Building New Models for Peroxisome Biogenesis

Electron micrograph showing the cytochemical localization of uricase in a peroxisome in mature cowpea nodules (from Fig. 3 in Webb and Newcomb, 1987; reprinted with permission). The intense staining of the peroxisome in the uninfected cell is the result of treatment with 3,3′-diaminobenzidine plus urate. Peroxisomes in infected cells of mature cowpea nodules do not contain uricase and do not show this dark staining (Webb and Newcomb, 1987). × 13,000; bar = 1 μm. M, Mitochondrion; N, nucleus; P, peroxisome; St, starch-containing plastid. Peroxisomes are unique organelles whose physiological functions vary depending on the type of tissue in which they are found, and also on the metabolic and developmental state of the organism. Plants have several classes of peroxisomes, each present at different stages in the life cycle and each sequestering different enzymes specific for the physiological role of the organelle. Leaves, roots, and young seedlings possess a distinct class of peroxisomes specialized to perform tissue-specific functions (Olsen and Harada, 1995). Leaf-type peroxisomes contain enzymes that are needed for photorespiration. Young seedlings have glyoxysomes, which provide nutrition for growth through the action of the glyoxylate cycle, until the plant becomes autotrophic. Peroxisomes in uninfected cells of root nodules (Fig. 1) contain enzymes, including uricase and allantoinase, that assist in nitrogen metabolism (Webb and Newcomb, 1987). The interconversion between peroxisome classes appears to be transcriptionally regulated; the mRNA expressed at a given time in a given tissue determines which enzymes are sequestered in the organelle (for review, see Olsen, 1998). Enzymes involved in jasmonic acid biosynthesis (Stintzi and Browse, 2000) and the metabolism of reactive oxygen species recently have been discovered in plants (Corpas et al., 2001), suggesting that peroxisomes play important roles in various biotic and abiotic stress responses. Peroxisomes have received considerable attention in mammalian systems primarily because of the diseases associated with defective peroxisomes. The first disease in humans to be linked to peroxisome biogenesis was Zellweger (cerebrohepatorenal) syndrome (Goldfischer et al., 1973). People afflicted with Zellweger syndrome lack functional peroxisomes and experience neonatal seizures, psychomotor retardation, and abnormalities of neuronal migration in the brain. Children born with Zellweger syndrome have a mean survival of 6 to 7 months. More than a dozen peroxisomal disorders have been characterized in humans. Most involve single enzyme defects where a protein is missing or unable to be normally imported into the peroxisome (Masters and Crane, 1995). Until recently, only a few mutants in plant peroxisome function had been identified. Screens for photorespiratory mutants yielded several lines that were characterized biochemically (for review, see Somerville and Ogren, 1982). The molecular cause of the photorespiratorysat mutant is a single nucleotide substitution in the Ala:glyoxylate aminotransferase1 gene (Liepman and Olsen, 2001). Defects in β-oxidation disrupt lipid mobilization (Hayashi et al., 1998; Lange and Graham, 2000) and inflorescence development in Arabidopsis (Richmond and Bleecker, 1999). Mutant Arabidopsis seedlings lacking the glyoxylate cycle enzyme isocitrate lyase are also unable to break down storage lipids (Eastmond et al., 2000). It is surprising that Arabidopsis mutants resistant to an endogenous auxin (Zolman et al., 2000), mutants lacking fatty acid oxidation (Hayashi et al., 2000), and mutants with defective seedling oil bodies (Lin et al., 1999) were each shown to be caused by mutations in genes required for peroxisome biogenesis. These mutants may allow researchers to apply genetic approaches to understand peroxisome assembly and function in plants. Unlike mitochondria and chloroplasts, peroxisomes do not possess their own genome. All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and imported posttranslationally into the organelle. That peroxisomal matrix proteins are synthesized in the cytoplasm suggests the need for peroxisome-specific mechanisms to target and translocate these proteins across the peroxisomal membrane and into the matrix (for review, see Olsen, 1998). Based on research from other posttranslational protein import systems such as mitochondria and chloroplasts, a very simple model was proposed for how peroxisomes compartmentalize all the enzymes they need. In this model, a matrix