The Budding Yeast Pex5p Receptor Directs Fox2p and Cta1p Into

RESEARCH ARTICLE The budding yeast Pex5p receptor directs Fox2p and Cta1p into peroxisomes via its N-terminal region near the FxxxW domain Łukasz Rymer*, Błażej Kempiński*, Anna Chełstowska and Marek Skoneczny‡

ABSTRACT the cell. A comprehensive account of peroxisome biogenesis and The import of most of peroxisomal proteins into the lumen of their functions can be found in the recent special issue of Biochimica et target organelle is driven by C-terminal (PTS1) or N-terminal (PTS2) Biophysica Acta (2016, vol. 1863, pp. 787-1069). When compared signals recognized by the Pex5p or Pex7p receptors, respectively. with the proteomes of other organelles, the peroxisomal proteome However, some proteins in budding yeast, such as acyl-CoA oxidase is relatively small. Currently, the number of known peroxisomal (AOx) and carnitine acetyltransferase (Cat2p), are imported into proteins in humans is 101, while that in budding yeast peroxisomes via an alternative route that does not rely on known (Saccharomyces cerevisiae Meyen ex E.C. Hansen) is 75 (http:// PTS signals and involves the Pex5p receptor N-terminal region. Here, www.peroxisomedb.org/, accessed 17 July, 2018) (Schlüter et al., we show that two other budding yeast peroxisomal proteins, a 2010). Of those proteins, only a subset is localized in the multifunctional enzyme from the β-oxidation pathway (Fox2p) and peroxisomal lumen; however, the mechanisms for the import of catalase A (Cta1p), both of which contain PTS1, can be imported these few proteins into peroxisomes are surprisingly diverse. independently of this signal. The I264K amino acid substitution in Peroxisomal matrix proteins are imported post-translationally, and Pex5p adjacent to its FxxxW diaromatic motif, previously shown to the specificity of transport depends on the peroxisome-specific abolish the import of AOx and Cat2p into peroxisomes, also affects targeting signal encoded in the protein sequence. Thus far, two such Fox2p and Cta1p import. Moreover, we demonstrate that Pex9p, a signals are known and have been well-characterized: peroxisomal newly discovered paralog of Pex5p that was recently implicated in the targeting signal 1 (PTS1) and peroxisomal targeting signal 2 (PTS2) import of malate synthases in budding yeast, also exhibits weak (Girzalsky et al., 2010). receptor activity towards Fox2p and Cta1p. These findings indicate The PTS1 targeting signal was established as a tripeptide located at the need to re-evaluate the peroxisomal import paradigm. the extreme C-terminus of proteins (Gould et al., 1989). While an SKL sequence appears to be the most typical, permutation analysis This article has an associated First Person interview with the first led to the formulation of a more flexible rule for the PTS1 signal: author of the paper. (S/A/C)-(K/H/R)-(L) (Swinkels et al., 1992). The comparison of many PTS1 sequences in mammals, plants, protozoa and yeast KEY WORDS: PTS3, Dual-function receptor, Peroxisome biogenesis, allowed the generalization of the PTS1 composition as follows: Peroxisome matrix proteins, Pex9p (small)-(basic)-(large non-polar) amino acids. In silico analysis generalized the consensus even further (Nötzel et al., 2016). The INTRODUCTION tripeptide PTS1-like sequence is present in the majority of Peroxisomes are small vesicular organelles that are present in almost peroxisomal matrix proteins and is necessary and usually sufficient all eukaryotic cells. They are surrounded by a single membrane to direct reporter proteins to peroxisomes. Interestingly, however, and contain diverse sets of enzymes that are involved in several some PTS1 variants are not active in a heterologous context, although metabolic pathways, including fatty acid β-oxidation and lipid they function in the proteins in which they naturally occur. This biosynthesis. Peroxisomes are characterized by the presence of a behavior implies that some additional information may be needed for number of oxidases that produce hydrogen peroxide, which is the import of these proteins. Therefore, the complete PTS1 signal decomposed by catalase (Tolbert, 1981). ensuring the proper recognition of peroxisomal proteins has been The comparison of peroxisomal protein contents in different proposed to include up to nine amino acid residues preceding the tissues and species demonstrates that some tissues and species C-terminal tripeptide (Brocard and Hartig, 2006). lack peroxisomal proteins that are present in others, which clearly A much smaller group of peroxisomal matrix proteins uses a reflects the ability of peroxisomes to adjust to specific metabolic PTS2 signal consisting of nine amino acids with the consensus functions. Even within evolutionarily related groups, such as sequence (R/K)-(L/I/V)-X5-(H/Q)-(L/A/F) located within the first mammals, peroxisome diversity is observed (Islinger et al., 2010). 40 N-terminal amino acids of the protein (Lazarow, 2006). To date, Moreover, the peroxisomal proteome may be modified to adapt to only two proteins with functional PTS2 sequences have been changes in environmental conditions and the metabolic state of identified in S. cerevisiae, 3-ketoacyl-CoA thiolase (Fox3p/Pot1p) (Erdmann, 1994) and glycerol-3-phosphate dehydrogenase (Gpd1p) (Jung et al., 2010). Curiously, the import of the Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy nicotinamidase Pnc1p into peroxisomes also depends on the of Sciences, Pawińskiego 5A, 02-106 Warszawa, Poland. *These authors contributed equally to this work PTS2 signal, but it relies on the signal in Gpd1p via a piggy-back import mechanism (Effelsberg et al., 2015; Kumar et al., 2016; ‡ Author for correspondence ([email protected]) Saryi et al., 2017), another feature that is characteristic of M.S., 0000-0001-9922-2276 peroxisome biogenesis. Two well-known peroxisomal proteins in S. cerevisiae function

