2508 Research Article Targeting of the tail-anchored peroxisomal membrane PEX26 and PEX15 occurs through C-terminal PEX19-binding sites

André Halbach1, Christiane Landgraf2, Stephan Lorenzen3, Katja Rosenkranz1, Rudolf Volkmer-Engert2, Ralf Erdmann1,* and Hanspeter Rottensteiner1,* 1Institut für Physiologische Chemie, Abt. Systembiochemie, Ruhr-Universität Bochum, 44780 Bochum, Germany 2Institut für Medizinische Immunologie and 3Institut für Biochemie, Universitätsklinikum Charité, 10115 Berlin, Germany *Authors for correspondence (e-mail: [email protected]; [email protected])

Accepted 16 March 2006 Journal of Cell Science 119, 2508-2517 Published by The Company of Biologists 2006 doi:10.1242/jcs.02979

Summary Tail-anchored proteins contain a single transmembrane from mislocalization to mitochondria. Its function as a domain (TMD) followed by a short C-terminal domain targeting motif was proved by its ability to insert a extending into the organellar lumen. Tail-anchored heterologous TMD-containing fragment into the proteins are thought to target to the correct subcellular peroxisomal membrane. Finally we show that PEX19 is compartment by virtue of general physicochemical essential for PEX26 import. Analysis of the yeast tail- properties of their C-termini; however, the machineries anchored Pex15p revealed that it also harbors a that enable correct sorting remain largely elusive. Here we luminal PEX19-binding site that acts as a peroxisomal- analyzed targeting of the human peroxisomal tail-anchored targeting motif. We conclude that C-terminal PEX19- protein PEX26. Its C-terminal-targeting signal contains binding sites mark tail-anchored proteins for delivery to two binding sites for PEX19, the import receptor for peroxisomes. several peroxisomal membrane proteins. One PEX19- binding site overlapped with the TMD, the other was Supplementary material available online at contained within the luminal domain. Although the PEX19- http://jcs.biologists.org/cgi/content/full/119/12/2508/DC1 binding site containing the TMD targeted to peroxisomes to some extent, the luminal site proved essential for correct Key words: Posttranslational protein import, Peroxisome biogenesis, targeting of the full-length protein, as it prevented PEX26 Peroxin, Endoplasmic reticulum, Peroxisomal targeting signal, FIS1 Journal of Cell Science

Introduction mitochondria and the ER, a concept emerged that explains Tail-anchored (TA) proteins are type II membrane proteins targeting specificity by general physicochemical rather than that harbor a single transmembrane domain (TMD) close to sequence-specific features of the targeting signal. These their C-termini and are thus equipped with a characteristic include the length and hydrophobicity of the TMD and the net short luminal domain. Members of this class of charges of its flanking residues. A mitochondrial targeting transmembrane proteins are found in the endoplasmic signal, for instance, exhibits a rather short TMD with only reticulum (ER) and its related endomembranes, the outer moderate hydrophobicity and flanking regions that are mitochondrial membrane, peroxisomes and in plants also in positively charged (Beilharz et al., 2003; Borgese et al., 2003; the outer membrane of plastids (Borgese et al., 2003). The Hwang et al., 2004; Rapaport, 2003). Insertion of a tail anchor methods by which TA proteins are correctly targeted to the into the ER membrane is thought to occur by default, i.e. in ER and the mitochondrial outer membrane, the principal sites the absence of a mitochondrial (or plastid) targeting signal of membrane integration, has attracted significant attention (Borgese et al., 2003), although SRP might support guidance since it became clear that the posttranslational import of these of at least some TA proteins (Abell et al., 2004). A number of proteins must occur by novel routes (High and Abell, 2004; TA proteins are initially targeted to the ER and subsequently Steel et al., 2002; Yabal et al., 2003). Machineries that would sorted to their final destination within the endomembrane enable the insertion of TA proteins into the target membranes system such as the Golgi or the nuclear envelope through remain elusive and the question as to whether membrane additional motifs that are located within their cytosolic domain insertion requires proteinaceous membrane components such (Beilharz et al., 2003). TA proteins destined for the as the signal recognition particle (SRP) is currently disputed peroxisomal membrane were also reported to use a bipartite (Abell et al., 2004; Brambillasca et al., 2005; Steel et al., system of targeting signals; an ER targeting signal to bring the 2002). protein to the ER, and a peroxisomal signal, which delivers the The targeting signals of TA proteins are generally located protein from the ER to the peroxisome (Elgersma et al., 1997; within their C-termini and include the TMD. From several Mullen et al., 1999; Mullen and Trelease, 2000; Nito et al., studies addressing the differential targeting of TA proteins to 2001). In this case, however, the tail-anchor itself rather than Tail-anchored PMP import requires PEX19 2509