protein is transported across the peroxisomal membrane following a signal-mediated interaction with a specific membrane receptor. It is not surprising that this naive model of peroxisomal protein import has been revised dramatically as new experimental data has emerged. This update will consider how the models proposed for peroxisome biogenesis have necessarily changed to accommodate new information. Although one might expect that all proteins going into peroxisomes would have the same targeting signal, nearly all matrix-localized enzymes contain one of two peroxisome-targeting signals (PTSs), each of which is necessary and sufficient to direct proteins from the cytosol into peroxisomes (for review, see Olsen, 1998; Subramani et al., 2000). Examples of PTSs in plant proteins are shown in TableI. PTS1, the first PTS identified, is a carboxyl terminal tripeptide consisting of the three amino acids Ser-Lys-Leu, or related variants (Subramani et al., 2000). It is interesting to note that though PTS1s are conserved across all eukaryotes, plant PTS1s apparently exhibit more variability in sequence compared with accepted signals in animals (Mullen et al., 1997a). Even though it may seem quite remarkable that such a short sequence motif can control import into a specific organelle, the majority of peroxisomal matrix proteins carry a PTS1 signal. Typical plant peroxisomal proteins with the indicated physiological functions and targeting signals PTS1 at extreme carboxyl terminus of protein. PTS2 in amino terminal portion of protein; Xn indicates the no. of residues between the amino terminus of the protein and the signal. TMD, Transmembrane domain. Typical plant peroxisomal proteins with the indicated physiological functions and targeting signals PTS1 at extreme carboxyl terminus of protein. PTS2 in amino terminal portion of protein; Xn indicates the no. of residues between the amino terminus of the protein and the signal. TMD, Transmembrane domain. The second type of PTS (PTS2) is present within the first 20 to 30 amino acids at the amino terminus and has a loosely conserved sequence of nine amino acids (Flynn et al., 1998; Olsen, 1998). In plants and mammals, the PTS2 signal is cleaved after the protein arrives in the peroxisomal matrix. Compared with the number of proteins that have PTS1 signals, few PTS2 proteins have been identified. The most well-characterized PTS2 protein is thiolase, a fatty acid β-oxidation enzyme found in plants, animals, and yeasts. Other known plant PTS2 proteins include isozymes of malate dehydrogenase, citrate synthase, amine oxidase, and Asp aminotransferase. In fact, there appear to be more PTS2 proteins in plants than in other eukaryotes. Several peroxisomal matrix proteins contain neither a recognizable PTS1 nor a PTS2 consensus sequence (Subramani et al., 2000). Internal targeting signals for these proteins have not been fully characterized, however, and no specific interactions between internal signals and receptors have been reported. Peroxisomal membrane protein (PMP) targeting and insertion requires other types of targeting signals that will be discussed later in this review. The discovery of multiple targeting signals for peroxisomal protein transport led to the prediction that multiple receptors interact with the matrix proteins and define separate import pathways. Support for the existence of multiple peroxisomal import pathways was provided by genetic screens for peroxisomal protein import mutants in yeasts. Several distinct classes of mutants were found: those that were defective in the import of PTS1 proteins, those that were defective in the import of PTS2 proteins, and those that were defective in import of both (for review, see Erdmann et al., 1997). The proteins initially identified in these screens to be required for peroxisome biogenesis have been termed peroxins (pex). More than 20 peroxins currently have been described; homologs for most, but not all, of the peroxins can be identified in the Arabidopsis genome. Separate receptors direct the import of peroxisomal matrix proteins with different targeting signals. Proteins possessing a carboxyl terminal PTS1 interact with a specific PTS1 receptor, Pex5p. Proteins possessing an amino-terminal PTS2, on the other hand, interact with a distinct PTS2 receptor, Pex7p. The soluble receptors converge on common docking complexes on the membrane after binding to their matrix protein cargo. The discovery of cytosolic receptors for peroxisomal matrix proteins was surprising because, at the time, most researchers expected the import pathways to