De novo peroxisome biogenesis: Evolving concepts and conundrums.

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Biochim Biophys Acta. Author manuscript; available in PMC 2017 May 01. Published in final edited form as: Biochim Biophys Acta. 2016 May ; 1863(5): 892–901. doi:10.1016/j.bbamcr.2015.09.014.

De novo peroxisome biogenesis: evolving concepts and conundrums Gaurav Agrawal and Suresh Subramani* Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, UC San Diego, La Jolla, CA 92093-0322, USA

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Abstract Peroxisomes proliferate by growth and division of pre-existing peroxisomes or could arise de novo. Though the de novo pathway of peroxisome biogenesis is a more recent discovery, several recent studies have highlighted key mechanistic details of the pathway. The endoplasmic reticulum (ER) is the primary source of lipids and proteins for the newly-formed peroxisomes. More recently, an intricate sorting process functioning at the ER has been proposed, that segregates specific set of PMPs first to peroxisome-specific ER domains (pER) and then assembles PMPs selectively into distinct pre-peroxisomal vesicles (ppVs) that later fuse to form import-competent peroxisomes. In addition, plausible roles of the three key peroxins Pex3, Pex16 and Pex19, which are also central to the growth and division pathway, have been suggested in the de novo process. In this review, we discuss key developments and highlight the unexplored avenues in de novo peroxisome biogenesis.

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1. An introduction to peroxisomes Early enzyme distribution studies in 1950s with animal cells led to the discovery of peroxisomes. Based on their appearance in liver parenchymal cells as an organelle with a single membrane surrounding a dense and granular matrix, peroxisomes were first named microbodies [1,2]. Later in 1960s, a thorough analysis of their enzymatic content revealed a unique sequestering of a variety of oxidases with catalase, an enzyme critical for the disposal of oxidase-generated hydrogen peroxide, and hence the term ‘peroxisome’ was proposed [3,4].

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Today peroxisomes are known as essential subcellular organelles that are ubiquitously present in all eukaryotes and with glycosomes (protozoan peroxisome) and glyoxysomes (plant peroxisomes) constitute the microbody family. Microbodies share their basic properties and are very adaptable organelles and often exhibit extraordinary specializations. Depending on the species, peroxisomes sequester specific enzymes for metabolizing distinct substrates, which often provide the organism vital adaptability, enabling it to survive in *

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Agrawal and Subramani

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unique environments. For example, in methylotropic yeasts, peroxisomes are essential for utilizing methanol as a carbon source [5]. In animal cells, peroxisomes are essential for the biosynthesis of cholesterol, dolichol, bile acids and most importantly in the β-oxidation of branched chain and very long-chain fatty acids and catabolism of polyamines and D-amino acids [6,7]. Peroxisomes are also essential for the biosynthesis of the glycerophospholipids: plasmalogens, which are highly abundant in the myelin sheath of neurons. Evidently, several peroxisomal biogenesis disorders (PBDs) are associated with the nervous system [8]. In plants, peroxisomes are essential for the glyoxylate cycle and photorespiration [9]. Interestingly, plant peroxisomal proteins are essential for synthesis and delivery of antifungal compounds outside the cell at the site of fungal infections [10,11].

2. Alternate pathways of peroxisome biogenesis Author Manuscript

2.1 Growth and division model

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Prevailing paradigms of peroxisome biogenesis continue to be actively debated [12]. Early characterization suggested that peroxisomes are like mitochondria and chloroplasts in functioning as autonomous organelles of endosymbiont origin that divide by growth and division of pre-existing organelles [13-15]. Supporting this view, cell fractionation experiments depicted several peroxisomal proteins in the soluble fractions, and some of the PMPs tested were synthesized on free ribosomes [14,16-18]. Peroxisomes divide and segregate between the dividing cells like other autonomous organelles. Moreover, peroxisomes have unique peroxisome targeting signals for matrix (PTS1 and PTS2) and membrane proteins (mPTS) and a unique membrane translocation machinery [19,20]. In mammalian cells, the peroxin (proteins involved in peroxisome biogenesis), Pex19, stabilizes newly-synthesized PMPs in the cytosol by binding them through their mPTS sequences [20,21]. Pex19, with its N-terminal Pex3-binding site [22-24], interacts with Pex3 on peroxisomal membrane, thereby inserting the PMPs into the membrane [25]. However, the growth and division pathway does not account for the source of membrane lipids required for the growth of dividing peroxisomes or the precise mechanism for membrane insertion of Pex3 or even other PMPs (Figure 1). 2.2 De novo peroxisome biogenesis

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Novikoff and colleagues were the first to demonstrate a connection between peroxisomes and the ER [26]. They identified stalk-like structures attaching peroxisomes to certain specialized areas of the smooth ER in the kidney tubule cells of guinea pig. Later in 1990s, the genetic studies identifying the essential genes for peroxisome biogenesis discovered two peroxins Pex3 and Pex19. Although, pex3Δ and pex19Δ cells were devoid of any detectable peroxisomes, peroxisomes reappeared upon reintroduction of a functional copy of the respective genes [27-30]. This propelled the idea of an alternate route for organelle formation. Today, in yeast and mammalian cells, several PMPs traffic via the ER to the peroxisome [31-33]. More importantly, the ER-derived biogenic route is capable of forming peroxisomes de novo [34,35]. Our current understanding recognizes the ER as an indispensable contributor towards peroxisome biogenesis and peroxisomes are considered as an integral part of the cellular endomembrane system [36].