the cytosolic domain contains the peroxisome-specific developed prediction program for PEX19-binding sites targeting information. (Rottensteiner et al., 2004), we were able to identify two Although PEX3 (Hoepfner et al., 2005) and probably a few potential PEX19-binding sites also in PEX26 (Halbach et al., other peroxisomal membrane proteins (PMPs) also first target 2005). The predicted scores for the entire PEX26 are shown to the ER (Geuze et al., 2003; Titorenko and Rachubinski, in Fig. 1A. One of the two peak-scoring peptides is located 1998), most PMPs are likely to be inserted into peroxisomes within the predicted single TMD comprising amino acids directly from the cytosol (Lazarow and Fujiki, 1985). This 253-267 (Matsumoto et al., 2003a) and the other one (amino latter process is facilitated by PEX19, a predominantly acids 276-290) C-terminally adjoins it (see Fig. 1D for a cytosolic protein that is also found at the peroxisomal graphic representation of PEX26). To test whether the membrane (Götte et al., 1998; Matsuzono et al., 1999). PEX19 predicted sites are indeed capable of binding PEX19, we interacts with virtually all PMPs (Fransen et al., 2001; synthesized synthetic 15-mer peptides covering the entire Sacksteder et al., 2000; Snyder et al., 2000) through a common PEX26 protein in an overlapping arrangement on a motif that is present at least once (Halbach et al., 2005; membrane. This membrane was then incubated with a Rottensteiner et al., 2004). This interaction on the one hand purified GST-fusion protein of human PEX19. As control, a prevents the PMP from aggregation before membrane insertion duplicate membrane was incubated with GST alone. Bound and on the other serves to guide the PMP to the peroxisomal PEX19 was visualized immunologically with a combination membrane (Jones et al., 2004; Shibata et al., 2004). PEX19- of monoclonal anti-GST antibodies and appropriate HRP- binding sites are sufficient for peroxisomal targeting provided conjugated secondary antibodies. Strikingly, two arrays of that one or more TMDs are additionally present to anchor the spots were recognized by GST-PEX19 (Fig. 1B), whereas the fragment within the membrane (Rottensteiner et al., 2004). A GST control did not bind significantly to any of the spots on distinct mode of PEX19 interaction applies for PEX3 (Fransen the membrane (not shown). The serial spots covered amino et al., 2005; Jones et al., 2004; Mayerhofer et al., 2002), acids 251-269 and 277-297, respectively, demonstrating the consistent with the proposed function of PEX3 as the docking existence of two neighboring PEX19-binding sites in the factor for PEX19 at the peroxisomal membrane (Jones et al., C-terminus of PEX26. Noticeably, the experimentally 2004; Muntau et al., 2003). Thus, current knowledge suggests determined PEX19-binding sites in PEX26 comprise peptides the presence of at least two distinct import pathways for PMPs: that lie within the highest-scoring peaks in the respective a PEX19-dependent one and one that involves the ER (Heiland regions (BS I, 248-270; BS II, 276-296, see Fig. 1A), and thus and Erdmann, 2005). reflect the conciseness of the prediction made. Quantification The mammalian PEX26 belongs to the class of peroxisomal of bound PEX19 revealed that the individual peptides from TA proteins. Despite the lack of sequence similarity, PEX26 the second binding site showed an interaction that was weaker might represent the functional orthologue of Pex15p in that by a factor of about three to four (Fig. 1C). Since four of these both proteins recruit PEX6 to the peroxisomal membrane peptides interacted comparably, whereas only three did so for (Birschmann et al., 2003; Matsumoto et al., 2003a). Patients the first binding site, it appears that the second PEX19- carrying loss-of-function alleles of PEX26 suffer from the binding site is slightly larger than the first. It is also worth

Journal of Cell Science typical symptoms ascribed to the Zellweger spectrum. On a noting that the first PEX19-binding site was devoid of the molecular level, the absence of PEX26 causes an import defect typical basic amino acids within its core region. of peroxisomal matrix proteins of type II (Matsumoto et al., Binding of PEX19 to PEX26 was also analyzed in vivo by 2003b) and to a large extent also of type I (Weller et al., 2005). using a yeast two-hybrid assay. Full-length PEX26 was fused Here we have studied the targeting of the mammalian TA to the GAL4 DNA-binding domain (BD) and expressed protein PEX26 and analyzed whether this occurs through the together with a GAL4 activation domain (AD) fusion of PEX19-dependent pathway. We show that the luminal domain HsPEX19. The transformed strain grew clearly on histidine- of PEX26 contained a typical PEX19-binding site that was adenine double-dropout plates, whereas coexpression of capable of peroxisomal targeting. Based on these observations, Gal4-BD-PEX26 with the GAL4-AD alone did not lead to we re-examined targeting of yeast Pex15p and found a similar growth (Fig. 2). This result showed that PEX19 also functional composition of its targeting signal. We discuss our interacted with PEX26 in vivo, as reported previously findings in terms of peroxisomal TA protein sorting that is (Fransen et al., 2005). We then analyzed two fragments of enabled through an appended C-terminal PEX19-binding site PEX26 for interaction with PEX19; one that comprised the and that does not necessarily require an interim transit through large cytosolic domain (amino acids 2-244) and one that the ER. included both PEX19-binding sites (amino acids 245-305). Fig. 2 shows that only the C-terminal fragment was tested Results positive in the assay. Further dissection of the C-terminus into Identification of two PEX19-binding sites in human the two individual binding sites revealed that the more C- PEX26 terminal, luminal PEX19-binding site (PEX26275-305) did, PEX26 from Homo sapiens is a tail-anchored protein of the albeit weakly, interact with PEX19, whereas the neighboring, peroxisomal membrane. It has been proposed that this class TMD-containing binding site (PEX26245-274) did not. These of PMPs might be targeted to peroxisomes by a pathway that data suggest that in vivo PEX19 might bind cooperatively to involves specialized regions of the ER (Elgersma et al., 1997; the two sites in PEX26, although it is also possible that Mullen and Trelease, 2000). Most PMPs, however are solubility or folding problems, particularly of the fragment inserted into the peroxisomal membrane directly from the harboring the TMD, have hampered the binding efficiency. cytosol. This process requires PEX19, which interacts with Nonetheless, the two-hybrid data correlated well with the all PMPs tested so far. Interestingly, by means of our result of the peptide scan. 2510 Journal of Cell Science 119 (12)

Fig. 2. Interaction of PEX26 with PEX19 in a yeast two-hybrid assay. The indicated PEX26 fragments were fused to the Gal4p binding domain (GAL4-BD) and expressed in combination with a HsPEX19 Gal4p activation domain fusion (GAL4-AD; left panel) or the Gal4p activation domain alone (right panel) in the yeast two- hybrid strain PJ69-4a. In this assay, protein-protein interactions were assessed by the ability of the double transformants to grow on histidine-adenine double-dropout medium plates. Two independent transformants are shown for each tested interaction.