resemble protein import into mitochondria and chloroplasts more closely. By now, it is widely accepted that Pex5p is the cytosolic receptor for PTS1 proteins and Pex7p is the PTS2 protein receptor. In Arabidopsis, PEX5 encodes a protein that contains seven tetratricopeptide repeat domains in its carboxyl terminus (Brickner et al., 1998); Pex5p has also been cloned from tobacco (Kragler et al., 1998) and watermelon (Wimmer et al., 1998). Proteins that possess a PTS1 tripeptide have been shown to interact with the TPR domains of Pex5p, whereas those lacking a PTS1 targeting signal do not (Terlecky et al., 1995). PEX7 encodes a protein composed almost entirely of WD-40 (β-transducin related) repeats. Pex7p, the PTS2 receptor, binds specifically to PTS2 signals and interacts with thiolase in both two-hybrid and co-immunoprecipitation experiments. In plants, Pex7p has also been cloned from Arabidopsis (Schumann et al., 1999). Pex5p and Pex7p also appear to interact directly or indirectly with each other during import—adding another layer of complexity to the import model. Two isoforms of Pex5p have been identified in mammals. Both serve as a PTS1 protein receptor, but the longer form has also been shown to interact directly with Pex7p (Otera et al., 2000). Yeasts appear to have only the short isoform, consistent with the absence of evidence of PTS1 and PTS2 pathway interactions. Plant Pex5p is most similar to the longer mammalian isoform, suggesting that the two soluble receptors will interact with each other during matrix protein import. It is not yet clear whether the two pathways converge at the membrane, as shown in Figure 2, or in the cytosol before docking on the membrane, as suggested in Figure 4. The exact localization of these two receptors has been somewhat controversial. Depending upon the species and the experimental techniques used, Pex5p and Pex7p have each been localized to the cytoplasm, to the peroxisomal membrane, and to the matrix (Olsen, 1998;Subramani et al., 2000). Because the predicted amino acid sequences of the two receptor proteins do not reveal obvious transmembrane domains, Pex5p and Pex7p do not appear to be typical integral membrane-bound receptors. Most researchers now believe that Pex5p and Pex7p are primarily cytosolic proteins that bind polypeptides destined for the peroxisomal matrix and that accompany their cargo to the peroxisomal membrane. Model of PTS1 protein import showing Pex5p shuttling between the cytosol and peroxisome. The PTS1 receptor, Pex5p, binds to the PTS1 protein in the cytoplasm. The Pex5p-PTS1 protein complex then docks on a membrane-bound peroxin complex, comprising at least Pex13p, Pex14p, and Pex17p. The Pex5p-PTS1 protein complex translocates across the membrane. Dissociation of the complex occurs in the matrix of the organelle; Pex5p releases its protein cargo in the matrix and may be recycled back to the cytoplasm through an entirely hypothetical export channel. An alternative model that is not shown proposes that Pex5p dissociates from the PTS1 protein and is released from the membrane before the PTS1 protein is translocated across the membrane. Summary model for peroxisomal matrix protein import. In the cytosol, a PTS1 protein interacts with Pex5p and a PTS2 protein interacts with Pex7p. The matrix protein receptor complexes may assemble directly in the cytosol or during docking on the membrane complex; each pathway can also occur independently. Pex7p binds to Pex14p; Pex5p binds to Pex13p and Pex14p. After docking, the receptor-cargo complexes are translocated across the membrane, through an entirely uncharacterized transport apparatus. One possibility is shown: translocation may occur through a channel formed by some or all of the zinc-binding proteins, Pex2p, Pex10p, and Pex12p. Inside the matrix, but close to the membrane, the PTS1 and PTS2 protein cargoes dissociate from their respective receptors. The PTS2 protein-targeting signal is cleaved off. Pex5p and Pex7p are recycled out of the peroxisome, perhaps through the zinc-binding proteins or another unidentified export complex (as shown in Fig. 3), for subsequent rounds of matrix protein binding and import. It was once thought that because there are separate PTS1 and PTS2 receptors, there might also be separate PTS1 and PTS2 import channels. Most of the evidence now supports a model in which both receptors dock at the same protein complex on the peroxisome membrane, as shown in Figure 4. Yeast peroxisome mutants again helped identify the players involved in recruiting the receptors to the membrane and then keeping