Received 18 February 2018; Accepted 7 August 2018 as import receptors: Pex5p with tetratricopeptide repeat (TPR) Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216986. doi:10.1242/jcs.216986 domains and Pex7p with WD-40 domains, which recognize PTS1 the PTS1 signal on its C-terminus, although the signal itself was and PTS2 signals, respectively. While Pex5p can function as an proven to be fully functional in a heterologous context (Elgersma autonomous receptor, Pex7p requires the co-receptors Pex18p and et al., 1995). These results were later corroborated by narrowing Pex21p (Purdue et al., 1998), which stabilize and target the down the region in the Pex5p polypeptide that is important for peroxisomal membrane cargo-bound Pex7p (Stein et al., 2002). the import of these two proteins to the 250-270 aa fragment Recently, another yeast protein, which for two decades was a encompassing the diaromatic motif FxxxW. Individual residues known paralog of Pex5p with no assigned function, was shown to indispensable for AOx and/or Cat2p recognition were identified participate in the import of a subset of PTS1-dependent peroxisomal within this motif and at neighboring positions (Klein et al., 2002). matrix proteins and was named Pex9p (Effelsberg et al., 2016; Therefore, the two budding yeast proteins AOx and Cat2p appeared Yifrach et al., 2016). However, its role in the peroxisomal import to utilize an alternative peroxisomal import pathway, which was machinery is still not fully understood. tentatively called the PTS3 route, even though its components The receptor-assisted translocation of proteins into the remain unknown to date. In particular, we do not know the PTS3 peroxisomal lumen occurs via a transient pore that is formed in signal itself – i.e. the consensus sequence – if one exists, of the the peroxisomal membrane by Pex5p and Pex14p peroxins and, amino acids that direct AOx and Cat2p into peroxisomes. depending on the cargo size, can be as wide as 9 nm (Meinecke Another question that remains is the number of proteins that et al., 2010). This mechanism distinguishes peroxisomes from other actually utilize this route. Are AOx and Cat2p the only budding cellular organelles, and the temporary nature of the import pores yeast proteins that are imported into peroxisomes via the explains past contradictory findings demonstrating that the hypothetical PTS3 route? Most peroxisomal matrix proteins peroxisomal membrane is able to maintain a pH gradient contain a PTS1 or PTS1-like tripeptide at their C-terminus. (Waterham et al., 1990) but is permeable to bulky particles Notably, however, many of them were assigned to the PTS1- (Walton et al., 1995). This unique mechanism allows the dependent import route solely because of the presence of the PTS1 peroxisome to internalize proteins in their native folded signal. The dependence of their import on this signal had been conformation and oligomeric protein complexes. As a implied but not proven directly. Therefore, a reasonable question consequence, proteins that lack their own PTS signal can be is whether the PTS1 signal is necessary and sufficient to drive imported into peroxisomes in association with PTS-containing these proteins into peroxisomes in all cases or if it is sometimes proteins via the piggy-back mechanism mentioned above. dispensable, as in the case of Cat2p. Finally, there are proteins that Curiously, two distinct types of membrane pores are created in the are functionally associated with peroxisomes but not shown to be peroxisomal membrane for the separate transport of Pex5p- or localized in this compartment and do not contain either PTS1 or Pex7p-dependent cargos (Montilla-Martinez et al., 2015). PTS2. Perhaps some of them do reside in the peroxisomal matrix Interestingly, this situation in S. cerevisiae is not universal. and are imported via the PTS3 route. Peroxisome import mechanisms appear to be subject to Although ambiguities in peroxisomal import signaling have been evolutionary diversification. Sometimes, the orthologous proteins recognized since exceptions from the strict rules regarding PTS1 from various systematic groups are imported via different routes. (and also PTS2) were reported, no attempt has been made to answer For example, while the import of thiolase is dependent on the PTS2 the above questions. This paper aims to address them. By employing signal and Pex7p receptor in most organisms, in Caenorhabditis several complementary approaches, we were able to demonstrate elegans, thiolase contains the PTS1 signal (Bun-Ya et al., 1997); a that in addition to AOx and Cat2p, at least two other proteins, PTS2-dependent import route and its components appear to be Fox2p and Cta1p, are partially dependent on interactions with the absent from this organism (Motley et al., 2000). N-terminal region of Pex5p for import. The Ile264 amino acid Another example is AOx, the key enzyme in peroxisomal residue in Pex5p, which is adjacent to the FxxxW diaromatic β-oxidation. These proteins in Yarrowia lipolytica, S. cerevisiae domain that was previously shown to be crucial for recognizing and many other yeast species have no known PTS signals AOx and Cat2p as cargos, is also important for the efficient import (Wang et al., 1999; https://portals.broadinstitute.org/cgi-bin/regev/ of Fox2p and Cta1p. Moreover, Pex9p appears to be able to orthogroups/show_orthogroup.cgi?orf=YGL205W, accessed 17 substitute, albeit weakly, for the absent Pex5p in the import of these July, 2018) but are efficiently imported into peroxisomes, whereas two proteins. That substitution most likely relies on the N-terminal in mammals and Pichia pastoris, they are imported via the PTS1 part of Pex9p. Although Pex9p does not contain the FxxxW route (Koller et al., 1999). Thus, the import mechanisms of diaromatic motif, it displays strong homology to the region of Pex5p peroxisomal matrix proteins appear to be as complex and diverse as that contains this motif. In light of