Peroxisomal Protein Import Research Articles

Two Independent Pathways Traffic the Intraperoxisomal Peroxin PpPex8p into Peroxisomes: Mechanism and Evolutionary Implications

Among peroxins involved in peroxisome biogenesis, only Pex8p is predominantly intraperoxisomal at steady state. Pex8p is necessary for peroxisomal matrix protein import via the PTS1 and PTS2 pathways. It is proposed to bridge two peroxisomal membrane subcomplexes comprised of the docking (Pex13p, Pex14p, Pex17p) and RING (Pex2p, Pex10p, Pex12p) peroxins and is also implicated in cargo release of PTS1 proteins in the matrix. We show that Pichia pastoris Pex8p (PpPex8p) enters the peroxisome matrix using two redundant pathways in a Pex14p-dependent, but Pex2p-independent, manner, showing that the intact importomer and RING subcomplex are not required for its import. One pathway depends on the TPR motifs in Pex5p, the C-terminal PTS1 sequence (AKL) in PpPex8p, and the intraperoxisomal presence of this peroxin. The alternative pathway uses the PTS2 receptor, Pex7p, its accessory protein, Pex20p, and a putative PTS2 motif in PpPex8p, but does not require intraperoxisomal PpPex8p. Pex20p interaction with PpPex8p is independent of Pex7p, but the interaction of PpPex8p with Pex7p requires Pex20p. These data suggest a direct interaction between PpPex8p and Pex20p. Our studies shed light on the mechanism and evolution of the dual import pathways for PpPex8p.

Identification and Functional Characterization ofArabidopsisPEROXIN4 and the Interacting Protein PEROXIN22[W

Peroxins are genetically defined as proteins necessary for peroxisome biogenesis. By screening for reduced response to indole-3-butyric acid, which is metabolized to active auxin in peroxisomes, we isolated an Arabidopsis thaliana peroxin4 (pex4) mutant. This mutant displays sucrose-dependent seedling development and reduced lateral root production, characteristics of plant peroxisome malfunction. We used yeast two-hybrid analysis to determine that PEX4, an apparent ubiquitin-conjugating enzyme, interacts with a previously unidentified Arabidopsis protein, PEX22. A pex4 pex22 double mutant enhanced pex4 defects, confirming that PEX22 is a peroxin. Expression of both Arabidopsis genes together complemented yeast pex4 or pex22 mutant defects, whereas expression of either gene individually failed to rescue the corresponding yeast mutant. Therefore, it is likely that the Arabidopsis proteins can function similarly to the yeast PEX4-PEX22 complex, with PEX4 ubiquitinating substrates and PEX22 tethering PEX4 to the peroxisome. However, the severe sucrose dependence of the pex4 pex22 mutant is not accompanied by correspondingly strong defects in peroxisomal matrix protein import, suggesting that this peroxin pair may have novel plant targets in addition to those important in fungi. Isocitrate lyase is stabilized in pex4 pex22, indicating that PEX4 and PEX22 may be important during the remodeling of peroxisome matrix contents as glyoxysomes transition to leaf peroxisomes.

Protein translocation machineries: How organelles bring in matrix proteins

Eukaryotic cells contain several thousands of proteins that have to be accurately partitioned over the components of the cytoplasm (cytosol or any of the known organelles) to allow proper cell function. To this end, various specific topogenic signals have been designed as well as highly selective protein translocation machineries that ensure that each newly synthesized polypeptide reaches its correct subcellular destination or, in case of secretory proteins, is exported to the cell exterior. This contribution gives an overview regarding the principles of the main examples of polypeptide sorting and translocation, with emphasis on the function of cofactor binding in peroxisomal matrix protein import.

