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Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by a peroxisomal targeting sequence (PTS) and ferry them to the peroxisomal membrane. Subsequently, the cargo is translocated into the peroxisomal lumen, whereas the receptor is released to the cytosol for further rounds of protein import. This cycle is controlled by the ubiquitination status of the receptor, which is best understood for the PTS1-receptor. While polyubiquitination of PTS-receptors results in their proteasomal degradation, the monoubiquitinated PTS-receptors are exported to the cytosol and recycled for further rounds of protein import. Here, we describe the identification of two ubiquitination cascades acting on the PTS2 co-receptor Pex18p. Using in vivo and in vitro approaches, we demonstrate that the polyubiquitination of Pex18p requires the ubiquitin-conjugating enzyme (E2) Ubc4p, which cooperates with the RING (really interesting new gene)-type ubiquitin-protein ligases (E3) Pex2p as well as Pex10p. Monoubiquitination of Pex18p depends on the E2 enzyme Pex4p (Ubc10p), which functions in concert with the E3 enzymes Pex12p and Pex10p. Our findings for the PTS2-pathway complement the data on PTS1-receptor ubiquitination and add up to a unified concept of the ubiquitin-based regulation of peroxisomal import.
Peroxisomes form a dynamic subcellular compartment in almost all eukaryotic cells . They can contain up to 50 different enzymes within their lumen, the peroxisomal matrix. These sets of enzymes link this organelle to distinct biochemical reaction pathways and physiological functions, which may vary depending on species, cell type or growth condition. The breakdown of fatty acids via beta-oxidation is considered to be a conserved function of peroxisomes, while other tasks linked to this organelle include the biosynthesis of plasmalogens and bile acid in mammals as well as the involvement in the photorespiration in plants or the biosynthesis of penicillin in certain fungi [2-4]. The vital role of peroxisomes is highlighted by the fact that dysfunction of human peroxisomes is associated with a spectrum of severe peroxisomal disorders like, e.g. Zellweger Syndome [5-7].
The functionality of this organelle is governed by dynamically operating import machineries for peroxisomal membrane and matrix proteins [8-10]. Matrix proteins usually harbor either a carboxy-terminal peroxisomal targeting sequence 1 (PTS1) or an amino-terminal PTS2 amino acid sequence, which is recognized by the soluble PTS1-receptor Pex5p or the PTS2-recognition factor Pex7p, respectively. The majority of peroxisomal matrix proteins is imported by the PTS1-receptor Pex5p. This cycling receptor binds its cargo in the cytosol and ferries it to a docking complex (Pex13p, Pex14p and Pex17p in yeast) at the peroxisome [8, 9]. The cargo is supposed to translocate over the membrane via a transiently opened import pore  and is released into the peroxisomal matrix. Finally, the PTS1-receptor is exported back to the cytosol in order to facilitate further rounds of matrix protein import. This final dislocation step is accomplished by the concerted action of certain peroxisomal membrane subcomplexes, collectively referred to as the exportomer . This molecular machinery comprises of enzymes required for the ubiquitination as well as the ATP-dependent extraction of the receptor from the membrane. A prerequisite for the dislocation step is the monoubiquitination of Pex5p on a conserved cysteine [13-16]. In yeast, this modification requires the presence of the peroxisomal RING (really interesting new gene)-finger complex, which consists of the E3 enzymes Pex2p, Pex10p and Pex12p, as well as the peroxisome-specific E2 enzyme Pex4p (Ubc10p) and its membrane anchor Pex22p [15-19]. The AAA (ATPases associated with various cellular activities)-type ATPases Pex1p and Pex6p dislocate the ubiquitinated Pex5p from the peroxisomal membrane back to the cytosol [13-15, 20, 21], where the ubiquitin is removed by deubiquitinating enzymes [22-24]. In case the export reaction is not working properly, Pex5p enters a quality control pathway, where it is polyubiquitinated on lysine residues and then degraded by the 26S proteasome. This alternative extraction pathway from the membrane depends on the formation of the polyubiquitin chain, which itself requires the presence of the RING-complex as well as the E2 enzyme Ubc4p or the partially redundant Ubc5p and Ubc1p [15, 16, 18, 25-27].