our data, the peroxisomal import the functions of this organelle and are definitely not fully mechanisms appear to be even more complex than previously understood. believed. After the PTS1- and PTS2-dependent import routes were basically determined, the import of the AOx enzyme, which in RESULTS several yeast species possesses neither of these signals, remained Approaches to identify the potential PTS3 pathway especially puzzling. However, studies trying to solve this puzzle cargo proteins have been scarce. Almost two decades ago, the import of AOx into We aimed to identify potential peroxisomal matrix PTS3-type peroxisomes in S. cerevisiae was shown to depend on Pex5p, but it proteins whose transport is dependent on the Pex5p receptor but not was also revealed that AOx interacts with the region between on its TPR domain, which is involved in the transport of proteins residues 136 and 292 in this receptor, which is outside the containing the classical PTS1 signal at the C-terminus. To assign a C-terminal half containing the TPR domain that is responsible for peroxisomal protein to this novel group of proteins, we established the recognition of the PTS1 signal. Moreover, the same 136-292 aa the following initial criteria: (1) a protein that is transported into fragment was found to bind the carnitine acetyltransferase Cat2p peroxisomes even though it does not contain the PTS1 signal, or the (Skoneczny and Lazarow, 1998). Curiously, the latter protein was signal can be removed without affecting protein peroxisomal shown to be imported into peroxisomes regardless of the presence of localization; (2) a protein that is not transported into peroxisomes in Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216986. doi:10.1242/jcs.216986 cells lacking the Pex5p receptor; and (3) a protein that is transported into peroxisomes in cells lacking the Pex7p receptor, thus showing its independence of the putative PTS2 signal recognition. Two approaches were undertaken to discover the novel target proteins transported via the PTS3 pathway and thus bearing this as yet unidentified PTS3 signal: an experimental approach and an ‘educated guess’ approach.

Fox2 protein is imported into peroxisomes regardless of its PTS1 signal As an experimental approach to identify further peroxisomal proteins whose import may depend on the PTS3 signal, we used metal affinity chromatography to identify proteins that physically interact with the N-terminal region of Pex5p encompassing the putative PTS3 recognition domain. Since it was previously demonstrated in a two-hybrid test and by metal affinity chromatography that AOx and Cat2p physically interact with a small fragment of Pex5p comprising 136-292 amino acid residues (Skoneczny and Lazarow, 1998), we used the same 6×His-tagged polypeptide (His-Pex5p136-292) as bait to affinity capture other proteins that might be imported into peroxisomes via the same route as AOx and Cat2p. Protein samples were extracted from S. cerevisiae cells grown in oleate-containing medium to induce peroxisome proliferation. Moreover, we used a pox1Δ,cat2Δ double-deletion strain to increase the purification yield of the prospective protein since we had previously noticed signs of competition between AOx and Cat2p for binding to the His- Pex5p136-292 bait. The resulting specifically bound protein was identified by mass spectrometry as Fox2p, a bifunctional enzyme with 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activity in the peroxisome fatty acid β-oxidation pathway that converts trans-2-enoyl-CoA esters to 3-ketoacyl-CoA esters (Hiltunen et al., 1992). The authenticity of the Fox2p retained on the affinity column was confirmed with cell extracts from the pox1Δ,cat2Δ,fox2Δ triple-deletion strain. No corresponding protein band was seen in the eluate from the affinity column loaded with the protein extract from the cells of this strain (data not shown). Thus, Fox2p could be another peroxisomal protein transported via the novel PTS3 pathway even though, like Cat2p, it possesses a PTS1 signal (-SKL). For that reason, it was commonly assumed to be targeted to the peroxisomal matrix via the PTS1 pathway; although, to the best of our knowledge, it was never demonstrated experimentally. To verify the functionality of the Fox2p C-terminal SKL sequence and the adjacent amino acid residues, hybrid genes were constructed encoding proteins in which the PTS1 sequence of Fox2p was removed and its C-terminus blocked by the green fluorescent protein (GFP) tag. Cells from the wild-type (WT) and mutant yeast strains were transformed with this plasmid and with the plasmid encoding the peroxisome localization marker mRFP-SKL. The localization of the fusion proteins was checked by fluorescence microscopy in cells grown in oleic acid-containing medium to induce peroxisome proliferation (see Materials and Methods for details of plasmid construction and yeast cell growth conditions). As Fig. 1. Fox2p and Cta1p are imported into peroxisomes in a PTS1- shown in Fig. 1A, Fox2p with a non-functional PTS1 was present in and PTS2-independent manner. The wild-type BY4741 strain and its yeast cells in the form of particles that colocalized with the pex7Δ,fox2Δ or cta1Δ knockout derivatives were transformed with peroxisomal fluorescence marker. plasmids encoding C-terminal fusions of the indicated proteins with GFP: (A) Fox2p-GFP, (B) Cta1p-GFP, (C) Cat2p-GFP or (D) AOx-GFP. The strains were also transformed with plasmids encoding the red PTS1 signal in budding yeast catalase A (Cta1p) is not fluorescent marker of peroxisomes mRFP-SKL. Transformant cells were absolutely necessary for its efficient targeting to grown overnight in oleate-containing medium and visualized under a peroxisomes fluorescence microscope as described in the Materials and Methods. An additional approach to find further peroxisomal matrix proteins Scale bars: 5 µm. imported by the PTS3-dependent route involved analysis of the Journal of Cell Science