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3. PMPs traffic through the ER Initially, the detection of PMPs in the ER [37] was sidelined as an artifact of overexpression [15,38]. However, the ER-routed trafficking of the N-glycosylated Pex2 and Pex16 in Y. lipolytica presented a strong case since these PMPs were expressed from their native promoters [39]. In addition, two ER-associated proteins functioning in the secretory pathway were implicated in the transit of these proteins from the ER. Since then, several studies have highlighted the trafficking of PMPs through the ER in yeast [31,32,40-42], mammalian cells [33,43-45] and plants [46-48]. These include members of both Type I PMPs (PMPs that require Pex19 for their targeting to the peroxisome; most PMPs), Type II PMPs (PMPs that do not require Pex19 for peroxisome targeting (Pex3 and Pex22) and the tail-anchored (TA) PMPs (Pex15, Pex26) [49] (Figure 1).

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3.1 Yeast PMP biogenesis Amongst the multiple organisms where this has been studied, the evidence for the role of ER in PMP biogenesis is strongest in yeast. These include S. cerevisiae [31,40,41,50], P. pastoris [32,37,42,51], Y. lipolytica [39,52] and H. polymorpha [53-56]. The intracellular trafficking of Pex3, a Type II PMP, has been extensively studied in several yeasts. This is because Pex3 (itself a PMP) along with Pex19 (a predominantly cytosolic peroxin) is reported to mediate post-translational (direct) insertion of other PMPs into the peroxisomal membrane [25,57], thus presenting an obvious dilemmas as to how Pex3 itself is incorporated into the peroxisomal membrane and how other PMPs follow the ER-route in yeast and direct post-translational insertion into pre-existing peroxisomes in mammalian cells.

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Three independent studies in S. cerevisiae, arrived at a unified conclusion that Pex3 was delivered to the peroxisomes through the ER [40,41,50]. Firstly, when Pex3 was deliberately targeted to the ER by fusion to an ER-signal peptide, Pex3 was still sorted to the peroxisomes [50]. Secondly, a strong visual proof for the ER-routed trafficking came when a fluorescently-labeled Pex3 was chased in real-time. When the expression of YFP-tagged Pex3 was induced, a perinuclear-ER localization was instantly apparent that became more focused in a punctate spot at the ER periphery. This spot was chased from the pER into functional peroxisomes [40-42]. The subdomain where the PMPs were accumulated before the budding happens is often referred to as pre-peroxisomal domain of the ER (pER) (discussed in section 5) [41,46]. More recently, in vitro assays in yeast found Pex3 in ERderived ppVs [32,58]. As the role of ER became apparent in PMP biogenesis, two immediate questions arose: 1. Whether the ER contributes PMPs and associated membrane lipids constitutively or is activated by specific environmental or cellular cues (such as cell division or defective organelle inheritance)? 2. Does the ER-derived route generate mature peroxisome de novo or feed into the growing and dividing peroxisomes or is it involved in both scenarios? Recent studies have attempted to address these conundrums. Interestingly, in yeast, a converging aspect for growth and division, as well as ER-derived pathways, has been highlighted. It was observed that growth and division is the default pathway for peroxisome biogenesis and the ER-derived route functions in replenishing the growing and dividing peroxisomes with PMPs and associated membrane lipids [34]. Biochim Biophys Acta. Author manuscript; available in PMC 2017 May 01.


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In previous studies, it is possible that the trafficking of PMPs to peroxisomes via the ER was missed in mammalian cells for technical reasons, such as lack of reliance on pulse-chase experiments and inducible promoters to drive the expression of the reporter protein being monitored. This situation has been rectified more recently in mammalian cells where the ER constitutively contributes various PMPs and membrane lipids to the pre-existing peroxisomes [33] (further discussed in section 3.2). Importantly, in these studies the sorting process for targeting these PMPs to the pre-existing peroxisomes was not identified. Later, when the vesicular pathway involved in trafficking of PMPs from the ER was established in yeast systems [32,58], two distinct routes for peroxisome biogenesis were described [35]. They demonstrated that the ER-derived ppVs do not feed the pre-existing peroxisomes but mature into new peroxisomes instead. However, an alternate view is that peroxisomes generated de novo only fuse with pre-existing peroxisomes and do not generate new ones, and that growth and division is the default pathway [34]. It should be noted, in this context, that when growth and division is blocked, then only the de novo pathway must generate all peroxisomes. Presumably, the signaling pathways, including the transcriptional responses, activate either pathway depending on the metabolic and environmental stimuli [59-61]. A recent study concluded that yeast cells switch from de novo biogenesis to fission for peroxisome formation when transferred from a glucose to fatty acid rich conditions [62]. Thus a cross-talk and collaboration between these pathways for maintaining a desired peroxisomal population in an energy-efficient manner is a more likely scenario. Identifying the components that recognize the environmental and cellular cues that selectively tip the balance towards one pathway over the other will be the key in understanding peroxisome homeostasis.


Previously, in certain pex mutants exhibiting impaired matrix protein import, characteristic peroxisomal membrane remnants were identified that were thought to furnish a structural framework for the incorporation of PMPs and matrix proteins [63,64]. This concept was recently validated in H polymorpha, when such remnants, described as reticular and vesicular structures, marked by the docking proteins, Pex13 and Pex14, and some, but not all, peroxisomal matrix proteins, Pex8 and alcohol oxidase, were shown to receive newlysynthesized Pex3 in pex3Δ cells, eventually restoring all matrix protein import [65]. Previously, this exact set of proteins was shown to be targeted via the ER before reaching the newly-formed peroxisomes in S. cerevisiae [31]. However, Knoops et al. [58] opposed de novo formation of peroxisome via the ER and suggested instead that PMPs were targeted directly from the cytosol to the peroxisomal membrane. Although this is an alternate route for peroxisome biogenesis, these authors did not eliminate the possibility that Pex3 itself might traffic via ER-derived ppVs that fuse with the reticular/vesicular structures to generate peroxisomes. The authors also speculated that these reticular/vesicular structures could be derived from pre-existing peroxisomes or from the ER. In favor of the latter scenario, ERderived ppVs were generated using ER from pex3Δ P. pastoris cells in an in vitro assay [32]. Thus, if the reticular/vesicular structures containing the small subset of PMPs observed in H. polymorpha cells were in fact ER-derived, then as in other yeasts, peroxisomes would be formed de novo upon reintroduction of Pex3 into pex3Δ cells [40,41,50]. This would also be in agreement with their previous study in H polymorpha itself that demonstrated the de novo formation of peroxisomes from the perinuclear-ER compartment [56].