Targeting of PEX26 depends on its two PEX19 binding sites We then analyzed whether the PEX19-binding sites of PEX26 are involved in the topogenesis of this tail-anchored PMP. Expression in human fibroblasts of PEX26 that had been fused to the C-terminus of GFP resulted in a punctate staining pattern that coincided with that of the peroxisomal integral membrane marker protein PEX14 (Fig. 3A), thereby demonstrating the peroxisomal localization of the GFP-PEX26 fusion protein. When PEX262-244, a fragment that lacked both PEX19-binding sites, was fused to GFP, a diffuse fluorescent staining pattern was obtained, indicating that this fusion protein was localized

Journal of Cell Science to the cytosol. By contrast, the PEX26245-305 fragment devoid of the entire cytosolic domain but containing both PEX19- binding sites and the TMD predominantly localized to peroxisomes (Fig. 3A). Thus, the peroxisomal membrane protein-targeting signal (mPTS) of PEX26 was narrowed down to a fragment that contains both PEX19-binding sites as well as its TMD. When the luminal PEX19-binding site was clipped from PEX26, the truncated protein (PEX262-274) was no longer Fig. 1. Prediction and identification of PEX19-binding sites in PEX26. peroxisomal but was localized to internal membranes instead. (A) Binding site prediction. The prediction matrix for PEX19-binding The observed mistargeting of PEX262-274 suggests that the sites (Rottensteiner et al., 2004) was applied to human PEX26 and the luminal PEX19-binding site contains peroxisomal targeting hits obtained are presented as dotted vertical lines. Two sites with a information. To prove this assumption, this binding site was high probability score are clearly visible. The positions of the high- scoring peptides within these sites as well as the amino acid sequence examined for its ability to direct an otherwise nonperoxisomal of each peak scoring peptide are indicated. (B) Peptide scan for fragment to peroxisomes. For this, a fragment of the mPTS of PEX26. 15-mer peptides with two amino acid shifts between the adrenoleukodystrophy protein (ALDP87-164) that contains neighboring peptides and covering the entire PEX26 protein were two TMDs but lacks its PEX19-binding site was chosen synthesized on a nitrocellulose membrane and tested for interaction (Halbach et al., 2005; Landgraf et al., 2003). Expression of a with GST-HsPEX19. Bound PEX19 was visualized by monoclonal fusion of the luminal PEX19-binding site of PEX26 with this anti-GST antibodies in combination with the ECLTM reaction system. ALDP fragment revealed a clear peroxisomal localization of Arrows mark the peak interacting peptide within each binding site, the the chimera, whereas the PEX19-binding site (PEX26275-305) sequences of which are indicated. (C) Relative PEX19-binding affinity alone was almost entirely localized to nonperoxisomal for the two binding sites in PEX26. Nine peptides per binding site structures (Fig. 3B), only in a few cells were peroxisomes also were synthesized and assayed for PEX19 interaction as described in B. The sequences of the strongly interacting peptides are indicated. The discernible (not shown). Thus, PEX26275-305 provoked a right panel shows the relative intensities of the signals obtained for redirection of ALDP87-164 to peroxisomes in human cells. each spot. (D) Graphic representation of PEX26. Denoted are the The fluorescence studies left unclear whether this chimera, positions of the PEX19-binding sites as determined in vitro and the as well as the identified mPTS fragment of PEX26, are only postulated TMD (Matsumoto et al., 2003a). targeted to peroxisomes or are also inserted into the membrane. Tail-anchored PMP import requires PEX19 2511

Fig. 4. The peroxisomally targeted PEX26 fragments are inserted into the peroxisomal membrane. Human skin fibroblasts expressing GFP fusions of PEX262-305, PEX26245-305, or PEX26275-305- ALDP87-164 were lysed in hypotonic buffer and membranes were separated from soluble proteins (SHyp) by centrifugation at 100,000 g. Membranes were then extracted with alkaline sodium carbonate buffer and again subjected to centrifugation to obtain a soluble fraction (SCarb) and a membrane pellet fraction (PCarb). Sample volumes were adjusted to that of the whole lysate (Lys) and each fraction was analysed by immunoblotting using antibodies against GFP, the integral PMP PEX14, and the soluble peroxisomal matrix enzyme .

Cells expressing GFP fusions of the respective protein fragments or, as control, full-length PEX26 were therefore