3 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216986. doi:10.1242/jcs.216986 available literature and amino acid sequence data. Candidate peroxisomes and a cytosol fraction, and the catalase activity proteins were selected based on the following principles: (1) there was measured in both fractions as described in the Materials are reports indicating or suggesting the peroxisomal localization of and Methods. The peroxisomal portion of catalase A without PTS1 the protein, and (2) the protein lacks both PTS1 and PTS2 targeting was ∼20% smaller than wild-type catalase A (Fig. 2C), so removal of signals, or it does have the PTS1 signal, but no literature data the PTS1 signal had only a minor effect on its import competence. or conflicting data exist regarding its indispensability for import. We also included S. cerevisiae proteins that are homologues of The import of Fox2p-GFP and Cta1p-GFP into peroxisomes peroxisomal proteins from other organisms that also lack known does not depend on the piggy-back mechanism peroxisomal targeting sequences. Finally, the list of candidate One of the features of peroxisome biogenesis is the ability of this S. cerevisiae proteins that could be transported via the PTS3 organelle to import mature oligomeric protein complexes. As a route included Pcd1p, Faa2p, Mdh2p and Cta1p; however, the consequence, polypeptides with no peroxisomal targeting signal preliminary localization experiments excluded the first three from can enter peroxisomes while bound or ‘piggy-backed’ to further analysis. Only the import of Cta1p, the peroxisomal catalase polypeptides that contain one. This workaround mechanism was A, was analyzed in more detail. Earlier attempts to characterize first demonstrated for thiolase (Glover et al., 1994), and evidence the mechanism of yeast Cta1p recognition and targeting to the has since accumulated that this mechanism is widespread in S. peroxisomes suggested that in addition to its C-terminal SKF cerevisiae (Yang et al., 2001; Effelsberg et al., 2015; Kumar et al., tripeptide, some other internal sequence(s) could be involved in its 2016; Saryi et al., 2017) and other species (Titorenko et al., 2002; import (Kragler et al., 1993). This possibility was not subjected to Lee et al., 1997). Therefore, we tested whether the observed import any further studies, but to us, the import of Cta1p appeared likely of Fox2-GFP and Cta1p-GFP, which had their C-termini blocked to depend at least in part on the same mechanisms as that of AOx with a GFP tag, might be due to the formation of complexes with the and Cat2p. respective endogenous native proteins. This could occur for Cta1p- To test this assumption, we constructed a plasmid containing the GFP, as native catalase A is known to be a tetramer (Seah et al., hybrid gene encoding Cta1p protein from which the SKF sequence 1973); however, to the best of our knowledge, no data are available was removed and with the C-terminus blocked by GFP tagging. on the oligomeric state of S. cerevisiae Fox2p protein. However, Cells from the WT and mutant yeast strains were transformed with the fruit fly peroxisomal multifunctional enzyme was shown this plasmid and with the plasmid encoding the peroxisome crystallographically to be a dimer (Haataja et al., 2011). If the localization marker mRFP-SKL. The localization of the fusion same is true for its budding yeast orthologue, the piggy-back import proteins was checked by fluorescence microscopy in cells grown in of PTS1-less Fox2p-GFP bound to native Fox2p is conceivable. oleic acid-containing medium to induce peroxisome proliferation. However, as shown in Fig. 1A, the efficiency of Fox2p-GFP import As shown in Fig. 1B, the Cta1p-GFP fusion protein was found in in fox2Δ cells lacking the native Fox2p appeared to be similar to that peroxisomes, which indicated that its import could be independent of WT cells. The same was true for Cta1p-GFP (Fig. 1B), which of the PTS1 signal. displayed a comparable efficiency of import in WT and cta1Δ cells. The piggy-back mode of import for plasmid-encoded Cta1p-YIS Comparison of the PTS1-independent peroxisomal import (Fig. 2C) was also excluded because of the lack of genome-encoded efficiency of Fox2p and Cta1p with that of AOx and Cat2p native Cta1p in the GC1-8B strain. Incidentally, as shown in The images shown in Fig. 1A-D revealed that the PTS1-independent Fig. 1C,D, the import of neither the C-terminally GFP-tagged AOx, import of Cta1p and Fox2p might not be as efficient as the import of known to be an octamer in its native state, nor that of Cat2p-GFP AOx and Cat2p, the two PTS3-dependent proteins discovered showed signs of dependence on the piggy-back mechanism, as both earlier. Both AOx and Cat2p were tagged with GFP at the C-terminus were equally efficient in WT cells and in the respective deletion and were observed almost exclusively in particles colocalizing with strains pox1Δ and cat2Δ. the peroxisomal marker, whereas Cta1p-GFP and Fox2p-GFP, in addition to peroxisomes, were also observed in the cytosol. This Import of Fox2p and Cta1p into peroxisomes does not difference could mean that these enzymes are natively present in the depend on the Pex7p PTS2 import receptor but does depend cytosol in addition to peroxisomes or that their C-terminal regions on the Pex5p peroxin, which is similar to AOx and Cat2p have some contribution to their peroxisomal import. To resolve this, Two known peroxisomal import routes relying on PTS1 and PTS2 we tested the efficiency of Fox2p import when it was tagged with targeting signals are handled by the Pex5p and Pex7p cargo GFP at the N-terminus (GFP-Fox2p). The tagged protein is virtually receptors, respectively. None of the studied proteins have ever been exclusively present in peroxisomes (Fig. 2A, top row), whereas N- associated with the PTS2-dependent peroxisomal import route, nor terminally tagged Fox2p lacking its C-terminal SKL tripeptide do they contain any recognizable PTS2 signals. However, for the (GFP-Fox2p-ΔSKL) is imported less efficiently (Fig. 2B, top row), sake of completeness, we tested the influence of the absence of similarly to the C-terminally GFP-tagged construct. either Pex5p or Pex7p on the import of GFP-tagged Fox2p and By exploiting the fact that enzymatic activity and, hence, its levels Cta1p into peroxisomes. As shown in Fig. 1A-D, the absence of in subcellular fractions can be measured easily, we quantified the Pex7p ( pex7Δ strain) had no influence on the import of these relative contributions of PTS1-dependent and PTS1-independent proteins, just as in the case of AOx and Cat2p. In contrast, as shown Pex5p-Cta1p interactions to the import of Cta1p into peroxisomes by in Fig. 2A,B,D,E, their import depended on the Pex5p receptor testing the native protein and the protein with its C-terminal SKF (compare rows labeled WT and pex5Δ in panels A, B, D and E in signal replaced with YIS. Both proteins were expressed from Fig. 2). This dependence classifies them into the same group as AOx centromeric, single-copy YCp50-derivative vectors (YCp-Cta1p and Cat2p. In addition to the images, we collected quantitative data and YCp-Cta1p-YIS, respectively). The GC1-8B yeast strain by counting cells expressing GFP-tagged Fox2p and Cta1p to transformed with these plasmids was devoid of endogenous Cta1p determine the fraction that showed at least partial particulate and Ctt1p catalases. Transformant cells grown in oleate-containing fluorescence (Fig. 2F). The data obtained for constructs encoding medium were fractionated into an organellar fraction containing Fox2p and Cta1p with their C-termini blocked by GFP (Fig. 2D,E), Journal of Cell Science

