Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi

Translocation of glycolytic enzymes to peroxisomes in fungi suggests broader metabolic role for this organelle. Peroxisome sites for key enzymes of glycolysis Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK) are highly expressed enzymes with a central function in glycolysis. Both proteins are generally considered to be cytoplasmic proteins. This paper shows that several fungal species translocate these glycolytic enzymes into peroxisomes — eukaryotic organelles known to play a part in fatty acid metabolism. In the plant pathogen Ustilago maydis and in Aspergillus nidulans , both enzymes contain cryptic peroxisomal targeting signals that are activated by alternative splicing or translational readthrough of a termination codon. The authors suggest that peroxisomes may have a broader metabolic function than was previously thought, and that other 'cytoplasmic' enzymes may have alternative undiscovered subcellular localizations. Peroxisomes are eukaryotic organelles important for the metabolism of long-chain fatty acids 1 , 2 . Here we show that in numerous fungal species, several core enzymes of glycolysis, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK), reside in both the cytoplasm and peroxisomes. We detected in these enzymes cryptic type 1 peroxisomal targeting signals (PTS1) 3 , which are activated by post-transcriptional processes. Notably, the molecular mechanisms that generate the peroxisomal isoforms vary considerably among different species. In the basidiomycete plant pathogen Ustilago maydis , peroxisomal targeting of Pgk1 results from ribosomal read-through, whereas alternative splicing generates the PTS1 of Gapdh. In the filamentous ascomycete Aspergillus nidulans , peroxisomal targeting of these enzymes is achieved by exactly the opposite mechanisms. We also detected PTS1 motifs in the glycolytic enzymes triose-phosphate isomerase and fructose-bisphosphate aldolase. U. maydis mutants lacking the peroxisomal isoforms of Gapdh or Pgk1 showed reduced virulence. In addition, mutational analysis suggests that GAPDH, together with other peroxisomal NADH-dependent dehydrogenases, has a role in redox homeostasis. Owing to its hidden nature, partial peroxisomal targeting of well-studied cytoplasmic enzymes has remained undetected. Thus, we anticipate that further bona fide cytoplasmic proteins exhibit similar dual targeting.