Journal of Cell Science lysed in hypotonic buffer and membranes were separated from the soluble proteins by centrifugation at 100,000 g. The resulting membrane pellet was treated with alkaline sodium carbonate buffer to extract all proteins barring integral membrane proteins. Finally, extracted proteins were separated from the membranes by centrifugation at 100,000 g. Immunoblot analysis of samples of each fraction revealed that the soluble peroxisomal matrix enzyme catalase was released after hypotonic lysis in all cells, whereas the integral PMP PEX14 was exclusively found in the membrane fraction (Fig. 4). Likewise, PEX26 , PEX26 -ALDP , as well Fig. 3. 245-305 275-305 87-164 Targeting of PEX26 is dependent on its PEX19-binding site. as full-length PEX26 were also resistant to carbonate (A) Identification of an mPTS in PEX26. Human fibroblasts were transiently transfected with plasmids designed to express GFP fusions extraction. It was therefore concluded that the analyzed PEX19 of the indicated PEX26 fragments. Thereafter, cells were processed for binding site-containing fragments behaved like integral indirect immunofluorescence using polyclonal anti-PEX14 antibodies. membrane proteins that were indeed sufficient for peroxisomal Merged images reveal eventual colocalization of the GFP fusion membrane insertion. proteins with peroxisomal PEX14 (Px). (B) The C-terminal PEX19- So far, the results showed that the luminal PEX19-binding binding site of PEX26 functions as a PMP-targeting motif. A GFP site of PEX26 represents a bona fide PMP-targeting motif. At fusion of a chimera of PEX26275-305 and ALDP87-164 was similarly the same time, the question of the role of the other PEX19- analyzed for subcellular location. In this chimera, the targeting motif is binding site in PEX26 arose. Since PEX262-274 was contributed by the PEX26 fragment, whereas the transmembrane mislocalized, it was conceivable that binding of PEX19 to the segment that is required for efficient membrane insertion is contributed TMD-containing PEX26 might be solely required to by the ALDP fragment. GFP fusions of the individual fragments 245-274 protect the protein from aggregation in the cytosol. To (PEX26275-305 and ALDP87-164) served as controls. (C) Function of the TMD-containing PEX19-binding site of PEX26 as a PMP targeting determine, nonetheless, whether this PEX19-binding site can signal. A GFP fusion of PEX26245-274 was inspected for colocalization in principle also serve as a targeting motif, the ability of with PEX14. This transmembrane segment with PEX19 binding PEX26245-274 to target GFP to peroxisomes was analyzed. This capability suffices for peroxisomal targeting. Bar, 10 ␮m. was indeed the case, even though significant cytosolic 2512 Journal of Cell Science 119 (12)

fluorescence was also visible (Fig. 3C). PEX26245-274 can thus most cells. It should be noted, however, that in some cells be regarded as a minimal mPTS that is composed of a PEX19- which exhibit a very strong GFP-derived fluorescence, even binding site coinciding with a TMD. In the physiological full-length PEX26 was localized to mitochondria. The context of the full-length protein, however, the luminal PEX19- fluorescence pattern of PEX26275-305 did not coincide with the binding site is additionally required for the correct targeting of mitochondrial marker, but revealed some overlap with the ER, PEX26. visualized with an antibody directed against calreticulin (Fig. Since peroxisomal TA proteins were suggested to harbor 5B). None of the other PEX26 fragments revealed any both a peroxisomal- as well as an ER-targeting signal (Borgese congruent staining with the ER (not shown). et al., 2003; Elgersma et al., 1997), we also determined to which organelle the nonperoxisomal PEX26 fragments PEX19 is necessary for targeting of PEX26 are targeted. Mislocalized PEX262-274 was targeted to The combined results strongly indicate that the TA protein mitochondria as evidenced by a staining pattern that was PEX26 follows the PEX19-dependent targeting route that is superimposable with that of the mitochondrial matrix used by most PMPs. To underscore this conclusion, the protein TRAP1 (Fig. 5A). In addition, the mPTS fragment subcellular localization of GFP-PEX26 was studied in PEX26245-305 was detectable in mitochondria to some extent, a PEX19-deficient cell line. Since these cells lack whereas full-length PEX26 was exclusively peroxisomal in morphologically detectable peroxisomes, PEX26 should accumulate in the ER if it indeed had to pass the ER en route to peroxisomes. However, colocalization studies with the mitochondrial marker TRAP1 clearly demonstrated that in the absence of PEX19, PEX26 was localized to mitochondria (Fig. 6A). The same fate was also reported to be met by other PMPs including PEX14, PEX12 or ALDP (Sacksteder et al., 2000). Thus, either the removal of the luminal PEX19-binding site or the absence of PEX19 resulted in a mitochondrial localization of PEX26. The results obtained are therefore consistent with the hypothesis that the luminal PEX19-binding site prevents PEX26 from mitochondrial mistargeting. To determine whether the remote possibility that the targeting motif of PEX26, which does bind PEX19, remains functional in the absence of PEX19 as long as peroxisomes are present, the subcellular location of PEX26 was analyzed after the expression of PEX19 had been transiently knocked down by siRNA. Transfection of human fibroblasts with a published PEX19 duplex siRNA (Jones et al., 2004) led to a drastic

Journal of Cell Science decrease in the protein concentration of PEX19 at day two after transfection and remained low until day five, whereas levels of the PMP PEX14 were hardly affected throughout the duration of the experiment (Fig. 6B, lower panel). A control knock down with PEX5-specific siRNA (Jones et al., 2004) that selectively inhibits peroxisomal matrix protein import did not lead to a significant decrease in protein levels of both PEX19 and PEX14 (Fig. 6B, upper panel). Sixty hours after the siRNA treatment, cells were transfected with a GFP-PEX26-expressing plasmid. After another 24 hours, localization of the newly synthesized fusion protein was inspected and compared with the fluorescence pattern of endogenous PEX14. Visualization of PEX14 revealed that peroxisomes persisted even after 4 days of incubation with the PEX19-specific siRNA in most cells, which was consistent with a previous observation (Jones et Fig. 5. Targeting of PEX26 fragments to nonperoxisomal al., 2004). Cells with a normal appearance of peroxisomes compartments. (A) Staining of mitochondria (Mc). Transiently were then scrutinized for the localization of PEX26. About transfected human fibroblasts were processed for indirect 60% of the cells showed at least a partial mislocalization of immunofluorescence using anti-TRAP1 antibodies to visualize PEX26 to mitochondria as exemplified in Fig. 6C,D, whereas mitochondria. The achieved staining pattern was compared with that in the PEX5 siRNA control, this was the case for only 35% caused by the GFP fluorescence of the indicated PEX26 fusion of the cells. The reason for the large number of control cells proteins. GFP-PEX262-274 and also some PEX26245-305 showed a congruent staining pattern with the mitochondrial marker protein. with mislocalized PEX26 needs to be addressed in future (B) Staining of the ER. The same kind of transfected cells were work but can in part be explained by the fact that strong labeled with an anti-calreticulin antibody and were similarly analyzed overexpression of PEX26 resulted in its targeting to for colocalization with the ER. The GFP-PEX26275-305 fusion protein mitochondria even in untreated cells. Nonetheless, the revealed some overlap with the ER marker. Bar, 10 ␮m. significant increase of mislocalized PEX26 in the PEX19 Tail-anchored PMP import requires PEX19 2513