4 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216986. doi:10.1242/jcs.216986

Fig. 2. The import of Fox2p and Cta1p depends on the Pex5p receptor but can also occur with the assistance of Pex9p. The wild-type BY4741 strain and its pex9Δ,pex5Δ single-knockout and pex5Δ,pex9Δ double-knockout derivatives were transformed with (A) GFP-Fox2p, (B) GFP-Fox2p-ΔSKL, (D) Fox2p-GFP or (E) Cta1p-GFP plasmids. The strains were also transformed with the PTS2-DsRed plasmid encoding the red fluorescent marker of peroxisomes. Transformant cells were grown overnight in oleate-containing medium and visualized under a fluorescence microscope as described in the Materials and Methods. Scale bars: 5 µm. The green channel images for pex5Δ cells were brightened with Adobe Photoshop CS6 (Adobe Systems Inc., San Jose, CA, USA) image processing software to expose residual peroxisomal localization of the respective proteins, which resulted in overexposure of the adjacent cells. (C) GC1-8B strains transformed with either the YCp-Cta1p plasmid encoding native catalase A or YCp-Cta1p-YIS encoding catalase A with its PTS1 inactivated were precultured in selective SC medium and grown overnight in YPEO medium and then spheroplasted, homogenized and fractionated. The catalase activity in each fraction was measured spectrophotometrically at 240 nm as H2O2 decomposed (see Materials and Methods for more details). The graph shows the distribution of catalase A activity between the organellar (dark gray boxes) and cytosolic (white boxes) fractions, expressed as the percentage of combined activity found in both fractions. The data from three experiments are averaged. Error bars represent the s.d. (F) The import efficiency of the plasmid-encoded GFP- tagged proteins in the tested strains was quantified and expressed as the percentage of cells (relative to the total number of cells analyzed) containing particles visible in the green fluorescence channel that colocalized with the red fluorescence peroxisome marker. Individual data points are the average (mean) of three replicates of 100 cells scored. No cells with foci colocalizing with peroxisomes were scored for the pex5Δ,pex9Δ double-knockout strain, and therefore, no data for this strain are displayed. Error bars represent the s.d. Statistical significance was calculated using Student’s t-test: **P<0.01 and *P<0.10. together with the data presented in Fig. 1A,B, clearly indicate that purification experiment for Fox2p and fully support the previously the import of these proteins into peroxisomes can be driven by postulated dual-domain nature of Pex5p. These results also suggest Pex5p without recognition of the PTS1 signal by its TPR domain. that, in addition to AOx and Cat2p, the import of Fox2p and Cta1p

These findings are consistent with the results from our affinity may depend, at least in part, on the hypothetical PTS3 route. Journal of Cell Science

5 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216986. doi:10.1242/jcs.216986

In the absence of Pex5p, its paralogous receptor protein, tagged GFP-Fox2p with its PTS1 signal exposed, the difference in Pex9p, displays weak receptor activity towards Fox2p the numbers of cells with GFP particles observed in the presence of and Cta1p WT Pex5p and in the presence of its I264→K-mutated version were Interestingly, although the importance of Pex5p for the import of less pronounced. Ile264 residue is located near the FxxxW motif Fox2p and Cta1p was beyond doubt, we could occasionally see known to be important for the interaction of Pex5p receptor with residual peroxisomal localization for both these proteins in the Pex14p, a subunit of the peroxisomal translocon (Williams et al., pex5Δ strain (see rows labeled pex5Δ in Fig. 2A,B,D,E). A paralog 2005). Therefore, its substitution could potentially influence the of Pex5p, Pex9p, has been recently characterized in S. cerevisiae interaction between these peroxins and in consequence affect the (Effelsberg et al., 2016; Yifrach et al., 2016), and its participation in Pex5p-dependent import as a whole, so we tested if substitution of the import of the subset of PTS1-dependent peroxisomal matrix the Ile264 residue in Pex5p affects import that relies on the PTS1 proteins was demonstrated. Therefore, we tested whether this protein targeting signal. As shown in Fig. 3B, substitution of the Ile264 contributes to Fox2p and Cta1p import and is therefore responsible residue in Pex5p had no effect on the proportion of cells displaying for their visible residual peroxisomal localization. As shown in Fig. 2, particulate localization of mRFP-SKL, a marker protein imported absence of the PEX9 gene had no noticeable effect on the import of into peroxisomes exclusively via a PTS1-dependent route. Notably, the studied proteins into peroxisomes, but the absence of both the in the absence of Pex5p, we did not observe any residual PEX5 and PEX9 genes had a stronger detrimental effect on the import peroxisomal localization that was similar to that seen for GFP of GFP-tagged Fox2p and Cta1p than the absence of the PEX5 gene fusions of Fox2p and Cta1p. alone; in the pex5Δ,pex9Δ double-deletion strain, both proteins were Analogous results were obtained in the experiment in which we observed exclusively in the cytosol. Note that while the images in the tested the influence of the I264→K mutation in Pex5p on the rows labeled WT, pex9Δ and pex5Δ,pex9Δ are representative of distribution of Fox2p-GFP (Fig. 3C) and native catalase A (Fig. 3D) whole-cell populations, the images in the rows labeled pex5Δ display between the organellar and cytosolic fractions. Fox2p-GFP was the rare instances of cells with particulate GFP-tagged Fox2p or quantified by western blotting with anti-GFP antibodies in the same Cta1p. Most of the cells from the pex5Δ strain looked exactly strain as used previously (Fig. 3A). Catalase A was quantified by its like those shown in the rows labeled pex5Δ,pex9Δ.Notably,the enzymatic activity in the pex5Δ derivative of the GC1-8B strain, colocalization of Fox2p or Cta1p with the PTS2-DsRed peroxisomal which is devoid of both yeast catalases, and in which we expressed marker was observed only in cells that displayed weak fluorescence in the native Cta1p protein and the WT or mutated I264→K-variant of the green channel, i.e. contained low levels of GFP-tagged proteins. Pex5p. In both cases, yeast cells were grown in YPEO medium and The quantitative data (Fig. 2F) clearly show that 5-10% of pex5Δ cells fractionated into an organellar fraction containing peroxisomes and expressing every GFP fusion construct used in this experiment a cytosol fraction. The distribution of both proteins between the displayed weak but visible particulate fluorescence colocalizing organellar and cytosolic fractions revealed that the substitution of with the peroxisomal marker, whereas in pex5Δ,pex9Δ cells, the the Ile264 residue in Pex5p disturbed the import of both native fluorescence was purely cytosolic. These data suggest that although Cta1p and Fox2p-GFP by ∼35%. Pex9p appears not to significantly contribute to the import of either protein in the presence of Pex5p, it can to some extent act as a DISCUSSION substitute receptor for them when Pex5p is missing. Notably, since The intriguing case of the budding yeast peroxisomal acyl-CoA this phenomenon is observed for C-terminally GFP-tagged Fox2p oxidase that somehow enters the lumen of its destined organelle and Cta1p (Fig. 2D,E), Pex9p, similarly to Pex5p, appears to be able despite possessing neither of the identified targeting signals has to drive the residual import of these proteins through a mechanism been known for several decades. While the PTS1- and PTS2- that does not involve the recognition of the PTS1 signal by its dependent peroxisomal import routes are well-characterized and are TPR domain. evolutionarily conserved from simple unicellular eukaryotes to mammals, the import of the budding yeast AOx is a curiosity A putative PTS3 receptor domain located in the N-terminal because its orthologues from many other organisms do have PTS1 part of the Pex5p protein participates in the import of Fox2p signals on their C-termini. In addition to S. cerevisiae, some other and Cta1p yeast species contain AOx proteins with no recognizable PTS1 or As previously postulated, Pex5p appears to have two domains PTS2 (Wang et al., 1999) that probably follow the same route into that interact with cargo proteins: the well-known TPR domain peroxisomes as the budding yeast AOx. recognizing the C-terminal PTS1 and the less well-defined internal No less intriguing were the cases of proteins such as S. cerevisiae region in the N-terminal half of its polypeptide. Amino acid residues Cat2p (Elgersma et al., 1995) and Hansenula polymorpha alcohol that are important for the Pex5p interaction with AOx and Cat2p oxidase (Gunkel et al., 2004), which do have a PTS1 signal able to were identified within this region, and one of them, Ile264, was direct reporter proteins to peroxisomes, yet they are seemingly found to be important for the interaction with both proteins but not dispensable within these proteins. These observations challenge the with the elements in the translocon complex consisting of Pex13p common belief, which has evidently been oversimplified, regarding and Pex14p (Klein et al., 2002). Therefore, we tested whether the the import mechanisms of peroxisomal matrix proteins. Once substitution of this residue affects Fox2p and Cta1p import. Fig. 3 PTS1 and PTS2 were discovered in the 1990s (Gould et al., 1989; shows the effect of replacing isoleucine 264 with lysine at the Swinkels et al., 1991), most peroxisomal proteins were attributed to corresponding position in the Pex5p polypeptide on the import of the PTS1- or PTS2-dependent route based on the mere presence of these proteins. Indeed, the substitution of Ile264 did affect the the respective signals in their amino acid sequences. The fact that import of Fox2p-GFP and Cta1p-GFP, both with their C-termini in budding yeast, PTS1-containing Cat2p and AOx, which lacks blocked by the GFP tag, diminishing the number of cells with PTS1, both share a mechanism for peroxisomal internalization that visible green fluorescence particles that colocalized with the involves interaction with the N-terminal region of Pex5p, especially peroxisomal marker by 20% and 35%, respectively (see Fig. 3A). with its diaromatic FxxxW motif, indicates the need to re-evaluate

