Ubiquitination on the Peroxisomal Membrane for Protein Transport in Plants

Abstract

Peroxisomes are ubiquitous organelles present in most eukaryotic cells that have important biological functions related to fatty acid metabolism and detoxification of reactive oxygen species. Disruption of peroxisomal function affects the survival of cells and organisms. Peroxisomes do not have their own genome, and peroxisomal proteins are encoded in the nuclear genome. Therefore, efficient and accurate posttranslational transport of peroxisomal proteins is necessary to maintain peroxisomal function. In mammals, yeast, and plants, many factors involved in protein transport to peroxisomes have been identified and their molecular mechanisms elucidated. In plants, analysis of Arabidopsis peroxisome mutants, such as apem (aberrant peroxisome morphology) and ibr (indole-3-butyric acid-response), enabled the identification of the factors mediating protein transport. Of these, several proteins, such as PEX1 (Peroxin 1), PEX2, PEX4, PEX6, PEX10, PEX12, PEX22, and APEM9, constitute the ubiquitin system on the peroxisomal membrane, and loss of function of each protein reduces the efficiency of protein transport to peroxisomes. This ubiquitin-dependent peroxisomal protein transport system is also present in yeast and mammalian cells and is an example of a type of ubiquitin modification that serves as a signaling tag rather than as a tag for protein degradation. This chapter introduces the factors involved in protein transport to the peroxisome via the ubiquitin system in plants and outlines their functions.

1. Introduction

Peroxisomes are ubiquitous organelles present in most eukaryotic cells; they were discovered in the early 1960s as organelles measuring approximately 1.0 μm in diameter [1]. Peroxisomes proliferate by division of preexisting peroxisomes, and abnormal peroxisomes that are no longer needed or have been oxidized are degraded via a peroxisome-specific autophagy process called pexophagy [2, 3, 4, 5, 6, 7]. Peroxisomes are involved in various biological functions. Of these, fatty acid metabolism and detoxification of reactive oxygen species are functions common to many organisms, whereas bile acid biosynthesis and alcohol metabolism are peroxisome functions specific to mammals and yeast, respectively. Photorespiration, which salvages byproducts of photosynthesis, and biosynthesis of phytohormones such as jasmonic acid and auxin are plant peroxisome-specific functions. Peroxisomes do not have their own genome and all peroxisomal proteins are encoded in the nuclear genome; therefore, posttranslational transport must be efficient and accurate to ensure peroxisomal protein function. Numerous studies in a variety of organisms have identified the processes involved in peroxisome biogenesis, such as protein transport, proliferation, differentiation, and inheritance, and have elucidated the molecular mechanisms [8, 9, 10, 11, 12]. In particular, the analysis of peroxisome biogenesis factors called peroxins (PEXs) revealed the molecular mechanism underlying peroxisome biogenesis including protein transport [8, 9, 10, 11, 12].

Peroxisomal protein transport can be divided into the following stages (Figures 1 and 2). (i) Cytosolic receptors recognize and bind peroxisomal proteins, and direct them to the docking site on the peroxisomal membrane. (ii) The peroxisomal protein-receptor complex associates with the docking complex and passes through the peroxisomal membrane. (iii) After the peroxisomal protein dissociates from the receptor, the receptor is returned to the cytosol to engage in the next transport cycle. Studies in mammals, yeast, and plants identified the factors involved in each stage: PEX5 and PEX7 in stage (i), PEX13 and PEX14 in stage (ii), and PEX1, PEX2, PEX4, PEX6, PEX10, PEX12, PEX22, and APEM9/Pex15p/PEX26 in stage (iii) (Figures 1 and 2) [9, 10, 11, 12]. Various protein modification systems involving PEXs are required for efficient protein transport to peroxisomes. For example, stage (iii) requires ubiquitination for receptors, such as PEX5, on the peroxisomal membrane, and several proteins necessary for ubiquitination have been identified and characterized at the molecular level [13, 14, 15, 16]. Defects in individual components of this ubiquitin system decrease the efficiency of protein transport to peroxisomes, leading to abnormalities such as dwarfism in plants, growth defects in yeast, and genetic diseases in humans [10, 14, 15, 16].

Figure 1.

Protein transport to peroxisomes in plants. Most peroxisomal proteins contain PTS1 or PTS2. PTS1 and PTS2 are recognized by the cytosolic receptors PEX5 and PEX7, respectively, and are directed to a docking complex containing PEX13 and PEX14 on the peroxisomal membrane. The peroxisomal protein-receptor complex passes through the peroxisomal membrane, and the peroxisomal protein and receptor are released. PEX5 and PEX7 return to the cytosol to engage in the next round of protein transport. Ubiquitination may be required for recycling the receptor in the cytosol, and proteins targeted for ubiquitination on the peroxisomal membrane are involved in the export apparatus. PEX4 has UBC activity and functions as an E2 enzyme, and PEX2, PEX10, and PEX12 are RING-finger proteins that function as E3 ligases. PEX1 and PEX6 are AAA+ ATPases involved in the dissociation of the receptor from the membrane. APEM9 is a functional homolog of yeast Pex15p and mammalian PEX26 that tethers the PEX1-PEX6 complex to the peroxisomal membrane. PEX22 is responsible for tethering PEX4 to the peroxisomal membrane. Whether the peroxisomal protein-receptor complex fully enters the peroxisome in plants remains unclear. Ubiquitination of PEX5 has been detected in yeast and animals, but not in plants.