The concept of the PTS2-pathway is in many aspects comparable to the PTS1-dependent matrix protein import [28, 29]. The PTS2-recognition factor Pex7p cycles between the cytosol and the peroxisomal compartment [30, 31]. However, unlike the PTS1-receptor, Pex7p is necessary but not sufficient to carry out all steps of the import cycle as it requires species-specific auxiliary proteins. These PTS2-co-receptors are the redundant Pex18p and Pex21p in Saccharomyces cerevisiae, the orthologous Pex20p in Yarrowia lipolytica, Neurospora crassa, Hansenula polymorpha and Pichia pastoris or Pex5L, the longer of two splice isoforms of Pex5p, in mammals and plants . This bipartite assembly of the PTS2-receptor module is also evident in the molecular mechanism of the PTS2-protein import. In S. cerevisiae, Pex7p first binds the PTS2-cargo in the cytosol and then interacts with Pex18p before it can efficiently reach the peroxisomal membrane . Interestingly, the cargo-bound Pex7p can only translocate across the membrane when Pex18p is monoubiquitinated on the conserved cysteine . These results are in line with the ‘Export-driven import model’ , which suggests that matrix proteins can only be translocated and imported when the receptor is ubiquitinated and exported. However, the enzymes that catalyze this reaction so far have not been identified.
Here we describe the discovery of two ubiquitination cascades acting on the PTS2-co-receptor Pex18p. We demonstrate that the polyubiquitin chains of Pex18p, which mediate the turn-over of Pex18p, are generated by the E2 enzyme Ubc4p in concert with the RING-ligases Pex2p and Pex10p, while the monoubiquitin signal, which is essential for matrix protein import and receptor recycling, is attached to the co-receptor by Pex4p and the RING-ligases Pex12p and Pex10p.
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- Materials and Methods
- Supporting Information
The PTS2-co-receptor Pex18p of S. cerevisiae was the very first peroxin that has been found to be ubiquitinated, when modified forms of Pex18p were described in 2001 . More recently, we were able to characterize this posttranslationally modified PTS2-co-receptor in more detail and discovered that Pex18p is polyubiquitinated on two lysine residues and monoubiquitinated on a cysteine residue . In this study, we describe two ubiquitination cascades that are responsible for these two different ubiquitin-modifications (Figure 5).
Figure 5. Model of the ubiquitination cascades acting on the PTS2-co-receptor Pex18p in Saccharomyces cerevisiae. Proteins harboring the PTS2 are ferried to the peroxisomal membrane by a PTS2-selective receptor complex which consists of the cargo recognition factor Pex7p and the regulatory co-receptor Pex18p. The receptor/cargo-complex associates with a docking complex at the peroxisome membrane (not depicted here). The PTS2-cargo is translocated to the peroxisomal matrix and the PTS2-receptor proteins are returned to the cytosol by the AAA-complex (Pex1p and Pex6p anchored by Pex15p) depending on the ubiquitination (Ub) status of Pex18p. Monoubiquitination of Pex18p on a cysteine is required for the recycling and further import rounds of import, while polyubiquitination on lysines results in proteasomal degradation. The data of this manuscript complement the central positions of the ubiquitin-based regulation of Pex18p. The ubiquitination cascade required for polyubiquitination of Pex18p consists of Uba1p (E1), Ubc4p (E2) as well as Pex2p and Pex10p (E3s). The ubiquitination cascade that catalyzes the monoubiquitination consists of Uba1p (E1), the Pex22p-anchored Pex4p (E2) as well as Pex12p and Pex10p (E3s).