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Recent studies suggest that the ER-derived biogenic route could be divided into five specific steps: (1) Import of PMPs into the ER membrane [31,37,66], discussed in section 4.1, 4.2, 4.3. (2) Intra-ER sorting of PMPs to specific pER domains [67], discussed in section 4.4. (3) Packaging of PMPs and budding into the vesicular carriers [31,32,58], discussed in section 5. (4) Fusion of specific vesicular carriers carrying a complementary set of PMPs [35,68] (5) Assembly of the importomer complex followed by matrix protein import [35,65,69] (Figure 1). It is imperative to design and develop systematic biochemical assays for each step and test and assign the function of the known and putative proteins more precisely towards a specific step(s) of this pathway. In addition, the biochemical assays could be coupled with specific genetic mutants to enhance the read-out or bypass essential functions of the protein being tested. For example, the effect of a protein on the ER-export of PMPs could be analyzed by performing ER-budding assays. When coupled with cells from a pex1Δ/pex6Δ genetic background, the vesicles would accumulate without fusing and the enriched ppVs would be easier to detect. Another example is the use of a version of Pex3 that does not require Pex16 for targeting to the ER, thus analyzing the function of Pex16 in the targeting of Pex3 to the peroxisomes [33]. Developing such ingenious methodologies would accelerate our mechanistic understanding of the de novo peroxisome biogenesis pathway. 3.2 Mammalian PMP biogenesis

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Mammalian PEX3 assists PEX19 in the incorporation of other Type I PMPs directly into the peroxisomal membrane from cytosol [25]. In addition, in vitro assays demonstrate that mammalian Pex3 was also incorporated directly into the peroxisomal membrane in a Pex16and PEX19-dependent manner [57]. However, other studies proposed trafficking of mammalian PEX3 via the ER [33,43,44]. Some reasons for the opposing conclusions regarding the route that PEX3 takes en route to the peroxisome membranes are described in the next paragraph.

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As in yeast [50], PEX3 deliberately targeted to the ER with a signal peptide restored peroxisome biogenesis in pex3Δ human fibroblasts by forming peroxisomes de novo [44]. However, the ER-transit of PEX3 required PEX16, but was independent of PEX19. PEX16 also recruited other PMPs to the ER, while Pex16 itself was targeted co-translationally into the ER in a PEX3- and PEX19-independent manner [43]. In addition to de novo peroxisome formation, similar experiments showed that an ER-derived PEX3 (ssPEX3), fused to an ERtargeting signal, was sorted to the preexisting peroxisomes in a PEX16-dependent manner [33]. However, in the same study, a newly-synthesized, version of overexpressed PEX3 was predominantly incorporated directly into the peroxisomal membrane again in a PEX16dependent manner. Interestingly, when the kinetics of ssPEX3 vs the normal PEX3 were compared, the direct incorporation of normal PEX3 (probably through the Type II pathway) was more efficient. Also, PEX16 was identified as the recruiting factor for PEX3 on both ER and peroxisomes and its cellular levels inversely affected the direct incorporation of PMPs into peroxisomal membranes. Most importantly, and in contrast to earlier assumptions [15,38], the direct incorporation of PMPs into the peroxisomal membrane was considered a phenotype of overexpression of PMPs, whereas the ER-routed PMP sorting was proposed to be the default pathway for PMP biogenesis [33] (Figure 2). This was based primarily on the fact that depleting endogenous levels of Pex16 itself, increased direct incorporation of



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PMPs, suggesting that these PMPs are routed to the ER before their sorting to the peroxisome. However, in contrast to yeast [32,58], the mode of ER to peroxisome PMP trafficking is not well understood. Intriguingly, in experiments where ssPEX3 was expressed in PEX16-deficient cells, ssPEX3 remained associated with the ER and was not released to the cytoplasm. This suggests a role for PEX16 in ER exit of ssPEX3, presumably in a vesicular compartment. Evidently, such a vesicular compartment in mammalian cells has been demonstrated. ΔPEX3 cells when cotransfected with PEX3 and PEX16 formed vesicular structures within 24h of after transfection [70]. At this time the vesicular structures contained PEX3 and PEX16 but not PEX13, PEX14 or PMP70. However, within 48-60h post-transfection the vesicular structures also acquired PEX13, PEX14 and PMP70. These vesicular structures were termed pre-peroxisomes. In course of 3-7 days the vesicular structures formed fully functional import competent peroxisomes. This was the first confirmed report for vesicular carriers in mammalian cells (Figure 2).

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3.3 Plant PMP biogenesis None of the plant peroxin mutants studied till date has been reported to result in complete loss of peroxisomes. Thus a de novo peroxisome biogenesis pathway is not yet established in plants. Nonetheless, contribution of the ER in plant peroxisome biogenesis has been well documented. The membrane-bound, peroxisomal enzyme, ascorbate peroxidase (APX), was localized to the reticular structures of the ER, as well as to the peroxisome [46]. The enzyme was incorporated exclusively in the ER membranes in vitro, but not to the peroxisome membrane, indicating that ER-transit might be a prerequisite for sorting to peroxisomes. In addition, trafficking of APX from the ER to peroxisomes was sensitive to BFA (toxin that inhibit vesicular export form the ER).