Figure 2.

Outline of PTS1- and PTS2-mediated transport in yeast and mammals. (A) In Pichia pastoris, proteins containing PTS1 and PTS2 are recognized by Pex5p and Pex7p, respectively, and they are transported independently. A co-receptor is required for efficient PTS2 transport. Pex18p and Pex21p in Saccharomyces cerevisiae and Pex20p in Pichia pastors are coreceptors of Pex7p. Pex5p and Pex20p are ubiquitinated and transported from peroxisomes to the cytosol. (B) In mammals, alternative splicing of the PEX5 gene generates two variants, PEX5S and PEX5L. PEX5S is involved in the transport of PTS1, whereas PEX5L binds to PEX7 to facilitate the efficient transport of PTS2. As in yeast, PEX5 is ubiquitinated and transported from peroxisomes to the cytosol. Proteins homologous to PEX4 are not present in mammalian genomes, and instead UbcH5a/b/c catalyzes the ubiquitination of PEX5. AWP1 functions as an adaptor for PEX6 and recognizes ubiquitinated PEX5. Pex15p, PEX26, and APEM9 in Figure 1 have the same function, namely, interacting with PEX6 to tether the PEX1- PEX6 complex to the peroxisomal membrane. However, the amino acid sequence identity between the three proteins is low except for the transmembrane region.

Ubiquitination on the peroxisomal membrane does not lead to protein degradation but has a signaling function. Ubiquitination is a posttranslational protein modification in which a ubiquitin protein of approximately 9 kDa binds to a target protein. The binding of ubiquitin to amino acids occurs through a three-step enzymatic cascade. A ubiquitin-activating enzyme (E1) activates ubiquitin via an AMP-bound intermediate. Ubiquitin then binds to the active-site cysteine in a ubiquitin-conjugating enzyme (E2) and is transferred to the target protein by a ubiquitin-protein ligase (E3). The E3 ligase can be a RING-type enzyme that binds to both the substrate and a ubiquitin-charged E2 enzyme and directly transfers the ubiquitin moiety to the target protein, or a thioester-bound HECT-type and RBR-type E3 that forms ubiquitin-E3 intermediates. The regulatory mechanism of the ubiquitination process has been described in other chapters and reviews. This chapter focuses on the ubiquitin system involved in protein transport to peroxisomes based on findings in Arabidopsis thaliana.

2. Components of the ubiquitin system on the peroxisomal membrane in plants

There are two types of protein transport to peroxisomes, peroxisome targeting signal (PTS) 1-dependent protein transport and PTS2-dependent protein transport. PTS1, which consists of 3–4 amino acid residues located at the C-terminal end of each protein, is recognized by the cytosolic receptor PEX5. On the other hand, PTS2-containing proteins are translated as larger precursors containing the extension sequence; PTS2 is present in the N-terminal extension sequence and is recognized by its receptor, PEX7. This extension sequence is cleaved after import into peroxisomes, and the PTS2-containing proteins become the mature form [17, 18, 19]. However, different species require different PEXs or use the same PEX in different ways. In yeast, the PTS1- and PTS2-dependent transport systems function independently from each other (Figure 2a). PTS2-dependent transport requires additional proteins that act as co-receptors to facilitate the import process. These include Pex18p and Pex21p in S. cerevisiae [20] and Pex20p in Pichia pastoris [21]; Pex5p and Pex20p are ubiquitinated and recycled to the cytosol [21, 22]. In mammals, two types of PEX5, PEX5L, and PEX5S are generated by alternative splicing from a single gene. PEX5L is involved in both PTS1- and PTS2-dependent protein transport, whereas PEX5S transports only PTS1 proteins (Figure 2b). As in yeast, ubiquitination of PEX5 is required for receptor export [23, 24], and the AWP1 (associated with PRK1) protein stimulates PEX5 export by interacting with PEX6 [25]. In plants, on the other hand, PTS1- and PTS2-dependent protein transports are interdependent; proteins containing PTS1 and PTS2 are transported as a single complex (Figure 1) [10, 11, 26]. Thus, although there are differences in the way PEXs are used by different organisms, they all share a common need for the ubiquitin system in protein transport. As detailed below, proteins involved in the ubiquitin system are present on the peroxisomal membrane and are required for protein transport to peroxisomes [10, 14, 15, 16]. Of these, PEX4 possesses ubiquitin-conjugating (UBC) activity [15, 16, 27, 28, 29, 30], and PEX2, PEX10, and PEX12 function as ubiquitin ligases [31, 32, 33]. PEX1 and PEX6, which belong to the ATPases associated with diverse cellular activities (AAA+) family (AAA ATPases), are responsible for the energy required for the receptor to dissociate from the peroxisomal membrane [34, 35]. PEX22 and Pex15p/PEX26/APEM9 tether PEX4 and the PEX1-PEX6 complex to the peroxisomal membrane, respectively [36, 37].