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The only other peroxisomal protein known to be posttranslationally modified in a similar manner is the PTS1-receptor Pex5p of S. cerevisiae. Interestingly, our recent data indicate that both receptor proteins share similarities concerning their ubiquitination requirements, but also display some differences. It has been noted before that the PTS2-co-receptor Pex18p resembles the amino-terminal half of the PTS1-receptor Pex5p. This was based on similarities in the amino acid sequence and shared binding partners at the peroxisomal docking complex  as well as the fact that a chimeric protein, consisting of the cargo-binding C-terminus of Pex5p and Pex18p lacking its Pex7p-binding site, can import PTS1-proteins . As shown recently, the stability of both Pex5p and Pex18p depends on polyubiquitination on two conserved lysine residues, while membrane topology and export are governed in both cases by the monoubiquitination on a conserved cysteine [15, 16, 33]. In this study, we shed more light on the similar and therefore mechanistic related modules of the PTS1- and PTS2-receptor ubiquitination machineries, by the discovery that both receptor proteins utilize the same ubiquitin-conjugating enzymes. We demonstrate that Ubc4p catalyzes the poly-ubiquitination of Pex18p both in vivo and in vitro. This is also the case for S. cerevisiae Pex5p [17, 18, 25, 26]. Even more significant is the finding that Pex4p is required for the monoubiquitination of Pex18p. After Pex5p [15, 16], Pex18p represents only the second known physiological target of this peroxisomal localized E2 enzyme that has been found to be a substrate both in vivo and in vitro.
However, there are also variations to this theme. In general, the presence of all three constituents of the peroxisomal RING-complex Pex2p, Pex10p and Pex12p is essential for the ubiquitination of Pex5p [17, 18, 25-27] as well as of Pex18p, as demonstrated here. On the basis of work with truncated versions that lacked the catalytic RING-domain in vivo as well as in vitro ubiquitination assays, Pex12p has been described to represent the E3 enzyme responsible for the monoubiquitination of Pex5p, while Pex2p was primarily found to be responsible for the polyubiquitination . Another in vivo study noticed that a Pex10p-mutant reduces the polyubiquitination of Pex5p , while a recent in vitro study suggested that Pex10p synergistically enhances the E3 activity of the Pex12p/Pex4p and Pex2p/Ubc4p enzyme pairs . To our surprise, we found that Pex18p can be directly monoubiquitinated by Pex4p in concert with Pex10p as well as Pex12p in vitro and that both E3 enzymes act redundantly also in vivo. Moreover, we could show that the Ubc4p-dependent polyubiquitination of Pex18p is carried out by Pex10p and Pex2p in vivo and in vitro.
The direct involvement of two E3 enzymes for each modification in the case of Pex18p is in contrast to the situation found for the PTS1-receptor and might be explained by one of the main regulatory differences between these two proteins. Pex5p is relatively stable under wild-type conditions and is only significantly polyubiquitinated when certain components of the exportomer are missing or non-functional . In contrast, Pex18p displays a high turn-over rate already under wild-type conditions [33, 40]. Thus, while the monoubiquitination of Pex5p is the dominant modification under normal conditions, the modifications of the Pex18p population is expected to be divided between recycling-related mono- and degradation-linked polyubiquitination. This circumstance might deliver an explanation and working model why two E3 enzymes are linked to each ubiquitination form of Pex18p.
The decision whether Pex18p enters the mono-ubiquitination-dependent recycling pathway or the polyubiquitination-dependent degradation pathway may be hypothetically related to the binding dynamics of Pex18p. In case Pex18p is functionally impaired, it may stay longer at the membrane complexes because it is not efficiently recognized by the recycling machinery and therefore may render it better accessible to the polyubiquitination machinery that normally should have a lower affinity to Pex18p. However, this model requires further elucidation.
The finding that the Pex10p(C301S) mutant does not complement the PEX10-deletion strain on oleate plates, even though its functional loss can be compensated by Pex12p for the essential monoubiquitination, indicates that Pex10p might have other important functions as well. One possibility could be that Pex10p ubiquitinates also other peroxisomal biogenesis factors. One hypothesis could even be that this important additional substrate is Pex10p itself, because it has been demonstrated to undergo autoubiquitination in vitro [18, 27, 47]. It is known from several RING-type ligases that their autoubiquitination is an important regulatory device in vivo [57, 58]. However, if this is also the case for Pex10p remains to be elucidated.