Interestingly, however, in yeast strains expressing N-terminally Pex5p- and PTS1-dependent import. This new mechanism itself Journal of Cell Science

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Fig. 3. Importance of the Ile264 residue located in the N-terminal region of Pex5p for the import of GFP-tagged Fox2p and Cta1p and native Cta1p. The pex5Δ derivative of the BY4741 strain (A-C) or the pex5Δ derivative of GC1-8B strain (D) were transformed with the pRS-Pex5pWT or pRS-Pex5pI264→K plasmids, encoding native and mutated versions of the Pex5p receptor, respectively. Additionally, as indicated, the strain was also transformed with GFP-Fox2p, Fox2p-GFP or Cta1p-GFP plasmids encoding N- or C-terminally tagged Fox2p or C-terminally tagged Cta1p cargo proteins or with mRFP-SKL plasmid encoding the marker protein imported into peroxisomes exclusively via the PTS1 route. (A,B) The peroxisomal import efficiency of GFP-tagged proteins and the mRFP-SKL marker protein was quantified and expressed as the percentage of cells (relative to the total number of cells analyzed) displaying peroxisomal localization of the fusion proteins. Individual data points are the average (mean) of three replicates of 100 cells scored. Error bars represent the s.d. Statistical significance was calculated with Student’s t-test: **P<0.01, *P<0.10; n.s., not significant. (C,D) Transformant cells precultured in selective SC medium were grown overnight in YPEO medium and then spheroplasted, homogenized and fractionated into cytosol and organellar fractions containing peroxisomes. (C) The import efficiency of the C-terminal Fox2p-GFP fusion was determined by western blotting with anti-GFP antibodies and analysis of protein levels as described in the Materials and Methods. (D) The catalase activity in each fraction was determined as described in the Materials and Methods. The graphs show the distribution of catalase A activity or Fox2p-GFP protein between the organellar (dark gray boxes) and cytosolic (white boxes) fractions expressed as a percentage of combined catalase activity or Fox2p-GFP protein content found in both fractions. Individual data points are the average (mean) of the values obtained from three experiments. Error bars represent the s.d. is unknown, as are the other elements of this hypothetical import canonical C-terminal PTS1 tripeptide SKL, yet in metal affinity route, such as the signal sequences within AOx and Cat2p chromatography, it bound the same 136-292 aa fragment of Pex5p as polypeptides, which, although currently unknown, are sometimes AOx and Cat2p did. This interaction also occurs in vivo, as Fox2p called PTS3 signals. Is this mechanism distinct enough to qualify it GFP-tagging at its C-terminus only partially affected its import (see as a separate peroxisomal import route? An attempt to answer these Fig. 1A). Cta1p, the candidate resulting from the analysis of the questions is the subject of a separate study. In this study, we set literature data and protein sequences, contains a working variant of another goal, to identify other peroxisomal proteins, in addition to PTS1, SKF, although its masking with the GFP tag or its removal had AOx and Cat2p, whose import into peroxisomes in budding yeast limited impact on the import of Cta1p into peroxisomes. C-terminally depends on the PTS3 route. Those could be the only two proteins GFP-tagged Fox2p and Cta1p proteins entered peroxisomes equally belonging to this group. Only three proteins in S. cerevisiae are efficiently in the presence and absence of their respective native, known to be imported via the PTS2 pathway (Erdmann, 1994; genome-encoded proteins, which excludes their piggy-back-assisted Effelsberg et al., 2015), yet the entire machinery consisting of three import. The fully efficient import observed in the absence of the peroxins is preserved for them (Purdue et al., 1998). Pex7p receptor also excludes their dependence on PTS2 (Fig. 1A,B). Nonetheless, we had reason to assume that the list of PTS3- Comparing these images with the ones in Fig. 1C,D revealed that the dependent peroxisomal proteins may indeed be longer. Other proteins efficiency of the import of Fox2p and Cta1p was lower than that of in addition to AOx are associated with peroxisomes but contain AOx and lower than that of Cat2p when the latter is not dependent on neither PTS1 nor PTS2 signals. These proteins could be imported the PTS1 signal. Similarly to AOx and Cat2p, the observed import of into peroxisomes but not previously categorized as such because of Fox2p and Cta1p occurs without interaction between their C-termini the absence of any recognizable PTS. Moreover, the case of Cat2p and the Pex5p TPR region, but it does depend on the presence of indicated that the presence of PTS1 is not the ultimate proof that the the Pex5p receptor (Fig. 2). This observation is compatible with the protein utilizes the PTS1-dependent import route. That appeared to be hypothesis of the dual functionality of Pex5p peroxin: (1) it contains a true for both proteins described in this study. Fox2p contains the PTS1 signal receptor within its TPR region located in the C-terminal Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216986. doi:10.1242/jcs.216986 half of the polypeptide and (2) it also contains a receptor for the with AOx and Cat2p, to the common group of peroxisomal proteins hypothetical PTS3 signal in AOx and Cat2p located in the region whose import into this organelle is dependent, at least partially, closer to its N-terminus, between residues 250 and 270. on their interaction with the specific region residing within By employing a mutated variant of Pex5p bearing the I264→K the N-terminal region of the Pex5p polypeptide that surrounds the substitution, which blocks the interaction of Pex5p with AOx and FxxxW diaromatic motif. Cat2p, the participation of the PTS3 pathway in the import of Fox2p Analysis of our data suggests that the importance of the and Cta1p can be estimated as between 20 and 35%, depending on interaction with this region for cargo recognition and/or import is the assessment method used (Fig. 3). somewhat different for Cta1p and Fox2p. While the substitution of As shown in Fig. 2C and Fig. 3D, substitution of the C-terminal Ile264 in Pex5p manifests in a similar way in the presence of the PTS1 of Cta1p-SKF with YIS lowered its import efficiency by SKF sequence at the Cta1p C-terminus and when its PTS1 is ∼20%, whereas substitution of Ile264 in Pex5p lowered the blocked by the GFP tag (compare Fig. 3A and D), peroxisomal import efficiency of native Cta1p by ∼35%. Superficially, one localization of Fox2p is affected more strongly by Ile264 could expect these two numbers to sum up to 100%, but the substitution when its C-terminus is blocked (see Fig. 3A). modifications introduced into Cta1p and Pex5p most likely only Our results revealed yet another interesting peculiarity regarding partially affect the PTS1- and PTS3-dependent import of Cta1p. the import of Fox2p and Cta1p into peroxisomes. The examination The region in Cta1p upstream from the SKF tripeptide probably of many pex5Δ cells expressing either of these proteins allowed us to also contributes to Cta1p interactions with the Pex5p TPR domain. notice an occasional particulate distribution of GFP fluorescence Likewise, the substitution of Ile264 in Pex5p may not completely that colocalized with the PTS2-DsRed marker imported by the abolish the interaction of Cta1p with the N-terminal part of Pex5p, PTS2 route, in addition to the uniform cytosolic fluorescence (see as other amino acid residues in this region were also shown to be rows labeled pex5Δ in Fig. 2A,B,D,E). The origin of this initially important for the interaction of Pex5p with AOx and Cat2p (Klein disturbing observation was clarified by the use of the pex5Δ,pex9Δ et al., 2002). double-deletion strain, in which no Fox2p or Cta1p particles could It is noteworthy that testing the importance of Ile264 for the be observed. For many years, S. cerevisiae Pex9p had been known import efficiency of Fox2p and Cta1p with the two methods only under the systematic name Ymr018wp and had no assigned gave similar results (compare Fig 3A,C and D). The percentage of function. Recently, however, the Mls1p and Mls2p proteins have cells displaying the peroxisome-located GFP fusion proteins is been shown to be imported into peroxisomes in oleate-induced cells considerably higher than the percentage of Fox2p-GFP or native in a Pex9p-dependent manner (Effelsberg et al., 2016; Yifrach et al., catalase A present in the organellar fraction, but this can be 2016). Our results demonstrated that in the absence of Pex5p, Pex9p explained by the presence of Fox2p-GFP and Cta1p-GFP in most can also exhibit some receptor activity towards Fox2p and Cta1p cells, both in particles and in cytosol (see Fig. 2D,E). (Fig. 2). Moreover, it must bind these proteins with the polypeptide Our data are also in agreement with previous reports on the region outside the TPR domain because the C-termini of these import of Cta1p that revealed the dispensability of its C-terminus for cargo proteins are completely blocked by the GFP tag. No firm its import into peroxisomes and revealed instead the importance of conclusions regarding the functional assignments of particular certain internal region(s) (Kragler et al., 1993). We demonstrated the regions of the Pex9p protein can be drawn yet. Nevertheless, the importance of the N-terminal region of Pex5p for the import of alignment of the N-terminal parts of Pex5p and Pex9p, as shown in Cta1p, which entitles us to assign Cta1p, as well as Fox2p, together Fig. 4, reveals regions of quite strong homology between them.

Fig. 4. Alignment of the Pex5p and Pex9p N-terminal regions. Identical and similar amino acid residues are enclosed in the black boxes. The two diaromatic WxxxF motifs and the region surrounding the conserved Cys6 amino acid residue that are common in both proteins, are enclosed in the green boxes. The region in Pex5p encompassing the third diaromatic motif important for interactions with AOx and Cat2p and with Pex14p and the corresponding region in Pex9p is enclosed in the red box. The alignment was created with ClustalW v.2.1 with the default settings and then graphically depicted with BioEdit v.7.2.5. Please note that the alignment algorithm inserted a gap in the Pex5p sequence, so the Ile264 residue is now at position 265. Journal of Cell Science