This section outlines the function of PEX proteins involved in ubiquitination on plant peroxisomal membranes, based primarily on the analysis of Arabidopsis mutants.

2.1 Advances in plant peroxisome research achieved using Arabidopsis mutants

Analysis of Arabidopsis mutants such as apem and ibr led to dramatic advances in plant peroxisome research. The apem mutants were isolated from a pool of ethyl methanesulfonate (EMS)-mutagenized Arabidopsis seeds expressing the peroxisome marker GFP-PTS1 as the parent plant. The apem mutants show alterations in the pattern of GFP fluorescence as follows: (i) elongated peroxisomes, (ii) enlarged peroxisomes, (iii) mislocalization of the GFP-PTS1 protein to the cytosol, and (iv) altered distribution of GFP-labeled peroxisomes [15, 38, 39, 40, 41, 42]. Among apem mutants, apem2, apem4, apem7, and apem9 show GFP fluorescence not only in peroxisomes but also in the cytosol, indicating that the efficiency of PTS1-dependent protein transport to peroxisomes is reduced (Figure 3) [15, 39, 41]. Furthermore, PTS2-dependent protein transport is disturbed in these mutants [15, 39, 41]. Identification of APEM genes and analysis of their gene products revealed that APEM2, APEM4, APEM7, and APEM9 encode PEX13, PEX12, PEX4, and the functional homolog of Pex15p/PEX26, respectively [15, 39, 41]. PEX4 and PEX12 function as a UBC enzyme and a ubiquitin ligase, respectively, and Pex15p/PEX26 is responsible for tethering the AAA ATPase complex, PEX1-PEX6, to the peroxisomal membrane [15, 39, 41]. APEM4/PEX12, APEM7/PEX4, and APEM9/Pex15p/PEX26 thus constitute a ubiquitin system on the peroxisomal membrane, which suggests that ubiquitination on the peroxisomal membrane is required for efficient protein transport to peroxisomes.

Figure 3.

GFP fluorescence patterns in root cells of the parent plant, GFP-PTS1, and apem mutants expressing the peroxisome marker GFP-PTS1. GFP is transported via a PTS1-dependent pathway, and peroxisomes are visualized as spherical structures. In the apem4, apem7, and apem9 mutants, GFP is detected in the cytosol in addition to peroxisomes. These phenotypes indicate a defect in PTS1-dependent protein transport. Bar, 50 μm.

A screening based on peroxisome function contributed to the identification of several peroxisome-related genes encoding proteins associated with the ubiquitin system. Mutants were isolated based on the conversion of indole-3-butyric acid (IBA) to indole-3-acetic acid (IAA), an endogenous auxin, by peroxisomal fatty acid β-oxidation, an exclusive function of peroxisomes in plants. The Arabidopsis pex1, pex4, pex6, pex12, and pex26 mutants were isolated because they exhibited abnormal conversion of IBA to IAA [30, 43, 44, 45]. A pex1-1 mutant was isolated as a suppressor that restores metabolic and physiological defects in the pex6 mutant [46]. Arabidopsis PEX2 was identified as a suppressor of the de-etiolated 1-1 (det1-1) mutant, which is defective in photomorphogenesis [47], and a different allele of pex6 was identified as a pfl (persistent GFP-ICL fluorescence) mutant showing a different pattern of GFP fluorescence from the parent plant expressing the GFP-ICL fusion gene [48].

Many plant PEX mutants with T-DNA insertions are lethal because they show complete protein dysfunction. However, the apem, ibr, and pfl mutants have a milder loss of function and thus provide a tool to analyze the function of the causative gene product because these are EMS-induced nucleotide substitution mutants. Analysis of these EMS-treated Arabidopsis mutants led to research into the role of peroxisomal membrane ubiquitin in plants. Below is an overview of the components of the ubiquitin system that regulate protein transport to peroxisomes.

2.2 PEX4 functions as a ubiquitin-conjugating enzyme

PEX4 has only been identified in yeast (also called Ubc10p in S.cerevisiae) and plants, and proteins with amino acid sequence homology to PEX4 have not been found in mammals. PEX4 genes were originally identified in a screening of mutants showing growth defects when cultured under carbon-limited conditions in yeast [27, 28, 29]. In plants, the Arabidopsis pex4 mutant was isolated separately as the pex4-1 and pex4-2 mutants and the apem7 mutant [15, 30, 49]. PEX4 proteins in yeast and plants have a putative active-site cysteine residue that is essential for forming a thioester bond with ubiquitin. The UBC activity of PEX4 was identified only in yeast, where it was reported to function as an E2 enzyme in the ubiquitination process [27]. A recent analysis using rabbit reticulocyte lysate and in vitro ubiquitin assays with recombinant wild-type PEX4 and mutant PEX4, which has an amino acid substitution at position 123 from proline to leucine, suggests that both PEX4 forms have UBC activities, although the activity and specificity of mutant-type PEX4 are reduced [15, 16]. However, the thioester bond between mutant PEX4 and ubiquitin is cleaved in vitro under reducing conditions, such as the addition of β-mercaptoethanol, whereas in vivo cleavage is incomplete [15]. This may be due to an alteration in the interaction of PEX4 with other factors on the peroxisomal membrane and/or in the conformation of PEX4 itself caused by the apem7/pex4 mutation. Although Arabidopsis PEX4 is difficult to purify as a soluble protein, it can be purified by expressing it as a PEX4-PEX22 fusion gene and can then be crystallized and have its structure elucidated (PEX22 is described in the next section) [16]. Crystallographic analysis indicates that the pex4-1 mutation site is located on a loop near the active-site cleft and it is thought to alter the residue immediately following the “gateway residue” involved in regulating ubiquitin access to the active site [16]. In other words, the proximity of the pex4-1 mutation site to the active-site cysteine likely affects the UBC activity of mutant-type PEX4.