A recent publication described the ubiquitination requirements of Pex20p in P. pastoris . While S. cerevisiae utilizes the partially redundant Pex18p and Pex21p as PTS2-co-receptors for Pex7p , P. pastoris contains Pex20p as sole PTS2-co-receptor protein . In contrast to the situation described for S. cerevisiae Pex18p, the P. pastoris study indicated that all three RING-peroxins are required for both the monoubiquitination as well as the polyubiquitination of Pex20p in vivo . One possible explanation for the observed different E3 requirement of these two PTS2-co-receptors may be species-specific differences in the regulation of the PTS2-pathway. One significant functional difference is the finding that Pex20p seems to cycle between peroxisome and cytosol even without Pex7p [41, 59], while Pex18p needs the presence of cargo-bound Pex7p to reach efficiently the peroxisomal membrane . Furthermore, Pex20p of H. polymorpha and Y. lipolytica has been reported to interact directly with the cargo [60, 61], which is not the case for Pex18p, because the association is always bridged by Pex7p [32, 39, 53]. A methodical difference between our study and the Pex20p study concerns the RING-mutants. While the Pex20p study uses RING-peroxins that carry two mutated cysteines in the first zinc-coordinating motif of each RING-domain, our study utilizes RING-variants that carry a single cysteine mutation in the second zinc-coordination sites of Pex2p and Pex10p or a single cysteine mutation in the single zinc-coordination motif of Pex12p (Figure 2A). We decided to introduce a single point mutation in the mentioned positions because this is supposed to inhibit the E3 activity without causing major structural damage to the protein . In fact, we observed before that the mutated RING-finger of Pex10p(C301S) has no E3 activity but still interacts with the RING-domains of Pex2p and Pex12p .
Another difference concerns the data that a pex4Δ strain of P. pastoris not only inhibits the monoubiquitination, but also leads to shorter polyubiquitin chains and slowed down degradation of the Pex20p cysteine-mutant. This led the authors to suggest that Pex4p might have a minor role in the polyubiquitination of Pex20p . However, we do not observe a direct role of S. cerevisiae Pex4p in the generation of polyubiquitin chains on Pex18p in vivo and in vitro.
Our findings indicate that the heterotrimeric RING-peroxin complex displays in part a differentiated responsibility of its three ligases toward the two known substrates, Pex5p and Pex18p. It is interesting to note that similar observations have been reported on the only other described heterotrimeric RING-protein complex with E3 activity, the Polycomb complex consisting of Ring1a, Ring1b and Bmi1. In this case, Ring1a, but not Ring1b, uses topoisomerase Top2alpha as target . Still, Ring1a is also capable to act on the histone H2A in a redundant manner to Ring1b . However, it is not known how the access of both Ring1a and Ring1b toward H2A is coordinated or how ubiquitination of Top2alpha by Ring1b is prevented.
Another interesting point concerns the finding that Pex10p is in principle capable to catalyze mono- as well as polyubiquitination of Pex18p. While RING-type ligases, like MDM2, facilitate the (multiple) monoubiquitination of their target at a low concentration, the same target gets polyubiquitinated at a high concentration of the ligase . Another example of a mode of action used by RING-ligases that catalyze different ubiquitin-modifications is the involvement of selective partners during catalysis, like different E2 enzymes . This latter scenario is comparable to the situation found for Pex10p in the Pex4p-dependent mono- and Ubc4p-dependent polyubiquitination of Pex18p. However, the molecular switch between the interplay with either Pex4p or Ubc4p remains to be defined.
Our data on the two ubiquitination cascades acting on Pex18p complement the findings regarding the ubiquitination requirements of Pex5p in the PTS1 pathway and therefore add up to a unified concept of the ubiquitination machinery of the peroxisomal import receptors. Concerning E2/E3 enzyme pairs, the Ubc4p/Pex2p axis is linked to the polyubiquitination and degradation of the receptor proteins, while the Pex4p/Pex12p axis plays a central role in monoubiquitination, receptor recycling and matrix protein translocation. It is interesting to note that Pex10p displays an additional function in the PTS2-pathway by directly ubiquitinating Pex18p. In the future, it will be important to elucidate how the additional Pex10p-requirement is mechanistically integrated in the interplay with the other E3 enzymes and how this process contributes to the translocation of folded matrix proteins over the peroxisomal membrane.