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Nonetheless, this study was conducted with overexpressed APX protein and previous work in yeast have cautioned against interpretations based on overexpressed proteins [38]. However, the ER localization of APX was authenticated when endogenous APX was found on validated rough-ER microsomes and further cell fractionation studies showed endogenous APX to be associated with both ER and peroxisome fractions [71].

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Interestingly, plant PMPs are sometimes detected in unusual compartments. APX was reported to localize to unknown membrane structures distinct from the ER and peroxisomal membranes [72]. Similarly, AtPex16 was found in punctate, non-ER, non-Golgi, preperoxisomal structures in cells recovering from BFA incubations or at low temperatures (Karnik et al., 2004). In another instance, plant peroxisomes have been observed within large pleomorphic clustered tubules, similar to the vesicular clusters that constitutes the ERGolgi intermediate compartment (ERGIC), which regulate the bi-directional secretory pathway traffic [73]. Thus such tubular plant peroxisomal structures were termed ERperoxisome intermediate compartment (ERPIC). It is hypothesized that ERPIC would contribute membrane and matrix proteins to the newly formed daughter peroxisomes. However, the precise functional significance of ERPIC is without any direct experimental evidence and is purely speculative (Figure 2). The ER-mediated trafficking in plants was further established when Pex10 and Pex16 were found in the ER of Arabidopsis cells grown in suspension cultures. However, unlike APX, Biochim Biophys Acta. Author manuscript; available in PMC 2017 May 01.


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Pex16 was localized throughout the ER and not to specific domains. At steady-state, Pex16 exhibited dual localization between the ER and peroxisomes [74]. Similar localization of Pex16 was reported in yeast and mammalian cells [39,43]. However, this is inconsistent with previous data where Pex16 was exclusively detected on peroxisomes in root hair and embryo cells in stably-transfected Arabidopsis plants [75]. Similarly, the cellular localization of Pex10 was inconsistent when analyzed in different cell types. Endogenous Pex10 in suspension cell cultures of Arabidopsis was ER-restricted and did not show any detectable peroxisomal localization [48]. However, when tobacco leaf epidermal cells were analyzed, transiently expressed Arabidopsis Pex10 was exclusively localized to peroxisomes [76].

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Perhaps the distribution of Pex10 and Pex16 suggests the diversity in plant peroxisome biogenesis depending on cell types and culture conditions. In agreement with this, the suspension cultures that show an ER-localization of PMPs, have predominantly rapidlydividing peroxisomes that require an active ER contribution, whereas the differentiated cells from plant tissues probably contain more mature or pre-existing peroxisomes. Evidently, in plants, peroxisomes often become specialized depending on the cell or tissue type, the developmental stage of plant and on the repertoire of enzymes they sequester [77]. For example glyoxysomes house the β-oxidation and glyoxylate cycle enzymes, while leaf-type peroxisomes carry out photorespiration in photosynthetically active cells [78]. Identifying the environmental and cellular cues determining such variations in intracellular PMP localization would be key in understanding peroxisome biogenesis in plants.


A novel peroxisome to ER trafficking pathway has been discovered in plants [79]. Tomato bushy stunt virus (TBSV) infection characteristically manifests a cytopathological feature where the infected cells contain hundreds of multivesicular bodies. These bodies originated from an inward vesiculation of peroxisomes and hence are often referred to as peroxisomal multivesicular bodies (pMVBs) [80] (Figure 2). The TBSV replication protein, p33, was essential for the formation of pMVBs [81,82]. The expression of p33 alone caused outward vesiculation of peroxisomal membranes and relocated p33 and other PMPs (PMP22 and APX) to the pER [79]. p33 was initially sorted from the cytosol to the pre-existing peroxisomes, however, the targeting pathway to the pER is still not fully understood. However, the ADP-ribosylation-factor 1 (ARF1) that promotes COPI-dependent retrograde vesicle trafficking of Golgi membranes back to the ER was implicated in p33 trafficking to the pER. In addition, the N-terminus of p33 possesses a targeting signal, similar to argininebased motif present in proteins of COPI-mediated retrograde trafficking. It is not clear how the retrograde targeting of PMPs serves the viral life-cycle or the normal de novo biogenesis pathway, but presumably, such reverse trafficking of selective peroxins (e.g. Pex16) could stimulate further proliferation of ER to form more peroxisomes and hence more pMVBs, which are critical for virus replication. Such pathways suggest deeper connections between the peroxisomal membrane system and the ER.

4. ER apparatus involved in peroxisome biogenesis As discussed above, the evidence for the role of ER in peroxisome biogenesis is mainly based on detection of PMPs in the ER, and their further trafficking via the ER to



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peroxisomes. Given our extensive understanding of the secretory pathway, it was expected that components required for the ER-associated machinery involved in ER-import, intra-ER sorting and budding of PMPs might have been discovered in genetic screens. However, except a handful of studies discussed below, the identity of such ER components remains elusive. This is likely because these genes might be essential, rendering gene knockouts inviable, or because the redundancy of components makes it hard to completely eliminate the process using single gene mutations. However, because many of the steps in the de novo peroxisome biogenesis pathway have been recently identified it is expected in coming years that the constituents of the ER apparatus will become apparent. 4.1 PMP import into the ER

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The first identification of ER-associated machinery came from the landmark study in Y. lipolytica, which proved that two ER-associated, secretory pathway proteins, Sec238 and Srp54, were involved in the trafficking of these PMPs [39]. However, their specific role at the ER in import, sorting or exit was not defined. Srp54 has a known function in targeting proteins to the ER [83,84], and presumably functions in ER targeting of PMPs as well. In agreement with this, mRNA encoding Pex3 was specifically targeted to the ER before translation and perhaps suggests the cotranslational import of Pex3 to the ER [85]. However, more direct in vitro experiments described below presents the post-translational model as the more likely one.