2.3 PEX22 functions in tethering PEX4 to the peroxisome membrane

Similar to PEX4, which has not been found in mammals, PEX22 has not been found in mammalian genomes. Analysis of the apem7 and pex4-1 mutants indicated that PEX4 localizes to the peroxisomal membrane [15, 30]. PEX4 does not have an obvious transmembrane domain, suggesting that it requires other factors for localization to the peroxisomal membrane. In yeast, Pex22p was identified as a factor that anchors Pex4p to the peroxisomal membrane [36, 37], and the cytosolic domain of Pex22p is involved in Pex4p activity [50]. In plants, Arabidopsis PEX22 was identified from the Arabidopsis cDNA library in a yeast two-hybrid analysis, which showed that it interacts with Arabidopsis PEX4 [30]. Arabidopsis PEX22 has high amino acid sequence homology with other plant homologs, but only 6–12% sequence identity with the yeast Pex22p protein; however, there are similarities in the sequence of the N-terminal transmembrane domain, the molecular weight, and the topology of the proteins [30]. S. cerevisiae Pex22p does not interact with Pichia pastoris Pex4p [36]. In addition, although coexpression of the Arabidopsis PEX4 and PEX22 genes complements the defects in the yeast pex4 pex22 double mutant, PEX4 or PEX22 alone do not restore the phenotype of the corresponding yeast mutant, indicating that Arabidopsis PEX4 does not interact with yeast Pex22p and vice versa [30].

The crystal structure of Pex4p bound to the soluble portion of Pex22p has been reported in S. cerevisiae, and binding of Pex4p to Pex22p increases the ability of Pex4p to transfer ubiquitin to its substrates [51, 52]. In plants, the Arabidopsis PEX4-PEX22 complex was crystallized and its structure was elucidated, as described in the previous section [16]. Comparison with yeast Pex22p shows that PEX22 maintains a similar Rossmann fold structure, with several salt bridges and long unstructured tethers in positions that contribute to the specificity of PEX22 for PEX4 and the ubiquitination of peroxisomal membrane targets away from PEX4 without dissociation from PEX22 [16]. These results indicate that the interaction between PEX4 and PEX22 has been acquired with species specificity.

2.4 PEX2, PEX10, and PEX12 function as ubiquitin ligases

There are three RING-finger domain-containing ubiquitin ligases on the peroxisomal membrane, PEX2, PEX10, and PEX12, and they have been identified in various organisms including plants [9, 10, 14]. They function as E3 ubiquitin ligases during ubiquitination on the peroxisomal membrane [14, 31, 32]. In plants, Arabidopsis PEX2, PEX10, and PEX12 possess in vitro ubiquitin ligase activity [33]. These three RING E3 ligases depend on each other for stability, and deletion or partial loss of function of one component induces instability of the other two components [44, 53, 54, 55].

Arabidopsis PEX2 was originally identified in an analysis of a suppressor of the det1-1 mutant [47]. DET1 is a global repressor of photomorphogenesis, and the det1 mutant grows in the dark with the phenotype of a light-grown plant [56]. The pex2 mutant, originally known as ted3 for reversal of the det phenotype, was identified as one of the ted mutants [47]. The ted3 mutant has an amino acid substitution at position 275 from valine to methionine in the RING-finger domain that suppresses the phenotypic defect of the det mutant [47]. Overexpression of the PEX2 RING domain in the det1 mutant also partially suppresses the det1 phenotype, suggesting that peroxisomes are involved in photomorphogenesis [57]. The Arabidopsis pex2-1 mutant was isolated by screening for abnormal degradation of the peroxisomal matrix protein [55]. Knockdown of the PEX2 gene by RNA interference [8] showed that the pex2-1 mutant is defective in protein transport to peroxisomes [55].

The Arabidopsis PEX10 gene was identified as a gene encoding a protein with 47–56% sequence similarity to the product of the PEX10 gene from mammals and yeast [58, 59]. Analysis of T-DNA insertion mutants of the PEX10 gene showed that loss of PEX10 affects normal embryo development and viability [58, 59]. Furthermore, the pex10-2 mutant was isolated as one of the ibr mutants, and further analysis showed that the pex2-1 pex10-2 double mutant has more severe growth defects than the respective single mutants, indicating that PEX2 and PEX10 function in a coordinated manner [55]. The pex10 mutant, which was generated by RNAi interference, shows defects in ER morphology and cuticular wax accumulation [60].