Yeast Pex14p Possesses Two Functionally Distinct Pex5p and One Pex7p Binding Sites

Current evidence favors a cycling receptor model for the import of peroxisomal matrix proteins. The yeast Pex14 protein together with Pex13p and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this cycle with both soluble import receptors Pex5p and Pex7p. In a first step of a structure-function analysis of Saccharomyces cerevisiae Pex14p, we mapped its binding sites with both receptors. Using the yeast two-hybrid system and pull-down assays, we showed that Pex5p directly interacts with two separate regions of ScPex14p, amino acid residues 1-58 and 235-308. The latter binding site at the C terminus of ScPex14p overlaps with a binding site of Pex7p at amino acid residues 235-325. The functional assessment of these two binding sites of ScPex14p with the peroxisomal targeting signal receptors indicates that they have distinct roles. Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in pex14delta cells expressing the Pex14-(59-341)-p fragment; however, it did not lead to a pex phenotype. In contrast, truncation of the C-terminal 106 amino acids of ScPex14p completely blocked this process. On the basis of these and other published data, we propose that the C terminus of Pex14p contains the actual docking site and discuss the possibility that the N terminus could be involved in a Pex5p-Pex14p association inside the peroxisomal membrane.

Peroxisome protein import: some answers, more questions

Recent advances in the study of plant peroxisomes are shedding new light on the importance of these organelles for plant development, and are revealing similarities and differences in peroxisome protein import pathways between plants, animals and fungi. For example, the import of matrix proteins that carry the PTS1 and PTS2 targeting signals is coupled in plants as it is in mammals, whereas these import pathways are separate in fungi. The expression of a human peroxisomal ATPase partially rescues the equivalent Arabidopsis mutant. Ubiquitination might play a role in receptor recycling in Saccharomyces cerevisiae and exciting progress is being made through studies of the targeting of membrane proteins.

Caudothalamic groove cysts in Zellweger syndrome

The cerebro-hepato-renal syndrome of Zellweger is the most severe peroxisome biogenesis disorder. Zellweger syndrome is caused by a disturbance in the peroxisomal protein import machinery and leads to multiple organ defects and death usually within the first year of life. Here we report a 3-month-old girl with Zellweger syndrome who was found to have cysts in the caudothalamic groove on cranial magnetic resonance imaging.

Biogenesis of peroxisomes

Genetic and proteomic approaches have led to the identification of 32 proteins, collectively called peroxins, which are required for the biogenesis of peroxisomes. Some are responsible for the division and inheritance of peroxisomes; however, most peroxins have been implicated in the topogenesis of peroxisomal proteins. Peroxisomal membrane and matrix proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu Rev Cell Biol1, 489-530). Progress has been made in the elucidation of how these proteins are targeted to the organelle. In addition, the understanding of the composition of the peroxisomal import apparatus and the order of events taking place during the cascade of peroxisomal protein import has increased significantly. However, our knowledge on the basic principles of peroxisomal membrane protein insertion or translocation of peroxisomal matrix proteins across the peroxisomal membrane is rather limited. The latter is of particular interest as the peroxisomal import machinery accommodates folded, even oligomeric, proteins, which distinguishes this apparatus from the well characterized translocons of other organelles. Furthermore, the origin of the peroxisomal membrane is still enigmatic. Recent observations suggest the existence of two classes of peroxisomal membrane proteins. Newly synthesized class I proteins are directly targeted to and inserted into the peroxisomal membrane, while class II proteins reach their final destination via the endoplasmic reticulum or a subcompartment thereof, which would be in accord with the idea that the peroxisomal membrane might be derived from the endoplasmic reticulum.

Identification of a Novel, Intraperoxisomal Pex14-Binding Site in Pex13: Association of Pex13 with the Docking Complex Is Essential for Peroxisomal Matrix Protein Import

