LETTER doi:10.1038/nature11051 Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi Johannes Freitag 1 , Julia Ast 1 & Michael Bo ¨lker 1 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 per- oxisomal isoforms vary considerably among different species. In the basidiomycete plant pathogen Ustilago maydis, peroxisomal target- ing of Pgk1 results from ribosomal read-through, whereas alterna- tive 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 dehydro- genases, 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. Peroxisomes are highly versatile organelles involved in many biological processes in addition to their common function in fatty-acid metabolism and hydrogen peroxide degradation 4 . In plants, specialized peroxisomes (glyoxysomes) are used during seed germination for breaking down storage lipids 5 . Trypanosomatide parasites contain glycosomes that harbour nearly all glycolytic enzymes, which in all other eukaryotes reside in the cytoplasm 6 . In filamentous ascomycetous fungi, peroxisome-derived Woronin bodies prevent cytoplasmic bleeding in disrupted fungal hyphae and thus allow multicellular development 7 . Despite the metabolic diversity of peroxisomes, their biogenesis is highly conserved 8 . Peroxisomes receive their membrane from the endo- plasmic reticulum and are matured in the cytoplasm 9,10 . Peroxisomal proteins are not processed during import and pass the membrane in the fully folded state, cofactor bound and even as oligomers 11 . Most peroxisomal proteins contain a carboxy-terminal PTS1 derived from the prototype tripeptide Ser-Lys-Leu (SKL) 3 . Further studies revealed that additional C-terminal residues modulate targeting efficiency, with the last 12 C-terminal residues being most important 12 . Some peroxisomal proteins contain an alternative signal (PTS2) near the amino terminus 13 . Import of proteins into peroxisomes is mediated by an evolutionary conserved set of proteins known as peroxins (Pex). PTS2-containing proteins are recognized by Pex7, whereas import of PTS1-containing proteins depends on the PTS1 receptor Pex5 (ref. 11). During a survey of differentially spliced transcripts in the plant pathogenic fungus U. maydis, we noticed that the glycolytic enzyme GAPDH (Enzyme Commission (EC) number 1.2.1.12) exists in two isoforms (MIPS Ustilago maydis database (MUMDB) accession numbers um02491 and um10167), of which one ends with a predicted 14 PTS1 motif (-SRL), suggesting peroxisomal localization of this isoform (Gapdh pex ) (Fig. 1a). Fusion of the 12 C-terminal amino acids of Gapdh pex to green fluorescent protein (GFP–PTS1 Gapdh ) resulted in punctate staining (Fig. 1a). These structures were also stained by the peroxisomal marker proteins mCherry–SKL and acyl-coA thioester hydrolase 15 (GFP–Pte1; Supplementary Fig. 1a). We could demonstrate peroxisomal localization of full-length Gapdh pex by expression of GFP–Gapdh pex , derived from the PTS1- containing splice variant (Supplementary Fig. 1b). In peroxisome- deficient mutants (Dpex6), GFP–Gapdh pex showed diffuse cytoplasmic staining (Supplementary Fig. 1c). To estimate the fraction of perox- isomal Gapdh pex , alternative messenger RNA splice variants were quantified by real-time PCR (rtPCR). Gapdh pex transcripts accounted for about 10% of total gapdh mRNA (Fig. 1a and Supplementary Fig. 2). This value is in agreement with the fraction of expressed sequence tags (ESTs) (4 out of 36) listed in the U. maydis database corresponding to Gapdh pex (Fig. 1a). Taken together, these data indi- cate that U. maydis expresses a peroxisomal isoform of GAPDH. The unanticipated peroxisomal localization of a central glycolytic enzyme prompted the screening of other glycolytic enzymes for cryptic PTSs. Analysis of the pgk1 gene (um04871) encoding PGK (EC 2.7.2.3) revealed that ribosomal read-through 16 of the termination codon results in an extended isoform (Pgk1 pex ), which contains a functional PTS1 (Fig. 1b). To mimic translational read-through, we replaced the termination codon (TGA) of pgk1 by a serine codon (TCA). GFP fused to this extended version (GFP–Pgk1 pex ) was found in peroxisomes (Supplementary Fig. 1d), and this localization was abolished in Dpex6 mutants (Supplementary Fig. 1c). Expression of C-terminal GFP fusion protein revealed that efficient translational read-through depended on mRNA sequences downstream of the termination codon (Supplementary Fig. 3a). Secondary structure prediction using RNAfold 17 indicated that this region can form a stable stem–loop structure (Supplementary Fig. 3b). To exclude overexpres- sion artefacts GFP–Pgk1 fusion protein was also expressed under control of its endogenous promoter (Supplementary Fig. 3c). Western blot analysis revealed that a considerable amount (20%) of GFP–Pgk1 was expressed as C-terminally extended protein, indicating translational read-through (Fig. 1b and Supplementary Fig. 3d). We also discovered cryptic peroxisomal targeting of GpdA (GAPDH) and PgkA (PGK) in the ascomycetous fungus A. nidulans (Fig. 2). Notably, A. nidulans uses reciprocal mechanisms for dual targeting of glycolytic enzymes. Peroxisomal GAPDH is generated by ribosomal read-through (Fig. 2b), whereas the peroxisomal isoform of PGK results from alternative splicing (Fig. 2c). The predicted C-terminal targeting motifs were fused to red fluorescent protein (RFP) and expressed in Saccharomyces cerevisiae. Peroxisomal local- ization of the corresponding fusion proteins (RFP–PTS1 GpdA and RFP–PTS1 PgkA ) demonstrated that both PTS1 signals are functional. Peroxisomal 2,4-dienoyl-CoA reductase encoded by the S. cerevisiae SPS19 gene served as a control 18 (Fig. 2b, c). Database searches revealed that many fungal genes coding for GAPDH and PGK contain cryptic peroxisomal-targeting motifs 1 Department of Biology, Philipps University Marburg, Karl-von-Frisch-Strasse 8, D-35032 Marburg, Germany. 522 | NATURE | VOL 485 | 24 MAY 2012 Macmillan Publishers Limited. All rights reserved ©2012