For PEX12, a T-DNA knockout mutant of PEX12 and a pex12 partial loss-of-function mutant were generated using RNA interference, and the phenotypes of individual plants were analyzed [61]. These mutants show developmental arrest, plant growth inhibition, and reduced lethality in early embryogenesis [61]. In addition, the apem4 (previously known as apm4) mutant, which shows a decrease of both PTS1- and PTS2-dependent protein transport to peroxisomes, was isolated as having a mutation in the PEX12 gene [39]. The apem4 and other Arabidopsis pex12 mutants show defects in PTS1- and PTS2-dependent protein transport [39, 44], suggesting that the ubiquitin ligase activity of PEX12 is required for efficient protein transport to peroxisomes. Indeed, Arabidopsis PEX12 can bind to PEX7 [62], suggesting that PEX12 is involved in receptor recycling from peroxisomes to the cytosol, as discussed below. The apem4 and pex12-1 mutants are characterized by the replacement of the arginine at position 170 by lysine and that of glutamic acid at position 171 by lysine, respectively [39, 44]. The mechanism by which these mutations affect the ligase activity and conformation of PEX12 remains to be elucidated.

2.5 PEX1 and PEX6 function as AAA ATPases

PEX1 and PEX6 are type 2 AAA+ ATPases containing two conserved ATPase domains, D1 and D2, preceded by an N-terminal domain that interacts with substrates and adaptor proteins. They assemble to form a heterohexamer in which nucleotide-binding pockets form at the interface between adjacent subunits in the ring [34, 35]. In mammals and yeasts, PEX1 and PEX6 form a heterohexamer that functions as an unfoldase for the retrotranslocation of PEX5 from the peroxisomal membrane [63, 64, 65]. Arabidopsis PEX1, an ortholog of S. cerevisiae Pex1p, was cloned and its cDNA was used to generate transgenic A. thaliana harboring luciferase under the control of the PEX1 promoter. The transgenic plants showed that PEX1 gene expression is upregulated in response to physiological levels of hydrogen peroxide, wounds, and pathogen infection [66]. The Arabidopsis pex1 mutant pex1-1 was isolated because of its ability to suppress the pex6-1 mutant phenotype (Arabidopsis pex6 mutants are described in the next paragraph) [67]. The pex1-1 mutant has an amino acid substitution from glutamic acid to lysine at amino acid 748, near the AAA1 domain, and this mutation ameliorates the metabolic and physiological defects of pex6-1 [67]. Two additional pex1 mutants, pex1-2 and pex1-3, were isolated from a pool of ibr mutants [45]. The mutations in both pex1-2 and pex1-3 are located in the AAA2 domain. The pex1-2 mutant has reduced PEX1 and PEX6 levels, suggesting that PEX1 enhances PEX6 stability and vice versa [45]. In the pex1-3 mutant, peroxisomes are larger than those in the wild type, and GFP-PTS1 is detected in the cytosol as well as in peroxisomes; it also displays embryonic lethality [45]. These findings indicate that PEX1 is involved in peroxisome biogenesis and protein transport and that peroxisome-related functions are required for embryogenesis in plants.

The Arabidopsis pex6-1 mutant was isolated as one of the ibr mutants [43]. The pex6 mutation results in an amino acid substitution from arginine to glutamine at position 766, which is conserved in plants, humans, and yeast. Introduction of human PEX6 into the Arabidopsis pex6 mutant functionally complements its phenotype [43]. The pex6-2 mutant was subsequently isolated as one of the pfl (persistent GFP-ICL fluorescence) mutants [48]. Isocitrate lyase (ICL) is a peroxisomal protein that is transported via a PTS1-dependent pathway. The pfl mutants show a GFP fluorescence pattern that differs from that of the parent plants expressing the GFP-ICL fusion gene [48]. Novel alleles of the pex6 mutant, pex6-3 and pex6-4, were also isolated from the pool of ibr mutants [46]. Of the four pex6 alleles, pex6-2 has a mutation near the N-terminus of AAA1, and pex6-1, pex6-3, and pex6-4 have mutations at or near the AAA2 domain. The mutant phenotypes affecting plant growth and peroxisomal protein transport are similar to those of the wild type in pex6-2, whereas pex6-1, pex6-3, and pex6-4 show more severe defects, indicating the importance of the AAA2 domain [46]. In PEX1 described in the previous paragraph, the importance of the AAA2 domain is suggested by the fact that of the three Arabidopsis pex1 mutants, pex1-2 and pex1-3, were isolated as mutants with mutations in the PEX1 AAA2 domain [45, 46].