The peroxisomal docking complex is a key component of the import machinery for matrix proteins. The core protein of this complex, Pex14, is thought to represent the initial docking site for the import receptors Pex5 and Pex7. Associated with this complex is a fraction of Pex13, another essential component of the import machinery. Here we demonstrate that Pex13 directly binds Pex14 not only via its SH3 domain but also via a novel intraperoxisomal site. Furthermore, we demonstrate that Pex5 also contributes to the association of Pex13 with Pex14. Peroxisome function was affected only mildly by mutations within the novel Pex14 interaction site of Pex13 or by the non-Pex13-interacting mutant Pex5(W204A). However, when these constructs were tested in combination, PTS1-dependent import and growth on oleic acid were severely compromised. When the SH3 domain-mediated interaction of Pex13 with Pex14 was blocked on top of that, PTS2-dependent matrix protein import was completely compromised and Pex13 was no longer copurified with the docking complex. We conclude that the association of Pex13 with Pex14 is an essential step in peroxisomal protein import that is enabled by two direct interactions and by one that is mediated by Pex5, a result which indicates a novel, receptor-independent function of Pex5.

Differential Contribution of Two Peroxisomal Protein Receptors to the Maintenance of Peroxisomal Functions in Arabidopsis

Peroxisomes in higher plant cells are known to differentiate in function depending on the cell type. Because of the functional differentiation, plant peroxisomes are subdivided into several classes, such as glyoxysomes and leaf peroxisomes. These peroxisomal functions are maintained by import of newly synthesized proteins containing one of two peroxisomal targeting signals known as PTS1 and PTS2. These targeting signals are known to be recognized by the cytosolic receptors, Pex5p and Pex7p, respectively. To demonstrate the contribution of Pex5p and Pex7p to the maintenance of peroxisomal functions in plants, double-stranded RNA constructs were introduced into the genome of Arabidopsis thaliana. Expression of the PEX5 and PEX7 genes was efficiently reduced by the double-stranded RNA-mediated interference in the transgenic Arabidopsis. The Pex5p-deficient Arabidopsis showed reduced activities for both glyoxysomal and leaf peroxisomal functions. An identical phenotype was observed in a transgenic Arabidopsis overexpressing functionally defective Pex5p. In contrast, the Pex7p-deficient Arabidopsis showed reduced activity for glyoxysomal function but not for leaf peroxisomal function. Analyses of peroxisomal protein import in the transgenic Arabidopsis revealed that Pex5p was involved in import of both PTS1-containing proteins and PTS2-containing proteins, whereas Pex7p contributed to the import of only PTS2-containing proteins. Overall, the results indicated that Pex5p and Pex7p play different roles in the maintenance of glyoxysomal and leaf peroxisomal functions in plants.

The Saccharomyces cerevisiae Peroxisomal Import Receptor Pex5p Is Monoubiquitinated in Wild Type Cells

Pex5p is a mobile receptor for peroxisomal targeting signal type I-containing proteins that cycles between the cytoplasm and the peroxisome. Here we show that Pex5p is a stable protein that is monoubiquitinated in wild type cells. By making use of mutants defective in vacuolar or proteasomal degradation we demonstrate that monoubiquitinated Pex5p is not a breakdown intermediate of either system. Monoubiquitinated Pex5p is localized to peroxisomes, and ubiquitination requires the presence of functional docking and RING finger complexes, which suggests that it is a late event in peroxisomal matrix protein import. In pex1, pex4, pex6, pex15, and pex22 mutants, all of which are blocked in the terminal steps of peroxisomal matrix protein import, polyubiquitinated forms of Pex5p accumulate, ubiquitination being dependent on the ubiquitin-conjugating enzyme Ubc4p. However, Ubc4p is not required for Pex5p ubiquitination in wild type cells, and cells lacking Ubc4p are not affected in peroxisome biogenesis. These results indicate that Pex5p monoubiquitination in wild type cells serves to regulate rather than to degrade Pex5p, which is supported by the observed stability of Pex5p. We propose that Pex5p monoubiquitination in wild type cells is required for the recycling of Pex5p from the peroxisome, whereas Ubc4p-mediated polyubiquitination of Pex5p in mutants blocked in the terminal steps of peroxisomal matrix protein import may function as a disposal mechanism for Pex5p when it gets stuck in the import pathway.