Fig. 6. PEX19 is required for the targeting of PEX26. (A) PEX26 localizes to mitochondria in PEX19-deficient cells. A GFP- PEX262-305-expressing plasmid was transfected into PEX19-deficient human fibroblasts and the localization of the GFP fusion protein was determined. Mitochondria were labelled by indirect immunofluorescence using anti-TRAP1 antibodies (Mc). (B) Expression of PEX19 is downregulated in human fibroblasts treated with PEX19 siRNA. Wild-type fibroblasts were transfected with PEX19-specific siRNA or with PEX5 control siRNA and incubated for 5 days at 37°C. Samples were taken every 24 hours and whole-cell lysates were prepared. Equal amounts of protein were analyzed by immunoblotting using antibodies against PEX19 and PEX14. (C,D) Inhibition of PEX19 affects targeting of PEX26. After 60 hours, PEX19 siRNA-treated cells were transfected with a GFP- PEX262-305-expressing plasmid. 24 hours later, cells were analyzed for the distribution of the fusion protein. Peroxisomes (Px) and mitochondria (Mc) were visualized by indirect immunofluorescence using anti-PEX14 (C) and anti-TRAP1 (D) antibodies, respectively. Bar, 10 ␮m.

and that the luminal tail (amino acids 353-383) directs Pex15p from the ER to peroxisomes (Fig. 7A). Our prediction for PEX19-binding sites within the mPTS of Pex15p suggested the existence of two binding sites that are distributed similarly to PEX26: one of them comprises the C-terminal 15 amino acids, the second predicted site roughly coincides with the putative TMD (Fig. 7B). Yeast Pex19p indeed interacted with a fragment of Pex15p that contained both predicted binding sites (Pex15p315-383) in a yeast two-hybrid assay, whereas the large cytosolic domain (Pex15p2-314) did not (Fig. 7C). The luminal tail of Pex15p alone (amino acids 350-383) interacted with Pex19p even more strongly, probably as a result of the absence of the TMD in this construct. The fragment Pex15p315-358, which only contained the PEX19-binding site proposed to coincide with the TMD, did not give rise to a positive signal

Journal of Cell Science when tested for interaction with Pex19p (Fig. 7C). Thus, the two-hybrid assay did not clarify whether the TMD would bind Pex19p, but did confirm the presence of a PEX19-binding site within the luminal domain of Pex15p. To address the role of the luminal PEX19-binding site in targeting of Pex15p, a fragment including this site as well as the TMD (amino acids 315-383) was fused to GFP. The fragment, which was slightly shorter than the previously published mPTS of Pex15p (Elgersma et al., 1997), gave rise to a fluorescence pattern that was superimposable with that of the peroxisomal marker protein PTS2-DsRed (Fig. 8A), indicating that Pex15p315-383 was correctly targeted to peroxisomes. As expected for a fragment lacking a TMD, the C-terminal PEX19-binding site alone (amino acids 361-383) siRNA-treated cells adds further support for the function of localized to the cytosol and to nonperoxisomal structures (Fig. PEX19 as a soluble import receptor for PEX26. 8B). However, a chimera composed of this binding site and the nonperoxisomal ALDP87-164 fragment regained the ability to The targeting signal of the tail-anchored S. cerevisiae target to peroxisomes (Fig. 8C,D). Likewise, the C-terminal Pex15p also contains a PEX19-binding site PEX19-binding site of PEX26 also redirected this ALDP We then analyzed whether the targeting signal of Pex15p, the fragment to yeast peroxisomes (Fig. 8E). Thus, the luminal only known tail-anchored PMP of the yeast S. cerevisiae, also PEX19-binding site of both TA proteins Pex15p and PEX26 contains PEX19-binding sites. The mPTS of Pex15p has been function as peroxisomal targeting motifs in yeast. localized to its C-terminal 80 amino acids, which includes the TMD and its luminal domain (Elgersma et al., 1997). Since Peroxisomal redirection of mitochondrial Fis1p by the deletion of the latter domain (30 amino acids) resulted in a PEX19-binding site of Pex15p mislocalization of Pex15p to the ER, the authors concluded that Finally, we analyzed whether Fis1p (Mozdy et al., 2000), a TA amino acids 302-353 of Pex15p contain an ER-targeting signal, protein of the outer mitochondrial membrane, is also present 2514 Journal of Cell Science 119 (12)