2.6 Pex15p/PEX26/APEM9 tether the PEX1-PEX6 complex to peroxisome membranes

The ubiquitination of proteins as a signal for protein transport requires energy from ATP hydrolysis on the peroxisomal membrane, which is provided by the PEX1-PEX6 complex. However, PEX1 and PEX6 do not have a membrane association domain. In yeast and mammals, Pex15p and PEX26, which are tail-anchored proteins, are located on the peroxisomal membrane and are involved in recruiting the PEX1-PEX6 complex to the peroxisomal membrane from the cytosol [68, 69]. The homolog of Pex15p/PEX26 in plants was identified after the isolation of the apem9-1 mutant and based on the sequence of APEM9, the T-DNA insertion mutant was identified as apem9-2. Similar to apem4 and apem7, the apem9 mutant phenotype is characterized by the accumulation of peroxisomal proteins in the cytosol and impaired PTS1- and PTS2-dependent transport (Figure 3) [41]. APEM9 encodes a protein of unknown function that is found only in plant genomes; however, hydropathy profile analysis of APEM9 suggests that it is similar to Pex15p and PEX26 [41]. Although APEM9, Pex15p, and PEX26 have low amino acid sequence similarity except in the transmembrane domain, their secondary structures are similar [41]. Analyses suggest that APEM9 tethers the PEX1-PEX6 complex to the peroxisomal membrane, and the apem9-1 mutation disrupts the peroxisomal localization of APEM9 and the PEX1-PEX6 complex because the mutation is located in the transmembrane domain of APEM9; this suggests that the role of APEM9 is the same as that of Pex15p in yeast and PEX26 in mammals [41]. The Arabidopsis dayu mutant is characterized by abnormal pollen maturation and germination [70]. DAYU encodes APEM9. DAYU/APEM9 binds to PEX13, a factor of the import complex (Figure 1), suggesting that DAYU/APEM9 is involved in the import of both PTS1- and PTS2-containing proteins in addition to mediating receptor export by the ubiquitin system [70]. Moreover, the pex26-1 mutant, which was isolated as one of the ibr mutants [46], shows a more severe phenotype than apem9-1. This is likely because the apem9-1 mutation causes an amino acid substitution, whereas the pex26-1 mutation triggers a splicing defect that prevents translation of the C-terminal transmembrane domain of PEX26, which in turn prevents the PEX1-PEX6 complex from localizing to the peroxisomal membrane [46].

3. Molecular mechanism underlying the role of ubiquitination in peroxisomal protein transport

Regulatory mechanisms for the ubiquitination of peroxisomal proteins as a signal for transport exist in mammals, yeast, and plants. However, their components and usage differ slightly, and there is no unified model that can be adapted to all organisms. For example, a protein with amino acid sequence homology to PEX4 has not been identified in animals; instead, UbcH5a/b/c act as UBC enzymes [23]. As shown in Figures 1 and 2, peroxisomal protein import systems differ between animals, yeast, and plants, suggesting that the mechanism underlying receptor export, the latter step of protein transport triggered by the ubiquitin signal, may also vary from organism to organism. This section outlines the similarities and differences in the regulatory mechanisms according to the components of the ubiquitin system introduced in Section 2, the proteins targeted for ubiquitination, and the protein degradation that occurs when ubiquitin signaling is disrupted by another type of ubiquitination on the peroxisomal membrane.

3.1 Similarities and differences in the proteins involved in the ubiquitin system in animals, yeast, and plants

The presence of three RING-finger ubiquitin ligases, PEX2, PEX10, and PEX12, as E3 enzymes is common to yeast, mammals, and plants. Another common mechanism among the three organisms is the formation of a heterohexamer between PEX1 and PEX6 with AAA ATPase activity; this PEX1-PEX6 complex is recruited to the peroxisomal membrane by a tail-anchored protein: Pex15p in yeast, PEX26 in mammals, and APEM9 in plants [41, 68, 69]. However, the three tail-anchored proteins show low amino acid sequence identity. Indeed, although APEM9 could not be identified by sequence comparison with Pex15p or PEX26, it was identified as a functional homolog of Pex15p and PEX26 in plants because its secondary structure is similar to that of Pex15p and Pex26, especially in the location of the predicted single transmembrane domain and the hydrophilic region consisting of 35–60 residues immediately before the transmembrane domain [41].

There are significant differences between mammals and yeast/plants with regard to the UBC enzyme, namely, the E2 proteins of the ubiquitin system. In yeast and plants, Pex4p and PEX4 serve as E2 enzymes. However, proteins with a similar amino acid sequence to that of Pex4p or PEX4 have not been found in mammals. Instead, three UbcH5-family proteins, UbcH5a, UbcH5b, and UbcH5c, act as UBC enzymes [23]. As discussed in the next section, the peroxisomal protein transport-related target of the UbcH5 family is PEX5; however, the UbcH5 family also targets proteins unrelated to peroxisomes, such as IκBα and BRCA1 [71, 72]. In yeast and plants, Pex4p and PEX4 are UBCs involved only in peroxisomal protein transport, whereas, in animals, UBCs involved in other biological processes also function in peroxisomal protein transport. Proteins corresponding to Pex22p in yeast and PEX22 in plants have not been identified in animals. This is not surprising in animals, where homologs of Pex4p and PEX4 do not exist, and considering that Pex22p and PEX22 are responsible for tethering Pex4p and PEX4 to the peroxisomal membrane.