them there during import. Besides the two receptors, at least six peroxins, Pex13p, Pex14p, Pex17p, Pex18p, Pex20p, and Pex21p, have been implicated primarily in the process of peroxisomal matrix protein import, though the latter three proteins listed may be specific for yeast peroxisomes (see below). Three membrane-bound proteins, Pex13p, Pex14p, and Pex17p, interact to form a docking site for the cytosolic receptors on the organelle membrane (Fig. 3). Pex14p is a cytosolic facing, peripheral membrane protein that interacts with both Pex5p and Pex7p, usually as a dimer.pex14 mutants are unable to import both PTS1 and PTS2 proteins (see references in Subramani et al., 2000). Based on these results, Pex14p is thought to be the primary membrane docking protein where the two import pathways might converge. Pex17p is another cytosolic facing, peripheral membrane protein that interacts directly with Pex14p, and therefore indirectly with Pex5p and Pex7p. Mutations in PEX17 inhibit both the PTS1- and PTS2- dependent import pathways (Huhse et al., 1998). Pex14p has been further shown to interact with Pex13p, an integral PMP that has an SH3 (Src homology 3) domain and is oriented in the membrane such that both the carboxyl terminus and the amino terminus face the cytosol. Pex14p and Pex13p are known to interact via the carboxyl-terminal SH3 domain because mutations in this domain result in the inactivation of Pex13p function (Elgersma et al., 1996). Pex13p is required for Pex14p to function at the membrane. Like pex14and pex17 mutants, pex13 mutants are unable to import both PTS1 and PTS2 proteins. The SH3 domain of Pex13p has also been shown to interact with the PTS1 receptor, Pex5p, but it is less clear whether Pex13p interacts with the PTS2 receptor, Pex7p (Erdmann and Blobel, 1996; Girzalsky et al., 1999). Pex18p and Pex21p are structurally and functionally related peroxins that interact with Pex7p and seem to be required for Pex7p's targeting to the membrane. In Saccharomyces cerevisiae cells lacking both Pex18p and Pex21p, Pex7p remained cytosolic and PTS2 targeting was completely abolished (Purdue et al., 1998). The peroxin Pex20p was discovered in the yeast Yarrowia lipolytica. Y. lipolytica has no known Pex7p-like PTS2 receptor. Instead, Pex20p acts as the PTS2 receptor in this organism (Titorenko et al., 1998). Pex20p exhibits no homology to Pex7p and binds PTS2 proteins independently of the amino-terminal targeting signal. Plant homologs of Pex18p, Pex20p, and Pex21p have not yet been identified, even after thorough searches through the completed Arabidopsis genome. Most of the components required for peroxisome matrix protein import have not been fully characterized in plants, in yeast, or in animals. In addition to the six major proteins discussed above, other peroxins including Pex2p, Pex4p, Pex10p, Pex12p, Pex19p, and Pex22p have even less well-defined roles in peroxisomal protein import. As indicated in Figure 4, Pex2p, Pex10p, and Pex12p are integral PMPs with a zinc RING finger motif at the carboxyl terminus. Their role in matrix protein import is completely unknown, but it is reasonable to propose a role for zinc binding to these proteins as a regulatory mechanism during Pex5p binding and subsequent PTS1 protein import. The zinc-binding domain of Pex12p has been shown to interact with both Pex5p and Pex10p (Okumoto et al., 2000). Recent evidence indicates that PTS1 protein import is stimulated by the addition of zinc (Terlecky and Fransen, 2000). In addition, protein import complex intermediates may provide powerful tools for identifying other components of the translocation machinery in plants (Pool et al., 1998). The peroxisome membrane forms an important barrier between the organelle and the rest of the cell. This membrane has been poorly characterized, mainly because of the difficulties encountered in isolating membranes free from contamination. The peroxisome membrane has a relatively low phospholipid to protein ratio and low levels of cholesterol. Although it is thinner than most other single membrane-bound its is to the membrane and Peroxisomal membranes contain that are to including and (for a thorough of peroxisomal see 2000). is relatively known the mechanisms for membrane proteins to peroxisomes, even though of the peroxins characterized to are As integral membrane proteins signals other than PTS1 or PTS2 for targeting to the organelle et al., 2001). consensus sequences for membrane protein targeting have been These signals, termed membrane PTSs seem to the carboxyl terminus of the membrane proteins, to