Cryptic peroxisomal targeting determinants in protistan glycolytic enzymes suggest a 'how' for glycosome evolution

In containing the first six-to-seven glycolytic enzymes within the organelle matrix, the glycosomes of trypanosomatid protists and their nearest relatives represent extreme forms of peroxisome specialisation. How or why such extreme peroxisomes evolved is not known. Many proteins are peroxisome targeted following recognition of a C-terminal type-1 peroxisome targeting signal (PTS1). Here, we identified in Naegleria gruberi, an amoeboflagellate distantly related to trypanosomatids, cryptic PTS1 motifs in a variety of metabolic enzymes, including in some glycolytic enzymes. These signals arise from stop-codon read-through or alternative splicing and are conserved in opportunistic pathogen N. fowleri. We show selected cryptic N. gruberi PTS1 motifs function in protein import into glycosomes of trypanosomatid Crithidia fasciculata. Further analysis revealed similar cryptic PTS1 motifs in evolutionarily diverse protists, including some from Discoba - the broad eukaryotic group to which Naegleria and trypanosomatids belong. Fragmentary data allowed only a cursory, equivocal glimpse of peroxisome biochemistry within Euglenida, the protists most closely related to trypanosomatids. Collectively, however, our data indicate Naegleria displays more versatility within its unusual metabolism than previously appreciated but moreover suggest dual protein localisation may have been used to diversify peroxisome function early in eukaryotic evolution and point towards at least two stages in glycosome evolution.