3.2 Proteins targeted for ubiquitination involved in peroxisomal protein transport

In yeast and mammals, the PTS1 receptor PEX5 is recycled from the peroxisomal membrane to the cytosol by monoubiquitination of the conserved cysteine near the N-terminus [24, 32, 73, 74, 75]. However, the final target proteins remain undefined in plants. Although Arabidopsis PEX5 also has a cysteine residue at position 13, there is no direct evidence indicating that PEX5 is recycled via monoubiquitination of the cysteine residue in plants. However, although PEX5 is detected in both the cytosolic and membrane fractions, membrane-associated PEX5 is slightly larger than soluble PEX5 [15]. Furthermore, membrane-associated PEX5 is increased in apem and ibr mutants, which are characterized by defects in the factors involved in ubiquitination related to peroxisome protein transport [15, 16, 39, 43, 67]. In addition, the mutant phenotype associated with protein transport defects can be partially rescued by overexpression of the PEX5 gene [43, 48, 67], supporting that PEX5 may be a target for ubiquitination in plants.

In yeast, the PTS2 receptor Pex7p requires a co-receptor such as Pex18p, Pex20p, and Pex21p to function in peroxisomal protein transport. The N-terminal cysteine residue of Pex18p is monoubiquitinated during the recycling process of Pex7p and Pex18p [76]. In mammals, PEX5L, one of the longer splice variants alternatively produced from the PEX5 gene, functions as a co-receptor for PEX7. The conserved cysteine at the N-terminus of PEX5L is also monoubiquitinated, resulting in the return of both PEX5L and PEX7 to the cytosol from the peroxisomes [77]. As shown in Figure 1, in plants, PEX5 and PEX7 are transported as a single complex after recognizing PTS1 and PTS2, respectively, suggesting that if PEX5 is ubiquitinated and recycled to the cytosol, PEX7 is also returned, similar to PEX5L and PEX7 in mammals.

3.3 A different ubiquitination process on the peroxisomal membrane

Abnormal peroxisomal protein transport is associated with a type of ubiquitination on the peroxisomal membrane that differs from the monoubiquitination of PEX5 and Pex18p. In yeast, inhibition of monoubiquitination-dependent protein transport induces the polyubiquitination of Pex5p [22, 78, 79]. In this case, ubiquitin is conjugated to the 2 N-terminal lysine residues, but not to the cysteine residue, by another UBC enzyme, Ubc4p (and the partially redundant UBCs, Ubc1p, and Ubc5p) rather than Pex4p [22, 78, 79]. Polyubiquitinated Pex5p is then degraded by the 26S proteasome. With regard to PTS2-dependent protein transport, Pex18p and Pex20p, co-receptors for Pex7p, are lysine polyubiquitinated and degraded [76, 80]. In mammals, dysfunctional PEX7 is degraded via a ubiquitin-dependent pathway for PEX7 quality control [81].

In plants, the ubiquitination-dependent degradation of PEX5 and PEX7 has not been identified to date. However, Arabidopsis PEX4 catalyzes the formation of lysine48-linked ubiquitin chains [16], suggesting that PEX4 is involved in the degradation as well as recycling of PEX5. Dominant suppressors of KAR 2a, DSK2a and DSK2b, are ubiquitin-binding receptor proteins that specifically bind to the RING domain of PEX2 and PEX12, suggesting that the E3 ligases associated with DSK2s are involved in ubiquitination on the peroxisomal membrane [33].

Suppressors of plastid protein import locus 1 (SP1) and SP1-like 1 (SPL1) are RING-type ubiquitin ligases that are located on the peroxisomal membrane [82, 83]. SP1 physically interacts with PEX13 and PEX14, components of the import machinery, and with PEX2, thereby contributing to the destabilization of the protein import machinery [82]. However, SP1 was originally discovered as a component of the chloroplast protein import machinery and is involved in the degradation of the chloroplast protein transport complex by the ubiquitin-proteasome system [84]. SP1 localizes to peroxisomes and mitochondria as well as chloroplasts [84, 85]. SPL1 is the closest homolog of SP1, and similar to SP1, localizes to chloroplasts, mitochondria, and peroxisomes; however, SP1 shows a stronger peroxisomal localization than SPL1 [83]. SPL1 antagonizes SP1 function during peroxisomal protein import, suggesting that the balance of SP1 and SPL1 activities regulates the protein import machinery [83]. Thus, ubiquitination is required for the import of peroxisomal proteins as well as the export of receptors in the protein transport process. A protein with amino acid sequence similarity to SP1 and SPL1 is the human mitochondrial anchored protein ligase (MAPL) protein [86]. MAPL has not been identified in yeast. In mitochondria, it functions in mitochondrial fission by regulating dynamin-associated protein 1. Whether it functions in peroxisomes such as SP1 and SPL1 as potential regulators of docking proteins remains unknown [86]. Peroxisomes interact with multiple organelles, such as chloroplasts and mitochondria, to form metabolic pathways. Therefore, E3 ligases localized to multiple organelles may play an important role in coordinately regulating processes related to organelle biogenesis, such as protein transport, by recognizing interactions between organelles.

4. Conclusions

Proteins involved in ubiquitination on the peroxisomal membrane have been identified in plants, and their molecular mechanisms have been elucidated. As described above, the regulatory mechanisms in plants are similar to those in animals and yeast, but with species-specific fine-tuning that may have been acquired during evolution. Disruption of this ubiquitin system can lead to abnormalities in plant processes such as growth and embryogenesis, similar to the inability of yeast to use carbon sources for growth and the occurrence of genetic diseases in humans.