terminal 61 amino acids, consistent with the fact that targeting signals for TA proteins are generally located at the C-termini and include the single TMD. Noticeably, however, whereas the luminal tails of most TA proteins are usually very short, that of PEX26 comprises 36 amino acids. We could show that this luminal tail of PEX26 harbors a typical peroxisomal targeting motif. PEX26275-305 was not only necessary for the correct targeting of PEX26 but also sufficient to redirect an otherwise nonperoxisomal ALDP fragment to peroxisomes. This chimera as well as the mPTS of PEX26 also correctly inserted into the peroxisomal membrane, because both fragments proved to be resistant to sodium carbonate extraction. Importantly, the sequence of the luminal domain of PEX26 was predicted to harbor a PEX19-binding site, which we could indeed demonstrate both in vivo and in vitro. The targeting signal of yeast Pex15p also contained a PEX19-binding site within its luminal tail. Alone, this PEX19-binding site failed to target GFP to peroxisomes, in line with the previous observation that the entire luminal domain of Pex15p (amino acids 350-383) is not sufficient for targeting (Elgersma et al., 1997). However, the C-terminal 23 amino acids of Pex15p provoked a redirection to peroxisomes of the human ALDP fragment and of the mitochondrial TA protein Fis1p (Mozdy et al., 2000). These results corroborate the requirement for a TMD to anchor a PMP in the peroxisomal membrane. More Fig. 7. Prediction and determination of PEX19-binding sites in S. importantly, they show that the luminal tails of PEX26 and cerevisiae Pex15p. (A) Graphic representation of the mPTS of Pex15p contain targeting motifs that remain functional in a Pex15p as proposed by Elgersma et al. (Elgersma et al., 1997). heterologous context. Both ALDP as a typical PMP and Fis1p Shown are the TMD as well as the proposed peroxisomal (Px-TS) as a mitochondrial TA protein are thought to be inserted into and ER (ER-TS) targeting signal sequences. (B) Location of the target membrane directly from the cytosol (Lazarow and predicted PEX19-binding sites in the mPTS of yeast Pex15p. The Fujiki, 1985; Purdue and Lazarow, 2001). It therefore also two PEX19-binding sites as predicted by our program (Rottensteiner follows that the motifs are not dependent on a precedent et al., 2004) are denoted. (C) Yeast two-hybrid assay for the interaction of Pex15p with Pex19p. The indicated fragments of integration of the target protein into the ER membrane. Pex15p were tested as GAL4-BD fusions in combination with yeast The current model for PEX19 function stipulates that PEX19

Journal of Cell Science Pex19p fused to the GAL4-AD (left panel) or the GAL4-AD alone acts as a soluble import receptor for a number of PMPs (right panel) in the yeast strain PJ69-4a. Two independent colonies of including ALDP (Halbach et al., 2005; Jones et al., 2004). each transformation were assessed for growth on histidine-adenine PEX19 thus recognizes its substrate in the cytosol and delivers double-dropout medium plates. it to the peroxisomal membrane, where it binds to its docking factor, PEX3 (Fang et al., 2004; Götte et al., 1998; Muntau et al., 2003). Our data can therefore be reconciled with a model in yeast peroxisomes, because the apparent mammalian in which the luminal domains of peroxisomal TA proteins are orthologue was recently reported to be dually localized to immediately recognized by PEX19 in the cytosol upon which mitochondria and peroxisomes (Koch et al., 2005). A GFP- the proteins are directly delivered to peroxisomes. In line with Fis1p fusion colocalized with the synthetic mitochondrial this concept, transient inhibition of PEX19 by siRNA resulted marker PrF0ATP9-DsRed, but only very rarely, if at all, with in a significant increase in newly synthesized PEX26 that was peroxisomal PTS2-DsRed (Fig. 9), indicating that yeast Fis1p targeted to mitochondria. It should be noted, however, that is probably not a peroxisomal TA protein. Nonetheless, because because of the fact that PEX19 siRNA-treated cells often mitochondrial TA proteins are necessarily inserted directly from showed a dual distribution of PEX26 to peroxisomes and the cytosol, we asked whether the luminal PEX19-binding site mitochondria, this latter experiment might also be interpreted of Pex15p would be able to redirect this mitochondrial TA to suggest that PEX19 is not essential for peroxisomal targeting protein to peroxisomes. Appending the luminal PEX19-binding of PEX26, but considerably increases the specificity of that site of Pex15p indeed provoked an almost exclusive localization process. This would be achieved by competition of PEX19 of Fis1p to peroxisomes (Fig. 9). The PEX19-binding site was with other cytosolic chaperones to bind to the luminal tail of therefore able to enforce peroxisomal targeting of an otherwise PEX26. However, because this fragment proved capable of predominantly mitochondrial protein. redirecting a nonperoxisomal ALDP fragment to peroxisomes, a direct role for PEX19 in targeting appears more likely. That Discussion PEX26 is a typical PMP binding partner for PEX19 is also Our study on the targeting of two peroxisomal TA proteins, supported by the fact that its interaction depends on the same human PEX26 and yeast Pex15p, provides unequivocal residues of PEX19 that are required for the interaction with evidence for a protein-based sorting machinery for TA multiple other PMPs (Fransen et al., 2005). Moreover, recent proteins. The mPTS of PEX26 turned out to comprise its C- results from Matsuzono and Fujiki (Matsuzono and Fujiki, Tail-anchored PMP import requires PEX19 2515

Fig. 8. The role of PEX19-binding sites in targeting of ScPex15p. S. cerevisiae wild-type strain yHPR251 harboring an integrated copy of the synthetic peroxisomal marker PTS2-DsRed (Px) was used to express the following protein fragments fused to GFP: the C- terminal Pex15p315-383 fragment containing both PEX19-binding sites (A); Pex15p361-383, comprising the single C-terminal PEX19-binding site (B); the ALDP87-164 fragment (C); Pex15p361-383 appended to the ALDP87-164 fragment (D); and PEX26275-305-ALDP87-164, the C- terminal PEX19-binding site of PEX26 fused to the same ALDP fragment (E). The transformed strains were grown on ethanol-containing plates for 2 days and inspected for GFP- and DsRed-derived fluorescence. The merged images of the two acquisitions reveal eventual colocalization of peroxisomal PTS2- DsRed with the GFP fusion proteins. DIC, differential interference contrast. Bar, 2 ␮m.