Two independent studies further connected-the-dots highlighting the role of the conserved ER protein translocation channel, the Sec61 complex [86] in the incorporation of PMPs into the ER [31,66]. This complex allows SRP-dependent (cotranslational) [87], as well as SRPindependent (post-translational; in association with Sec63 complex) [84,88] protein import into the ER. Sec61 protein is an essential and major component of the heterotrimeric Sec61 translocon (Sec61, Sbh1, Sss1). During cotranslational translocation, the SRP complex binds and targets the nascent polypeptide emerging form the translating ribosomes to the SRPreceptor on the ER-membrane. Further steps of membrane insertion and translocation are performed by the Sec61 translocon [89]. In contrast, during post-translational protein insertion into the ER, the fully synthesized precursor proteins are targeted with the assistance of cytosolic heat-shock protein chaperones. At the ER, the Sec63 complex (Sec62, Sec63, Sec66) recognizes the signal sequence (like the SRP-receptor in cotranslational import) and subsequent membrane integration and translocation is accomplished by the Sec61 translocon [90]. Evidently, depleting Sec62 and Sec63 proteins by using repressible promoters inhibited the incorporation of several PMPs (Pex8, Pex13 and Pex14) into the ER membrane and the PMPs remained in the cytosol [31]. Because Sec63 has been demonstrated to function in both co and post-translational insertion pathways [91], the authors could not cleanly distinguish between the two pathways. The second study further provided several lines of evidences to establish the role of Sec61 in the ER-import of Pex3 in S. cerevisiae cells. Firstly, using a temperature-sensitive mutant of Sec61 (Sec61-2), a drastic effect on peroxisome formation was observed at the nonpermissive temperature [66]. This Sec61-2 mutant was demonstrated to have a shorter halflife at a non-permissive temperature [92,93] and thus the number of ER-translocons were



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reduced. Similar results were obtained when Sec61 was depleted using a repressible promoter. However, these assays only showed a role of Sec61 towards peroxisome biogenesis but not necessarily in the insertion of PMPs in the ER. More conclusive proof for the insertion of PMPs in the ER came when yeast Pex3, carrying a glycosylation-tag, that was glycosylated in wild-type cells, failed to show this ER-derived modification in Sec61-2 cells at the non-permissive temperature or when Sec61 was depleted [66]. The role of Sec61 in ER-import of PMPs was further supported by in vitro assays demonstrating the posttranslational incorporation of PEX3 into the ER-derived microsomes. In contrast to an earlier study [57], here the in vitro translated Pex3 was incorporated not only in yeast but also in mammalian ER membranes (dog pancreatic cells). Based on these data, it seems more likely that yeast Pex3 is inserted post-translationally into the ER. However, one cannot rule out a difference between yeast and mammalian Pex3 in the mode of import into the ER.

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In contrast to the situation for yeast Pex3, in mammalian cells, PEX16 was targeted to the ER cotranslationally in vitro [43]. This process was independent of Pex3 and Pex19 but most likely involves the Sec61 translocon. At the ER, PEX16 recruits many other PMPs including PEX3 to the ER. Consistent with this idea is the mislocalization of PEX3 to mitochondria in the absence of PEX16 [43]. This mislocalization of PEX3 was lost when PEX16 was reintroduced into these cells. How PEX16 helps in importing these PMPs to the ER is not clear. It is reasonable to formulate a model where PEX16, like the Get1/Get2 or Sec63 complex, forms a specific receptor at the ER for importing PMPs post-translationally. This was also evident from the observation that PEX16 binds the mPTS domain of PEX3 [57] and this interaction recruits PEX3 to the ER [70]. Although, PEX19 qualifies as the cytosolic chaperone that could deliver PMPs to the ER, PEX3 was still targeted to the ER in the absence of PEX19 [43,70].

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4.2 Targeting of Tail-Anchored PMPs

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The ER-incorporation and trafficking of tail-anchored (TA) PMPs via the ER is not consistent in yeast and mammalian cells. It is known that TA proteins are targeted to the ER through the Get pathway. The cytosolic Get3 binds the hydrophobic or the transmembrane region of the newly-synthesized TA protein and transports it to the ER-membrane where Get1 and Get2 proteins receive the Get3-TA cargo and incorporate it into the ER membrane [90]. TRC40 is the mammalian homologue of Get3. Initial reports for the targeting of a TA PMP, Pex15 through the ER [37] was debated [38] since Pex15 was overexpressed. However, a subsequent study showed the direct targeting of endogenous Pex15 to peroxisomes [94]. Later reports in S. cerevisiae showed a Get complex requirement for targeting Pex15 via the ER [31,58,95]. Pex15 was observed to form cytosolic aggregates or mislocalized to the mitochondria in get1Δ/get2Δ cells [95]. The kinetics of aggregation and mislocalization were pronounced in the absence of Get3, suggesting a role for the Get pathway in targeting of Pex15 to peroxisomes. In a later study, although a severe affect on peroxisomal targeting of Pex15 was observed in Δget3 cells, but unlike Δpex15 cells, these cells still formed fewer matrix-protein import competent peroxisomes [31]. However, Pex15 was rarely detected on these peroxisomes and it was presumed that Pex15 was degraded in the cytosol as its targeting to the ER was blocked in Δget3 cells. Recently in Neurospora, the TA protein Pex26 (homologue of yeast Pex15) was directly inserted into the peroxisomal