Obstruction of polyubiquitination affects PTS1 peroxisomal matrix protein import

Pex4p is an ubiquitin-conjugating enzyme that functions at a late stage of peroxisomal matrix protein import. Here we show that in the methylotrophic yeast Hansenula polymorpha production of a mutant form of ubiquitin (Ub K48R) has a dramatic effect on PTS1 matrix protein import. This effect was not observed in cells lacking Pex4p, in which the peroxisome biogenesis defect was largely suppressed. These findings provide the first indication that the function of Pex4p in matrix protein import involves polyubiquitination. We also demonstrate that the production of Ub K48R in H. polymorpha results in enhanced Pex5p degradation. A similar observation was made in cells in which the PEX4 gene was deleted. We demonstrate that in both strains Pex5p degradation was due to ubiquitination and subsequent degradation by the proteasome. This process appeared to be dependent on a conserved lysine residue in the N-terminus of Pex5p (Lys21) and was prevented in a Pex5p K21R mutant. We speculate that the degradation of Pex5p by the proteasome is important to remove receptor molecules that are stuck at a late stage of the Pex5p-mediated protein import pathway.

Ubiquitination of the Peroxisomal Targeting Signal Type 1 Receptor, Pex5p, Suggests the Presence of a Quality Control Mechanism during Peroxisomal Matrix Protein Import

PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.

NovelPEX1 coding mutations and 5′ UTR regulatory polymorphisms

Zellweger syndrome and its milder variants--neonatal adrenoleukodystrophy and infantile Refsum disease--comprise a clinical continuum of diseases referred to as the Zellweger spectrum. Mutations in the PEX1 gene, which consists of 24 exons and encodes a AAA ATPase protein required for peroxisomal protein import, account for approximately two-thirds of the known Zellweger spectrum patient mutations. In this paper, we report on four novel PEX1 mutations and two polymorphisms in an Australasian cohort. Two of the mutations--c.1108_1109insA and c.2391_2392delTC--that lead to the introduction of a premature termination codon in exons 5 and 14, respectively, are associated with the severe Zellweger phenotype. One patient with a milder disease phenotype was a compound heterozygote for two missense mutations (I989T and R998Q), both affecting amino acids in the second, C-terminal AAA domain of the protein. PTS1 protein import levels in cultured skin fibroblasts from this patient were almost 20% of normal control levels. We have also characterized two co-segregating polymorphisms in the 5' UTR of the PEX1 gene. Based on reporter assays, the c.-137T>C polymorphism leads to reduced PEX1 expression, whereas the c.-53C>G polymorphism leads to increased expression. When present together, these regulatory polymorphisms lead to near-normal PEX1 expression. Altered PEX1 expression due to the presence of either the c.-137T>C or the c.-53C>G variant could impact on residual PEX1 function if another co-allelic mutation was present which did not completely abolish PEX1 function. It also follows that the presence of polymorphisms in the PEX1 promoter region could have implications for patients with mutations in other PEX proteins known to interact with PEX1, such as PEX6. Thus, although not deleterious in control individuals, these polymorphisms could contribute to phenotypic heterogeneity among Zellweger spectrum patients.

Ubiquitination of the peroxisomal import receptor Pex5p.

Proteins harbouring a peroxisomal targeting signal of type 1 (PTS1) are recognized by the import receptor Pex5p in the cytosol which directs them to a docking and translocation complex at the peroxisomal membrane. We demonstrate the ubiquitination of Pex5p in cells lacking components of the peroxisomal AAA (ATPases associated with various cellular activities) or Pex4p-Pex22p complexes of the peroxisomal protein import machinery and in cells affected in proteasomal degradation. In cells lacking components of the Pex4p-Pex22p complex, mono-ubiquitinated Pex5p represents the major modification, while in cells lacking components of the AAA complex polyubiquitinated forms are most prominent. Ubiquitination of Pex5p is shown to take place exclusively at the peroxisomal membrane after the docking step, and requires the presence of the RING-finger peroxin Pex10p. Mono- and poly-ubiquitination are demonstrated to depend on the ubiquitin-conjugating enzyme Ubc4p, suggesting that the ubiquitinated forms of Pex5p are targeted for proteasomal degradation. Accumulation of ubiquitinated Pex5p in proteasomal mutants demonstrates that the ubiquitination of Pex5p also takes place in strains which are not affected in peroxisomal biogenesis, indicating that the ubiquitination of Pex5p represents a genuine stage in the Pex5p receptor cycle.