2005) that are complementary to our approach revealed Recent work by Hoepfner et al. (Hoepfner et al., 2005)

Journal of Cell Science evidence for a PEX19-dependent in vitro insertion of PEX26 showed that some PEX19 is also localized to distinct foci into isolated peroxisomes. emanating from the ER. These foci contain PEX3 and are Peroxisomal TA proteins are anticipated to traverse the ER thought to represent peroxisomes early in their development. en route to peroxisomes. Support for this assumption was It is therefore entirely possible that TA proteins are targeted to provided by an in vitro import experiment that suggested these PEX3-enriched ER subdomains rather than to mature preferential import of Pex15p into ER versus peroxisomal peroxisomes, but this would not change our concept of a direct membranes (Mullen et al., 1999). Furthermore, overexpressed and PEX19-dependent delivery of peroxisomal TA proteins to Pex15p appeared to be O-glycosylated, accumulated in the ER peroxisomes. In fact, if peroxisomal TA proteins were first and caused a massive proliferation of the compartment targeted to the ER by a PEX19-independent mechanism, then (Elgersma et al., 1997). However, it is also possible that in both PEX19 would be able to interact with its cognate luminal site cases the artificial excess of Pex15p prevented insertion into only from within the ER, which would require translocation of the correct target membrane because the amount of PEX19 had PEX19 across the ER membrane. become limiting. Another argument in favour of a bipartite We also detected a second PEX19-binding site within the C- targeting signal was the observed ER localization of a Pex15p terminal region of PEX26. Binding of PEX19 to this second devoid of its luminal domain (Elgersma et al., 1997). We found binding site, which coincides with the TMD of PEX26, could here that a similarly truncated PEX26 was also mistargeted, but be demonstrated in vitro by using a PEX26 peptide scan. to mitochondria. Moreover, in the absence of PEX19, even full- Interestingly, a GFP fusion of this binding site (PEX26245-274) length PEX26 inserted into the mitochondrial membrane. was clearly detectable also in peroxisomes, thereby proving Thus, it seems more likely that binding of the luminal domains function of this second PEX19-binding site as a peroxisomal of peroxisomal TA proteins by PEX19 prevents their insertion targeting motif. That the TMDs of a TA protein can possess into spurious membranes. The difference in targeting of the sequence-specific sorting information is not without two truncated PMPs is probably due to the different extreme precedence, as has been demonstrated recently for a plant C-terminal residues that emerged from clipping the PEX19- mitochondrial cytochrome b5 isoform (Hwang et al., 2004). On binding site. A number of studies have shown that modifying the other hand, the site was not sufficient for targeting in the a few C-terminal amino acids of TA proteins can suffice to alter presence of the N-terminal cytosolic domain of PEX26. It their subcellular localization (Borgese et al., 2003; Hwang et might be that in this construct (PEX262-274) the binding site is al., 2004). not accessible for PEX19 and that appending the luminal 2516 Journal of Cell Science 119 (12)

reactions, as well as the vectors and restriction sites used for cloning the PCR fragments are listed in Table S1 in supplementary material. The sequences of the primers are shown in Table 2 in supplementary material. The sequences of all PCR- generated fragments of this study were verified by automated sequencing (MWG Biotech, Ebersberg, Germany).