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membrane in a Pex19-dependent, but Get/TRC40 independent manner [96]. Similarly, in mammalian cells as well, Pex26 was targeted to the peroxisomal membrane in a TRC40independent manner [97]. Therefore, homologous proteins appear to be targeted by different pathways. Another peroxisomal TA PMP, Fis1, was also inserted directly into the peroxisomal membrane in a Pex19-dependent manner [98]. These studies clearly show a potential role of ER-associated protein complexes in import of PMPs into the ER membrane prior to their proper peroxisomal targeting. In addition, when fused with signal peptides or glycosylation tags, PMPs were processed or modified, respectively, at the ER and targeted to the peroxisomes without affecting the protein function [43,44,50,58]. In summary, the ER import of only handful of PMPs has been demonstrated using the Sec61 translocon or through Get pathway, but some of these PMPs, like Pex3 and Pex16, are capable of driving peroxisome biogenesis de novo.

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4.3 ER targeting signals

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In contrast to the PTSs, ER-targeting signals are not readily apparent in PMPs. Other than the hydrophobic amino acids at the C-terminal end of tail-anchored proteins that target them to Get/TRC40 pathway (discussed in section 4.2), other ER targeting signals remained elusive on non-TA proteins [31]. The N-terminal ∼1-45aa of Pex3, comprised of luminal, transmembrane and a small cytosolic regions, was sufficient for its transport to peroxisomes in yeast, plants and mammalian cells [30,41,99-102]. Recently an N-terminal ER targeting signal was discovered within the mPTS of Pex3, which was distinct from typical ERretention signals. Using an algorithm to specifically identify signal anchors (non-cleaved ER targeting signals), an N-terminal signal anchor was found in Pex3 in many different species [66]. It was characterized by a small stretch of positively charged amino acids followed by a longer hydrophobic amino acid stretch [103]. Mutations in the hydrophobic region disrupted the ER-targeting of Pex3 [66]. Nonetheless, how such targeting signals on PMPs are recognized and how these proteins are subsequently targeted to the ER post-translationally is not understood. 4.4 Intra-ER sorting of PMPs to pre-peroxisomal domain of the ER

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There are several reports highlighting a specific domain in the ER where PMPs are localized before their targeting to the peroxisome [31,40,41,104,105]. These ER-associated domains are often referred as pre-peroxisomal domains or templates or simply peroxisomal ER (pER) [41,46,104,106]. In mammalian cells, such specialized ER domains nucleate the formation of peroxisomes by acquiring other PMPs and matrix proteins, while still attached to the ER lamellae that later detach and form a new peroxisome [104,105] (Figure 2). In contrast to the rough-ER associated usual pER structures seen in yeast and plant cells, these specialized ER structures were associated with the smooth-ER and were enriched in Pex13 and PMP70. It was postulated that these ER-lamellae after detaching from the ER, forms a ‘peroxisomalreticulum’ from which mature peroxisomes are formed (Figure 2). Alternatively, other studies have identified a vesicular pathway where PMPs are exported in vesicular carriers from a pER domain (template), that later fuse to form import-competent peroxisomes [32,35,58,68] (discussed in section 5).



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It is controversial, however, whether the import of matrix proteins begins at the pER, or whether this happens after budding of ppVs from the pER - the latter mechanism has the advantage of avoiding the premature sorting or missorting of peroxisomal matrix proteins to the ER and appears more consistent with the fact that in mutants affecting the peroxisomal translocation machinery, matrix proteins are found to be cytosolic and not in the ER lumen.

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The localization patterns of PMPs in the ER often differ from each other. For instance, Pex16 in mammalian and plant cells is distributed all over the ER [74] while APX and Pex10 in plants are found in specific domains of the ER [46,48]. However, this was even more prominent in yeast where PMPs were either 1) distributed throughout the ER (both peripheral and perinuclear) or 2) focused in a punctate dot-like structure associated with the peripheral-ER, representing the pER domain. For example, the PMPs, Pex2 and Pex11, were dispersed along the entire ER, including the perinuclear ER, while other PMPs (Pex13, Pex14 and Pex15) were focused in a single punctate structure associated with the peripheralER (pER) in pex3Δ or pex19Δ cells [31]. Our unpublished results in P. pastoris also reveal similar peripheral and perinuclear-ER distribution for Pex2 and Pex12, while Pex3 and Pex17 were localized in punctate structures associated with the peripheral-ER (pER) in pex3Δ or pex19Δ cells (Agrawal et al, unpublished results). So far, any structural or functional attributes of the PMPs have not been correlated with this distinction in the intra-ER distribution of specific PMPs. However, it is tempting to speculate that a PMP-specific, intra-ER sorting process exists at the ER that spatially segregates these PMPs and thereby prevents their premature assembly.

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One such intra-ER sorting signal was recently identified in S.cerevisiae Pex3 [67]. This intra-ER sorting signal at the N-terminus (1-17aa) of Pex3 was found to be both necessary and sufficient in that it sorts a non-peroxisomal protein to the pER. Interestingly, the fusion protein was not targeted further to the peroxisome since the transmembrane region of Pex3 was essential for peroxisomal targeting. In addition, the intra-ER targeting signal is evolutionary conserved as human or Drosophila Pex3 signals could functionally replace the yeast signal. However, such intra-ER sorting signals, which remain to be discovered in other PMPs might direct different PMPs into distinct pER domains that will exit the ER in distinct ppVs [68,106].

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Interestingly, other studies highlight the role of the transmembrane domain of secretory proteins as an important determinant for intra-ER sorting and exit through ER exit sites (ERES) [107,108]. Distinct ER localization was observed for two identical proteins except for the length of their transmembrane segments [107]. The protein with a longer transmembrane was sorted to the ERES and subsequently exported in vesicular carriers, while the shorter transmembrane proteins was held back in the ER. It was presumed that the longer transmembrane would seek stiffer lipids, often found at the ERES, while thinner lipid membranes will better fit the smaller transmembrane segment. It would be interesting to investigate if the certain features in transmembrane segments of PMPs (like Pex3) determine their intra-ER sorting.



