi. Here, we identified an uncharacterized Arabidopsis thaliana protein with predicted structural similarity to Saccharomyces cerevisiae Pex8. Like yeast Pex8, Arabidopsis PEX8 is primarily composed of predicted HEAT repeats and has 2 predicted peroxisomal targeting signals. pex8 insertional and frameshift mutations were lethal, whereas expressing an artificial microRNA targeting PEX8 impaired lumenal protein import into peroxisomes and conferred physiological defects indicative of peroxisome dysfunction. Fluorescent reporters fused to the N terminus of PEX8 localized within peroxisomes in puncta associated with peroxisomal membranes. Our data show that Arabidopsis PEX8 is functionally equivalent to yeast Pex8, revealing the conservation of peroxisomal protein import machinery across eukaryotes and raising the intriguing possibility that other "yeast-specific" peroxins have eluded discovery in plants and mammals because of low primary sequence conservation.