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These regions not only surround the first two WxxxF diaromatic from Open Biosystems, Huntsville, USA) were used in most experiments motifs and the conserved Cys6 amino acid residue (marked with described in this paper. To study the intracellular distribution of plasmid- green boxes) but also the AOx- and Cat2p-interacting 250-270 aa encoded catalase A with or without the PTS1 signal in the presence of Pex5p region of Pex5p containing the third FxxxW diaromatic motif protein variants, the GC1-8B (MATa leu2-3 leu2-112 ura3 trp1 ctt1-1 cta1- (marked with red box) that, in light of our present results, also 2) strain devoid of both catalase activities (Cohen et al., 1985) and its pex5Δ derivative were used as hosts. Double-deletion strains with the BY appear to be important for the interaction with Fox2p and Cta1p. genetic background were constructed by crossing appropriate single- Therefore, we might speculate that the respective homologous deletion parental strains, followed by sporulation and tetrad analysis using region in the Pex9p polypeptide is also involved in interactions with standard yeast genetics procedures (Sherman, 2002). The pex5Δ derivative Fox2p and Cta1p. of the GC1-8B strain was made by transformation of the parental strain with Another line of speculation may be based on the fact that the the kanMX deletion cassette, which was PCR-amplified from the pex5Δ region surrounding the FxxxW diaromatic motif in Pex5p was knockout collection strain with the PEX5PRO and PEX5UTR primer pair previously shown to be important for the interaction of this peroxin (see Table S1 for a full list of the primers). with Pex14p, a subunit of the peroxisomal translocon (Williams Yeast cells were grown on standard YPD medium (1% yeast extract, 2% et al., 2005). One could argue that when Ile264 is missing from peptone and 2% dextrose) at 28°C. To induce peroxisome proliferation, Pex5p, the import of AOx, Cat2p, Fox2p and Cta1p is compromised oleate-containing YPEO medium was used (1% yeast extract, 2% bactopeptone, 2% ethanol, 0.25% Tween 80 and 0.1% oleic acid). Liquid due to an impaired interaction of Pex5p with Pex14p and not with its (0.67% yeast nitrogen base and 2% glucose) or solid (+2% agar) minimal cargo proteins. However, the interaction of three of these cargo SC medium supplemented with appropriate amino acids was used for yeast proteins with the discussed Pex5p region had been demonstrated transformant selection and for growing yeast cells whenever plasmid directly. Moreover, the substitution of Ile264 did not affect the maintenance was required. Pex5p- and PTS1-dependent import of mRFP-SKL. Nevertheless, it is conceivable that the competition between these cargo proteins Plasmid construction and Pex14p for binding to the same region in Pex5p does exist. To construct the single-copy plasmid containing the native CTA1 gene, a Moreover, it is not unthinkable that the displacement of cargo by 2779 bp EcoRI DNA fragment encompassing the CTA1 open reading frame Pex14p binding is a necessary step involving Pex14p, Pex5p and its together with upstream 818 nucleotides and downstream 413 nucleotides cargo protein en route to the peroxisomal lumen. Notably, this was cut out from the Yep13-CTA1-III plasmid (Cohen et al., 1985) and competition has been demonstrated for mammalian proteins. It was cloned into the EcoRI site in the YCp50 vector (Rose et al., 1987), thus shown that interactions between human catalase and human Pex5p creating the YCp-CTA1 plasmid. The YCp-CTA1-YIS plasmid containing the mutated CTA1 gene encoding catalase A with the C-terminal SKF is disrupted by the binding of the Pex14p N-terminal fragment to the (Ser-Lys-Phe) sequence replaced by YIS (Tyr-Ile-Ser) was constructed by Pex5p N-terminal domain (Freitas et al., 2011). That would also site-directed mutagenesis (see Table S1 for primer sequences). The 1147 bp suggest that the binding of peroxisomal cargo proteins to Pex5p fragment of the CTA1 coding region down to the STOP codon was PCR- N-terminal region is more common not only among various Pex5p- amplified with the CTAupAge and CTAs_rev primer pair. A 500 bp CTA1 dependent S. cerevisiae proteins but also among proteins from other gene 3′-untranslated region fragment, including the very end of its open systematic groups. If that were the case, then the question arises of reading frame, was amplified with the CTAs_for and CTAdwHin primer why only two PTS1-containing proteins, interacting with both TPR pair. The two PCR products were combined and used as a template in the and FxxxW regions, were identified in our study. Was our search not final PCR reaction with the CTAupAge and CTAdwHin primers. The thorough enough? While we cannot answer this question yet, it resulting 1647 bp fragment was digested with AgeI and HindIII and cloned should be mentioned that only Fox2p, not Cta1p, interacted with the into the YCp-CTA1 plasmid digested with the same enzymes. The resulting YCp-CTA-YIS plasmid-encoded Cta1p with its C-terminal tripeptide Pex5p N-terminal domain in the metal affinity chromatography replaced by the amino acid residues YIS. approach. Therefore, the interaction of other PTS1-containing To obtain plasmids encoding fusion proteins with green fluorescent proteins with the Pex5p N-terminal region and the partial dependence protein (GFP) located at the N- or C-terminus, either possessing or lacking of their import on this region cannot be ruled out. the C-terminal PTS1 signal, the respective sequences encoding the open In summary, our results clearly show that the Pex5p receptor- reading frame or the open reading frame and 300-500 base pairs from the dependent import of peroxisomal matrix proteins is more promoter sequence (with or without the last three codons) were PCR- convoluted than that described in current models of peroxisome amplified from yeast genomic DNA with the appropriate primer pairs biogenesis, perhaps not only in S. cerevisiae but also in other yeast containing restriction sites (see Table S1 for primer sequences). species. The results also show that the newly discovered Pex9p Amplification products were digested with the appropriate restriction receptor has a more universal role in peroxisomal matrix protein enzymes and cloned into the pUG36 (for N-terminal fusions) or pUG35 or pUG23 (for C-terminal fusions) plasmids (Güldener and Hegemann, http:// import than recently reported. The data presented here, together mips.helmholtz-muenchen.de/proj/yeast/info/tools/hegemann/gfp.html) with previous findings about AOx and Cat2p, expand our linearized with the same enzymes. By this strategy, the GFP-Fox2p, GFP- knowledge on Pex5p-dependent peroxisomal import routes, which Fox2p-ΔSKL, Fox2p-GFP and Cta1p-GFP plasmids were constructed. To justifies opening a new chapter in the studies of peroxisomal protein create Cat2p-GFP encoding GFP-tagged Cat2p with the N-terminal import mechanisms. They also contribute to better understanding mitochondrial leader removed, two primer pairs were used (see Table S1) the still unresolved issue of the PTS1- and PTS2-independent to amplify the CAT2 gene promoter and the CAT2 open reading frame (ORF) import of proteins into the peroxisomal matrix. starting at the second downstream ATG codon. Both fragments were inserted sequentially into the pUG35 vector linearized with the appropriate restriction enzymes. To obtain the AOx-GFP plasmid, the entire POX1 ORF MATERIALS AND METHODS together with 454 bp of its promoter (up to the BglII restriction site) was Yeast strains and growth conditions seamlessly fused to the GFP ORF ending with the SacI restriction site. The The wild-type BY 4741 (MATa leu2Δ0ura3Δ0 his3Δ1 met15Δ0) strain hybrid gene with a blunt-ended BglII site was inserted at the EcoRV and (Brachmann et al., 1998) and its derivatives of the MATaorMATα mating SacI sites in the pRS316 vector. To construct the pRS-Pex5pI264K type, as necessary, from the Saccharomyces Genome Deletion Project plasmid encoding Pex5p with the I264K amino acid substitution within (http://www-sequence.stanford.edu/group/yeast_deletion_project/) carrying the putative PTS3-recognizing domain, the PEX5 ORF was subjected to the pox1Δ, fox2Δ, cat2Δ, pex5Δ, pex7Δ or pex9Δ deletion alleles (obtained site-directed mutagenesis by PCR amplification with the I264K_up and Journal of Cell Science