Morphological analysis Live yeast cells were analyzed for enhanced green fluorescent protein (GFP) and DsRed fluorescence as described (Rottensteiner et al., 2004). Human fibroblast cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100,000 U/l penicillin and 100 mg/l streptomycin at 8% CO2. The human skin fibroblast cell line GM5756T was grown for 1 day in 12-well plates before it was transfected with the pEGFP-C1-derived plasmids. Transfection was performed with 0.5 ␮g plasmid DNA and 1.5 ␮l FUGENE 6 according to the manufacturer’s instruction (Roche Diagnostics, Mannheim, Germany). The patient-derived PEX19-deficient skin fibroblasts (primary cell line RW/mf/0854872, kindly donated by Ron Wanders, AMC, Amsterdam, The Netherlands) were similarly transfected, but were grown for 3 days before transfection. 2 days after the transfection, cells were fixed on cover glasses with 3% formaldehyde in phosphate-buffered saline (PBS), permeabilized with 1% Triton X-100 in PBS and subjected to immunofluorescence microscopy using polyclonal rabbit anti-PEX14 antibodies (Will et al., 1999) in conjunction with Alexa Fluor 594-conjugated antibodies. Samples were also inspected for GFP fluorescence. Fig. 9. Peroxisomal redirection of the mitochondrial TA protein To visualize the ER and mitochondria, rabbit polyclonal anti-calreticulin and Fis1p by the luminal PEX19-binding site of Pex15p. A GFP fusion mouse monoclonal anti-TRAP1 antibodies (Affinity Bio Reagents, Golden, CO), of Fis1p was coexpressed in the S. cerevisiae wild-type strain with respectively were used in combination with the appropriate Alexa Fluor 594- or the synthetic mitochondrial marker PrF ATP9-DsRed (Mc, A) or Alexa Fluor 568-conjugated secondary antibodies. All micrographs were recorded 0 on a Zeiss Axioplan 2 microscope with a Zeiss Plan-Apochromat 63ϫ/1.4 oil PTS2-DsRed (Px, B). GFP- and DsRed-derived fluorescence was objective and an Axiocam MR digital camera and were processed with AxioVision analyzed after strains had been grown on ethanol-containing plates 4.2 software (Zeiss, Jena, Germany). for 2 days. Localization of GFP-Fis1p-Pex15p361-383 was similarly determined. DIC, differential interference contrast. Bar, 2 ␮m. Alkaline sodium carbonate extraction Human fibroblasts (GM5756T) were cultivated in T75 flasks, transfected as described above and incubated for two additional days. For whole cell lysates, cells were washed with Hank’s buffered salts (BSS; PAA Laboratories, Pasching, PEX19 binding site is required to achieve an efficient Austria), treated with trypsin, resuspended in 5 ml Hank’s BSS containing 1 mM association with PEX19. It should be noted that our studies left PMSF and harvested by a 200 g centrifugation step. After washing the cells with PBS, pH 7.3, samples were resuspended in standard Laemmli SDS-PAGE buffer. open whether binding of PEX19 to the second site is essential For the membrane localization analysis, cells were disintegrated in hypotonic lysis for targeting of full-length PEX26, because this site overlaps buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, CompleteTM protease inhibitor with the TMD, which by itself is an essential part of the cocktail from Roche Diagnostics, Mannheim, Germany) and by passing the suspension through a 20G syringe needle five times. Membranes were separated targeting signal. This issue could be resolved in future work by from the soluble fraction by a 100,000 g ultracentrifugation step for 1 hour. Journal of Cell Science exchanging the TMD of PEX26 for one that does not bind Subsequently, membranes were resuspended in 100 mM Na2CO3 for 30 minutes at PEX19. 4°C with gentle agitation and the extracted proteins were separated from membranes In summary, we show in this work that peroxisomal TA by repeating the ultracentrifugation step. Finally, aliquots of each fraction, adjusted to equal amounts of cells, were resuspended in standard Laemmli SDS-PAGE buffer. proteins are targeted to peroxisomes in a PEX19-dependent Samples were analyzed by immunoblotting for the distribution of endogenous manner and that targeting specificity is achieved by the PEX14, catalase, and the transiently expressed GFP fusions of PEX26 fragments. presence of a C-terminal PEX19-binding site that largely Monoclonal anti-GFP (JL-8) antibodies were obtained from BD Biosciences, Pharmingen, Germany; polyclonal antibodies against human catalase were prevents insertion into other intracellular membranes. Thus, purchased from The Binding Site, Schwetzingen, Germany. our data lend credence to the existence of protein-based targeting machineries for TA proteins. Since the PEX19- PEX19 inhibition by siRNA binding site of PEX26 and Pex15p functioned as typical The sequences of the PEX19 and PEX5-specific siRNA oligonucleotides were adopted from Jones et al. (Jones et al., 2004). The oligonucleotides were obtained targeting motifs that insert PMPs directly from the cytosol, we as duplex RNA from Perbio/Dharmacon RNA Technologies, Bonn, Germany also suggest that topogenesis of peroxisomal TA protein does (PEX19: 5Ј-GAG AUC GCC AGG AGA CAC U dTdT-3Ј and 5Ј-AGU GUC UCC not normally require an additional ER-specific targeting signal. UGG CGA UCU C dTdT-3Ј; PEX5: 5Ј-AGA AGC UAC UCC CAA AGG C dTdT- 3Ј and 5Ј-GCC UUU GGG AGU AGC UUC U dTdT-3Ј). For analysis of siRNA-dependent repression of PEX19, human fibroblasts Materials and Methods (GM5756T) were grown on coverslips in a 12-well format for 24 hours. Transfection Strains and plasmids with siRNA was done with 50 ␮l of duplex PEX19 siRNA (2 ␮M) and 2 ␮l Escherichia coli strain DH5␣ was used for all plasmid amplifications and isolations. Dharmafect 4 reagent (Perbio/Dharmacon) according to the manufacturer’s E. coli strain BL21(DE3) (Merck, Darmstadt, Germany) was used for heterologous instructions. Cells were cultivated for 5 days without addition of antibiotics and expression of recombinant GST-HsPEX19. Two-hybrid assays were performed with every 24 hours a sample was withdrawn and prepared for immunoblot analysis. Saccharomyces cerevisiae strain PJ69-4A (P. James, Madison, USA). The S. Cells were washed with Hank’s BSS, trypsinized, suspended in Hank’s BSS plus 1 cerevisiae wild-type strain UTL7-A and its derivative yHPR251 harboring an mM PMSF and collected by centrifugation. Cells were washed in PBS pH 7.3 and integrated copy of a PTS2-DsRed construct were used for the expression of transferred in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, enhanced green fluorescent protein (GFP) fusions. Standard media for the CompleteTM protease inhibitor cocktail, 0.5% Triton X-100). The protein cultivation of yeast and bacterial strains were prepared as described (Sambrook et concentration was determined by Coo Protein Assay Reagent (Uptima-Interchim, al., 1989). Montlucon, France). Equal amounts of protein were analyzed by immunoblotting PEX26, the adrenoleukodystrophy protein (ALDP), and all fragments using anti-PEX14 and monoclonal anti-PEX19 antibodies (BD Biosciences, thereof were amplified from the commercially obtained cDNA clones Pharmingen, Germany). IMAGp998J1311625Q3 and IRAKp961I0514Q2, respectively (RZDP, Berlin, To investigate PEX19-dependent peroxisomal import of PEX26, cells were Germany). FIS1 and all PEX15 fragments used in this study were amplified from transfected with pAH13 (GFP-PEX262-305) 60 hours after cells had been treated genomic DNA of S. cerevisiae UTL-7A. The primer pairs used in these PCR with siRNA. 24 hours after transfection, cells were prepared for Tail-anchored PMP import requires PEX19 2517

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