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5. Pre-peroxisomal membrane intermediates In mammalian and plant cells, PMPs are sorted to pre-existing peroxisomes from the ER, nonetheless, vesicular carriers exporting these PMPs out of the ER, are not (yet) identified. However, in yeast, several studies have discovered small ppVs carrying PMPs and even matrix proteins [32,58,68]. The first such report of ppVs came from an extensive biochemical study in Y. lipolytica [68] that defined two distinct vesicle types (P1 and P2) derived from the ER, carrying Pex2 and Pex16 but distinct matrix proteins. The presence of matrix proteins in the lumen of these vesicles would suggest that these P1 and P2 fractions were already import competent. These two fractions were shown to fuse in a Pex1/Pex6 dependent manner to form a higher density vesicle (P3) that gradually imports other matrix proteins (forming P4 and then P5) and eventually forms a mature peroxisome.

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The ER origin of some ppVs was established later in cell free in vitro assays in S. cerevisiae [58] and P. pastoris [32]. Pex3 and an opsin-tagged Pex15 were followed as marker proteins budding from the ER in S. cerevisiae, while in P. pastoris Pex11 and Pex3 were followed. Both studies identified a requirement for Pex19, along with unknown cytosolic proteins and ATP, for the budding of such ppVs. However, in P. pastoris, Pex3 was dispensable for ppV budding and this was later confirmed in H. polymorpha, where ppVs were identified in pex3Δ cells [65]. However, these ppVs are not fully import competent, which explains why Pex3 is vital for peroxisome biogenesis in all organisms. In conclusion, only Pex19, thus far is the only peroxin, identified for ER-exit of PMPs from the ER.

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More recently, the ER-derived ppVs were further characterized into two distinct carriers, each trafficking distinct constituents of the peroxisomal importomer [35]. This multi-protein complex [109,110] is comprised primarily of the docking complex proteins (Pex13, Pex14 and Pex17) functioning in docking of PTS receptor/cargo complexes and membrane translocation of matrix proteins and the second major component is the subcomplex consisting of RING-domain proteins (Pex2, Pex10 and Pex12) critical for ubiquitination and subsequent recycling of cargo receptors from the peroxisome membrane to the cytosol. As noted above (section 4.4), for such specific packaging, an efficient intra-ER sorting would be required. In addition, biochemical requirements for budding of these ppVs from the ER were not described. It would be interesting to note if Pex19 alone is responsible for the budding of both vesicle types or Pex3 plays an additional or exclusive role for the budding of either vesicle type.

6 Concluding remarks Author Manuscript

In the past decade, our understanding of the role of ER in peroxisome biogenesis has significantly evolved. The contribution of ER in de novo, as well as in the growth and division, pathways is evident in yeast, mammalian cells and plants and is emerging as a unified paradigm. However, critical mechanistic insights into certain aspects of the ERderived route are still lacking. One such avenue is the poorly understood sorting process at the ER. How PMPs are sorted at the ER for separate exits in vesicular carriers and how they are sequestered away from the secretory pathway cargo are completely unknown. Mechanistic understanding is scarce as to how PMPs are targeted to the ER. Our knowledge



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of ER-targeting, as well as intra-ER targeting, signals is very limited and the ER-associated components that could recognize these signals are yet to be discovered. In addition, how the ppVs are pinched out from the ER-membrane is not understood. Most genetic screens have identified non-essential genes that are involved in peroxisome biogenesis and it could be highly plausible that the ER-associated sorting pathway components are essential for cell survival. Thus, to identify the essential genes, screening of temperature sensitive mutants is necessary.

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Our understanding of the ER-derived route has often resulted in controversies and disagreement. One major area of contention is the fate of the ER-derived route. Whether the ER-derived vesicles form new peroxisomes or replenish the pre-existing peroxisomes or accomplish both tasks, remains unclear. If the former is exclusively true, then why would peroxisomes need to divide at all, when the ER-derived route stocks the cell with new peroxisomes? Another possibility is that the fate of this route would depend on the cell's metabolic state and could be tipped either way. If true, studying the biogenesis process under different defined growth conditions is imperative to address these discrepancies.

Acknowledgments This work was supported by NIH grant RO1DK41737 to SS.

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Abbreviations Author Manuscript Author Manuscript Author Manuscript

aa

amino acids

ARF1

ADP-ribosylation-factor 1

APX

ascorbate peroxidase

BFA

brefeldin A

BY-2

bright yellow-2

COP

coatomer protein

ER

endoplasmic reticulum

ERES

endoplasmic reticulum exit sites

ERPIC

ER-peroxisome intermediate compartment

ERGIC

ER-golgi intermediate compartment

GFP

green fluorescent protein

HA

hemagglutinin

p33

33-kDa replication protein

Pex

peroxin

PBD

peroxisome biogenesis disorder

PMP

Peroxisomal membrane protein

pER

per-peroxisomal ER

pMVBs

peroxisomal multivesicular bodies

PTS

peroxisome targeting signal

TA proteins

Tail anchored proteins

TBSV

tomato bushy stunt virus

TMD

transmembrane domain



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Highlights •

Peroxisomes are formed from pre-existing peroxisomes but also originate de novo by budding from the ER.

•

The ER contributes to the formation of new peroxisomes as well as to the maintenance of pre-existing peroxisomes.

•

The Sec61 and the Get pathway proteins are involved in import of peroxisomal membrane proteins (PMPs) into the ER membrane.