Several issues related to the role of ubiquitin signaling in peroxisomal protein transport in plants remain to be addressed. Proteins targeted for ubiquitination in this system remain to be identified in plants. Whether PEX5 is ubiquitinated as in plants is a subject for further study. In yeast and animals, ubiquitinated PEX5 is released from the peroxisomal membrane to the cytosol, where it is deubiquitinated before engaging in the following transport cycle. The enzyme responsible for PEX5 deubiquitination is ubiquitin-specific protease 15 (Ubp15p) in yeast and ubiquitin-specific protease 9X (USP9X) in mammals [87, 88]; however, the deubiquitinase in plants has not been identified. Moreover, the mechanism underlying polyubiquitination-induced protein degradation on the peroxisomal membrane, when protein transport fails, is not as well understood in plants as in yeast and animals, including whether a similar mechanism exists in the first place. Next-generation sequencing and mass spectrometry will contribute to the identification of ubiquitin-related factors and ubiquitinated proteins in various nonmodel plants. Analysis using a variety of plants, including the liverwort Marchantia polymorpha, mentioned in the next section, will facilitate the elucidation of the molecular mechanisms of the ubiquitin system in plants and help clarify the issues discussed above.

5. Future perspectives

The current knowledge of peroxisomal protein transport in land plants presented in this chapter is based primarily on studies using Arabidopsis. However, these findings may not apply to all plants. In yeast, although the ubiquitin system involved in peroxisomal protein transport has been preserved through the course of evolution, the factors involved and their functions have changed; for example, proteins from S. cerevisiae do not complement their corresponding mutants in Pichia pastoris. Similarly, ubiquitin factors may have diverged during plant evolution. In this regard, the liverwort M. polymorpha is a good experimental plant for the study of peroxisomes. M.polymorpha is an early diverging land plant and thus retains features of ancestral land plants. The genome of M. polymorpha has been determined and compiled into genome databases (https://marchantia.info) [89]. Methods for genetic transformation, as well as various resources such as vectors suitable for M. polymorpha and genome editing by CRISPR/Cas9 have been established, which has enabled the collection of information on different mutants [90]. One of the advantages of using M. polymorpha is that it has a low gene duplication rate. The details of this process are beyond the scope of this chapter, and several reviews are available [12, 90, 91]. We have successfully visualized the peroxisome of M. polymorpha using a fusion gene of PTS1 or PTS2 and a fluorescent protein [12, 92]. The results show that both PTS1- and PTS2-dependent protein transport have been present from the beginning of the evolution of land plants. Bioinformatics analysis detected the presence of genes orthologous to Arabidopsis PEX1, PEX2, PEX4, PEX6, PEX10, PEX12, PEX22, and APEM9 in the genome of M. polymorpha [12], indicating that the ubiquitin system is involved in peroxisomal protein transport in M. polymorpha [the PEX genes we have identified, including the ubiquitin system-related PEX genes, are registered in MarpolBase (https://marchantia.info)]. However, the molecular mechanisms underlying the roles of the gene products need to be analyzed. Genome editing of ubiquitin-related genes identified by bioinformatics analysis of M. polymorpha transgenic plants with visualized peroxisomes will help clarify the ubiquitin signaling pathways regulating protein transport to peroxisomes in land plants.

Acknowledgments

We thank the staff at the Model Organisms Facility, Trans-Omics Facility, Optics, and Imaging Facility at the NIBB Trans-Scale Biology Center for technical support. We are also grateful to Ms. Chihiro Nakamori, Masami Araki, and Azusa Matsuda for supporting the experiments and taking care of the plants as technical staff in our laboratory.

This work was supported in part by JSPS KAKENHI (Grant Numbers 20059035, 22112523, 17 K07457, and 20 K06711).

Conflicts of interest

The authors have no conflicts of interest to declare.

Author contribution

Shoji Mano, Conceptualization; Investigation, Writing manuscript, Project administration, Funding acquisition

Kazumi Hikino, Investigation

Masatake Kanai, Investigation, Writing manuscript

Abbreviations

apem	aberrant peroxisome morphology
AWP1	associated with PRK1
BRCA1	breast cancer susceptibility gene 1
CRISPR/Cas9	clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins
dayu	dau, after the Chinese legendary hero
det1	de-etiolated 1
DSK2a	dominant suppressor of KAR 2a
EMS	ethyl methanesulfonate
GFP	green fluorescent protein
HECT	homologous to the E6-AP carboxyl terminus
IAA	indole-3-acetic acid
IBA	indole-3-butyric acid
ibr	iba-response
ICL	isocitrate lyase
IκBα	NF-κB inhibitor α
MAPL	mitochondrial anchored protein ligase
pfl	persistent GFP-ICL fluorescence
RBR	RING-in-between-RING
PEX	peroxin
PTS	peroxisome targeting signal
RING	really interesting new gene
SP1	plastid protein import locus 1
ted	reversal of the det phenotype
UBC	ubiquitin conjugating
Ubp15p	ubiquitin-specific protease 15
USP9X	ubiquitin-specific protease 9X
AAA ATPase	ATPases associated with diverse cellular activities