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I264K_lo primer pair. The mutagenized PEX5 ORF was digested with fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) with an Bsu36I and BstAPI and cloned into the pRS-PEX5WT plasmid (Kerssen EC Plan-NEOFLUAR 100× objective and 38HE-GFP or 20HE-rhodamine et al., 2006) linearized with the same restriction enzymes. The PTS2-DsRed filter sets and with Nomarski optics for bright-field imaging. plasmid expressing the peroxisomal marker was constructed by the insertion of the XbaI-PstI fragment cut out from the pHPR131 plasmid (Stein et al., Reagents 2002), which encodes the DsRed protein with the attached PTS2 signal from The iProof™ High-Fidelity DNA Polymerase was purchased from Bio-Rad S. cerevisiae thiolase, into the same sites in the pRS415 vector. The mRFP- (Hercules, CA, USA). All other reagents were of analytical grade and purity. SKL plasmid expressing the peroxisomal marker was constructed by the insertion of the XhoI-XbaI fragment cut out from pRS316 mRFP-SKL Competing interests (Fagarasanu et al., 2009), which encodes the mRFP protein ending with the The authors declare no competing or financial interests. PTS1 signal, into the same sites in the pRS415 vector. The iProof™ High-Fidelity DNA Polymerase (Bio-Rad, Hercules, CA, Author contributions USA) was used to amplify the PCR fragments, and the accuracy of Conceptualization: M.S.; Methodology: Ł.R., B.K., M.S.; Validation: M.S.; Formal all constructs was verified by sequencing. The XL-1 Blue [MRF’Δ analysis: Ł.R.; Investigation: Ł.R., B.K., A.C., M.S.; Resources: M.S.; Data curation: Ł (mcrA)183Δ (mcrCB-hsdSMR-mrr)173 endA1 supE44] E. coli strain from M.S.; Writing - original draft: .R., A.C., M.S.; Writing - review & editing: M.S.; Ł Stratagene was used for DNA cloning and plasmid propagation. Plasmids Visualization: .R., B.K.; Supervision: M.S.; Project administration: M.S.; Funding acquisition: M.S. were introduced into yeast cells using the high-efficiency lithium acetate transformation method (Gietz et al., 1995). Funding This work was supported by the Polish National Science Center (2013/08/M/NZ3/ Cell fractionation 01028). Yeast cells were grown overnight in liquid YPEO medium to induce the expression of genes encoding peroxisomal proteins. Cells from 200 ml of Supplementary information culture were spun down, washed with demineralized water, suspended in TD Supplementary information available online at buffer (100 mM Tris-HCl pH 8.0, 5 mM EDTA and 5 mM dithiothreitol; 3 ml http://jcs.biologists.org/lookup/doi/10.1242/jcs.216986.supplemental volume per 1 g of cell wet weight) and incubated for 30 min at 28°C with gentle shaking. The cells were then spun down, washed as before and References Beers, R. F. and Sizer, I. W. (1952). A spectrophotometric method for measuring suspended in Zymolyase digestion buffer (1.3 M sorbitol, 5 mM MOPS pH the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133-140. 7.2, 1 mM EDTA, and 0.05% Zymolyase 100 T; 2 ml of buffer per 1 g of yeast Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P. and wet weight) and incubated for 45 min at 34°C. The resulting spheroplasts were Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces spun down at 800 g, resuspended in homogenization buffer (0.65 M sorbitol, cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene 25 mM MES-NaOH pH 6.0, 0.5 mM EDTA and Pierce™ Protease Inhibitors disruption and other applications. Yeast Chichester Engl. 14, 115-132. (1 tablet per 50 ml of buffer); 2 ml per 1 g of starting cell wet weight) and Brocard, C. and Hartig, A. (2006). Peroxisome targeting signal 1: is it really a simple tripeptide? Biochim. Biophys. Acta 1763, 1565-1573. gently disrupted by using a Potter homogenizer (approximately 10 strokes of a Bun-Ya, M., Maebuchi, M., Hashimoto, T., Yokota, S. and Kamiryo, T. (1997). pestle rotating at 1000 RPM). The cell homogenate was separated by A second isoform of 3-ketoacyl-CoA thiolase found in Caenorhabditis elegans, centrifugation at 3000 g for 5 min into a fraction containing unbroken cells and which is similar to sterol carrier protein x but lacks the sequence of sterol carrier nuclear membranes and a supernatant containing other organelles. The protein 2. Eur. J. Biochem. 245, 252-259. supernatant was centrifuged at 25,000 g for 30 min to yield a pellet containing Cohen, G., Fessl, F., Traczyk, A., Rytka, J. and Ruis, H. (1985). Isolation of the peroxisomes and mitochondria and a soluble fraction containing the catalase A gene of Saccharomyces cerevisiae by complementation of the cta1 mutation. Mol. Gen. Genet. MGG 200, 74-79. cytoplasm. The organellar pellet was resuspended in the same volume of Effelsberg, D., Cruz-Zaragoza, L. D., Tonillo, J., Schliebs, W. and Erdmann, R. homogenization buffer as that used in the initial homogenization step. (2015). Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae. J. Biol. Chem. 290, 25333-25342. Quantification of GFP fusion proteins Effelsberg, D., Cruz-Zaragoza, L. D., Schliebs, W. and Erdmann, R. (2016). Pex9p is a new yeast peroxisomal import receptor for PTS1-containing proteins. Equal volumes of organelle suspensions and supernatants were separated by J. Cell Sci. 129, 4057-4066. SDS-PAGE on 10% gel and the separated proteins were transferred onto Elgersma, Y., van Roermund, C. W., Wanders, R. J. and Tabak, H. F. (1995). Immobilon®-P nitrocellulose membrane (Millipore, Burlington, MA, USA) Peroxisomal and mitochondrial carnitine acetyltransferases of Saccharomyces using the semi-dry method. GFP was detected using mouse anti-GFP cerevisiae are encoded by a single gene. EMBO J. 14, 3472-3479. primary antibody (11814460001, Roche Applied Science, Mannheim, Erdmann, R. (1994). The peroxisomal targeting signal of 3-oxoacyl-CoA thiolase Germany; 1:5000) and goat anti-mouse HRP-conjugated secondary from Saccharomyces cerevisiae. Yeast Chichester Engl. 10, 935-944. Fagarasanu, A., Mast, F. D., Knoblach, B., Jin, Y., Brunner, M. J., Logan, M. R., antibody (P 0447 from Dako Denmark A/S, Glostrup, Denmark). Blots Glover, J. N. M., Eitzen, G. A., Aitchison, J. D., Weisman, L. S. et al. (2009). ® were incubated with the SuperSignal West Femto (Thermo Scientific, Myosin-driven peroxisome partitioning in S. cerevisiae. J. Cell Biol. 186, 541-554. Rockford, IL, USA) and the chemiluminescence signal was digitized with Freitas, M. O., Francisco, T., Rodrigues, T. A., Alencastre, I. S., Pinto, M. P., Fluorchem SP CCD camera (Alpha Innotech, San Leandro, CA, USA). The Grou, C. P., Carvalho, A. F., Fransen, M., Sá-Miranda, C. and Azevedo, J. E. protein bands were quantified with ImageQuant 5.0 software (Molecular (2011). PEX5 protein binds monomeric catalase blocking its tetramerization and Dynamics Inc., Sunnyvale, CA, USA). releases it upon binding the N-terminal domain of PEX14. J. Biol. Chem. 286, 40509-40519. Gietz, R. D., Schiestl, R. H., Willems, A. R. and Woods, R. A. (1995). Studies on Measurement of catalase activity the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast The catalase activity was followed spectrophotometrically at 240 nm per the Chichester Engl. 11, 355-360. Girzalsky, W., Saffian, D. and Erdmann, R. (2010). Peroxisomal protein decomposition of H2O2. The activity was expressed as µmol of H2O2 −1 −1 translocation. Biochim. Biophys. Acta 1803, 724-731. decomposed×min ×mg of protein (Beers and Sizer, 1952). Glover, J. R., Andrews, D. W. and Rachubinski, R. A. (1994). Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer. Proc. Natl. Acad. Sci. USA Fluorescence microscopy 91, 10541-10545. To visualize the protein localization, yeast strains were transformed with Gould, S. J., Keller, G. A., Hosken, N., Wilkinson, J. and Subramani, S. (1989). plasmids expressing GFP-tagged proteins and co-transformed with A conserved tripeptide sorts proteins to peroxisomes. J. Cell Biol. 108, 1657-1664. plasmids expressing the mRFP-SKL or PTS2-DsRed protein, which is a Gunkel, K., van Dijk, R., Veenhuis, M. and van der Klei, I. J. (2004). Routing of Hansenula polymorpha alcohol oxidase: an alternative peroxisomal peroxisome marker imported independently of the Pex5p receptor. protein-sorting machinery. Mol. Biol. Cell 15, 1347-1355. Transformants were grown on YPEO medium to induce peroxisome Haataja, T. J. K., Koski, M. K., Hiltunen, J. K. and Glumoff, T. (2011). Peroxisomal proliferation. 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