•

Biochemically distinct pre-peroxisomal vesicles traffic PMPs out of the ER.

•

Pex19, a cytosolic peroxin is required for the budding of pre-peroxisomal vesicles from the ER.

Author Manuscript Author Manuscript Author Manuscript Biochim Biophys Acta. Author manuscript; available in PMC 2017 May 01.


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Author Manuscript Author Manuscript Author Manuscript

Figure 1.

Author Manuscript

Schematic representation of peroxisome biogenesis pathways 1[ PMPs are both translated in the cytosol on free ribosomes or on the ER-associated ribosomes and incorporated posttranslationally or cotranslationally in the ER-membrane. The ER-translocon, Sec61, is important for the PMP incorporation process. Similarly, TA-proteins are imported into the ER-membrane via the GET pathway. 2[ Subsequently, an intra-ER sorting process targets the PMPs to respective pER domains. 3[ The PMPs are exported from the ER in vesicular carriers and require Pex19. Pex16 is also important for the exit of Pex3 and other PMPs from the ER in mammalian cells. 4[ The vesicular carriers containing complementary sets of PMPs fuse to assemble the importomer complex. The fusion process requires peroxins Pex1 and Pex6. 5[ This assembly enables the nascent peroxisome to import matrix proteins and become a metabolically active organelle. 6[ Type II PMPs are imported directly into the peroxisome membrane with the assistance of Pex3 and Pex19 (Inset). 7[ The e novo route



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involving the ER also contributes to the cellular peroxisome population, thus sustaining the growth and division pathway and substituting for it when it is blocked or impaired. Using this backup pathway, in mutant cells (such as pex3Δ and pex19Δ cells) lacking functional pre-existing peroxisomes, reintroduction of the missing gene will form peroxisomes e novo and the new peroxisomes that are generated will restart growth and division pathway.



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Author Manuscript Author Manuscript Figure 2.


Comparative account of the ER-derived route of peroxisome biogenesis in yeast, mammalian cells and plants: The contribution of the ER to peroxisome biogenesis is evident in different model organisms, however, there are distinct mechanistic aspects specific for each system. In yeast, more PMPs have been documented to enter the ER than in any other system (section 3.1). As noted in Figure 1, these PMPs are sorted to the pER compartment. Recent results show that components of the peroxisomal RING and docking subcomplex proteins are sorted into distinct carriers (ppVs) [35]. In mammalian cells, such ER-derived ppVs are not yet described, however, pre-peroxisomal structures have been identified that sequentially import membrane proteins when peroxisome biogenesis is restored upon introduction of the missing protein in mutant cells [70] (section 3.2). In addition, these preperoxisomal structures presumably fuse with the pre-existing peroxisomes to replenish the growing and dividing peroxisomes. Various morphological studies depicted a close association of peroxisomes with specialized smooth-ER extensions also known as lamellarER [104,105]. This is distinct from the pER structures, which are associated with rough-ER. Pex13 is sorted to these lamellar-ER extensions and later detaches from the ER to form tubular structures. Once formed, additional integral membrane proteins are recruited leading to the assembly of the peroxisomal protein import apparatus. The formation of functional peroxisomes is concluded with the import of matrix enzymes and other Type II PMPs from the cytosol. Plants are the least studied system and trafficking of just a handful set of PMPs, including APX, Pex16 and Pex10, via the ER has been examined (section 3.3). However, neither ER-import requirements nor any peroxisome-specific vesicular carriers have been Biochim Biophys Acta. Author manuscript; available in PMC 2017 May 01.


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precisely defined in plants. None of the plant peroxin mutants have resulted in the complete loss of peroxisomes, thus the only proven pathway for peroxisome biogenesis is the growth and division model. A few morphological studies have noted that a non-ER, non-Golgi and non-peroxisomal compartment in plant cells receives Pex16 in cells recovering from BFA. This compartment is termed as ERPIC (ER-peroxisomes intermediate compartment), which in most ways act as a pre-peroxisomal compartment and is expected to be an intermediate in the formation of downstream peroxisomes. However, no concrete evidence exists delineating the functional significance of this compartment. In addition, during unique peroxisome to ER retrograde trafficking pathway has been identified in plants. Upon TBSV infection, plant peroxisomes undergo inward vesiculation resulting in the formation of pMBVs that traffics PMPs back to the pER. The mechanism by which these vesicle traffic PMPs back to the ER is unclear.


De novo peroxisome biogenesis revisited.

Glycosome biogenesis in trypanosomes and the de novo dilemma.

Peroxisome biogenesis in yeast.

Peroxisome biogenesis in mammalian cells.

Peroxisome biogenesis in Saccharomyces cerevisiae.

Somatoparaphrenia: evolving theories and concepts.

Hypertension and ischemia: evolving concepts.

Peroxisome biogenesis and function in Hansenula polymorpha.

Evolving Concepts of Asthma.

Evolving concepts in migraine.

Idiopathic pulmonary fibrosis: evolving concepts.

Evolving concepts of tumor heterogeneity.

The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis.

Lifelong learning: Established concepts and evolving values.

Eosinophilic esophagitis: current understanding and evolving concepts.

Peroxisome biogenesis in Hansenula polymorpha: different mutations in genes, essential for peroxisome biogenesis, cause different peroxisomal mutant phenotypes.

De novo AVM formation.

Novel variation and de novo mutation rates in population-wide de novo assembled Danish trios.

Translocation Mongolism Arising de Novo?

E3 ubiquitin ligase SP1 regulates peroxisome biogenesis in Arabidopsis.

De novo selection of oncogenes.

Multiple cerebral de novo aneurysms.

de novo computational enzyme design.

De-novo histoid hansen cases.