at least one transmembrane domain (see The most common of the few characterized of a of or amino acids et al., and 2000). The role of the in targeting membrane proteins to the peroxisome. PMPs are synthesized on soluble in the cytosol. of the PMPs and appears to be directly into the membrane from the cytosol. The PMPs peroxisomal Pex2p, and are first localized to a domain on the from which a might with other or with peroxisomes mature the membrane proteins to the mature peroxisome. peroxisomes by evidence of matrix protein through the has yet been It is not clear whether matrix proteins be imported into That on and where the membrane translocation complex As as six separate classes of peroxisome have been from the yeast Y. each class appears to be import for distinct of matrix proteins (Titorenko and 2001). It has also been proposed that the PMP from the into recognizable peroxisomes by the insertion of other proteins into the membrane et al., 1997). This in would allow the assembly of the matrix protein import the subsequent import of matrix proteins, and the of mature peroxisomes. An Arabidopsis of has been identified (Lin et al., 1999). It is interesting that it has been implicated in protein and oil but its role in peroxisome biogenesis has not been have on peroxisome-specific components required for organelle however, components that are involved in peroxisome biogenesis. usually proteins, are required for protein import into organelles and peroxisomes appear to be no such as class of bind to proteins during and after to the protein from and to the protein in an This is usually necessary for the protein to be to the membrane. however, have the to import proteins that are fully in or even to (for review, see and Olsen, 1999). are needed for peroxisomal protein import fully proteins can the from that protein can be imported in import of proteins is more and Olsen, 1998). cytosolic have been shown to inhibit the import of peroxisomal matrix and membrane proteins et al., and Olsen, 1998; et al., 1999). have also to peroxisomal proteins, suggesting that and peroxisomal proteins interact role might be to the matrix protein in an perhaps by with other in the of a cytosolic complex similar to the one required for the of the receptor in et al., or by the proteins in a loosely may protein import into plant peroxisomes at several and may perform different Two isoforms have been identified in the matrix of peroxisomes and 2000). is to the but its function is (Corpas and 1997). is also evidence the of other than in peroxisomal protein import in plants. are that and both a membrane-bound and a cytosolic have been identified in plants et al., and 2000). of the which transported polypeptides into after translocation across the and has been to in the peroxisome matrix et al., 1995). The first of in organelle biogenesis from showing that inhibit the in import of PTS1 proteins and Olsen, 1998). function in a complex with to mammalian receptors for binding and et al., 2001). also the assembly of protein The role of in peroxisomal import therefore be to the cytosolic PTS receptors for binding to peroxisomal proteins. In fact, the PTS1 receptor, Pex5p, and have been shown to interact in co-immunoprecipitation et al., 2001). This because has a single binding site for tetratricopeptide repeat domains et al., and Pex5p from Arabidopsis has seven tetratricopeptide that the carboxyl terminal of the protein (Brickner et al., 1998). In an might control the state of the proteins or of the import complex to membrane apparently in the form of is required for import of proteins into peroxisomes. was also shown to be sufficient to the in import of at least one PTS1 protein into peroxisomes (Brickner and Olsen, 1998). has been implicated in the and function of complexes et al., 2001). The exact role of in peroxisomal protein import has not been but it is reasonable to that some is required for the It is also that or binds to one or more of the components of the translocation apparatus. the models to peroxisome biogenesis have in more complex than The models include multiple targeting signals to direct proteins to the peroxisome, multiple cytosolic receptors that interact with the proteins, membrane components that interact with the receptors and proteins, other membrane components that have roles in that import, and complex targeting mechanisms to membrane proteins. however, peroxisome biogenesis that from researchers mammals, and plants will provide with new to of these complex and that will cause to to and new models for peroxisome biogenesis.