2508 Research Article Targeting of the tail-anchored peroxisomal membrane proteins 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 protein 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.