PEX1 deficiency presenting as Leber congenital amaurosis

Peroxisome biogenesis disorders result from defects in peroxin proteins involved in peroxisomal matrix and membrane protein import. Peroxins are encoded in peroxin protein genes; to date, the PEX genes responsible for all 12 peroxisome biogenesis disorders complementation groups are known. Peroxin protein 1 deficiency associated with complementation group 1 is responsible for disease in approximately two thirds of all patients with a peroxisome biogenesis disorder. Their phenotypes range from severe to mild, and it appears to be a phenotype-genotype relationship. This case report describes a patient with peroxin protein 1 deficiency presenting as Leber congenital amaurosis, in whom the diagnosis was questioned at the age of 2 years when seizures first appeared and mild facial dysmorphia became evident.

Functional role of the AAA peroxins in dislocation of the cycling PTS1 receptor back to the cytosol

Peroxisomal import receptors bind their cargo proteins in the cytosol and target them to docking and translocation machinery at the peroxisomal membrane (reviewed in ref. 1). The receptors release the cargo proteins into the peroxisomal lumen and, according to the model of cycling receptors, they are supposed to shuttle back to the cytosol. This shuttling of the receptors has been assigned to peroxins including the AAA peroxins Pex1p and Pex6p, as well as the ubiquitin-conjugating enzyme Pex4p (reviewed in ref. 2). One possible target for Pex4p is the PTS1 receptor Pex5p, which has recently been shown to be ubiquitinated. Pex1p and Pex6p are both cytosolic and membrane-associated AAA ATPases of the peroxisomal protein import machinery, the exact function of which is still unknown. Here we demonstrate that the AAA peroxins mediate the ATP-dependent dislocation of the peroxisomal targeting signal-1 (PTS1) receptor from the peroxisomal membrane to the cytosol.

A new definition for the consensus sequence of the peroxisome targeting signal type 2.

All organisms except the nematode Caenorhabditis elegans have been shown to possess an import system for peroxisomal proteins containing a peroxisome targeting signal type 2 (PTS2). The currently accepted consensus sequence for this amino-terminal nonapeptide is -(R/K)(L/V/I)X(5)(H/Q)(L/A)-. Some C.elegans proteins contain putative PTS2 motifs, including the ortholog (CeMeK) of human mevalonate kinase, an enzyme known to be targeted by PTS2 to mammalian peroxisomes. We cloned the gene for CeMeK (open reading frame Y42G9A.4) and examined the subcellular localization of CeMeK and of two other proteins with putative PTS2s at their amino termini encoded by the open reading frames D1053.2 and W10G11.11. All three proteins localized to the cytosol, confirming and extending the finding that C.elegans lacks PTS2-dependent peroxisomal protein import. The putative PTS2s of the proteins encoded by D1053.2 and W10G11.11 did not function in targeting to peroxisomes in yeast or mammalian cells, suggesting that the current PTS2 consensus sequence is too broad. Analysis of available experimental data on both functional and nonfunctional PTS2s led to two re-evaluated PTS2 consensus sequences: -R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)-, describes the most common variants of PTS2, while -(R/K)(L/V/I/Q)XX(L/V/I/H/Q)(L/S/G/A/K)X(H/Q)(L/A/F)-, describes essentially all variants of PTS2. These redefined PTS2 consensus sequences will facilitate the identification of proteins of unknown cellular localization as possible peroxisomal proteins.

PEX3 functions as a PEX19 docking factor in the import of class I peroxisomal membrane proteins.

PEX19 is a chaperone and import receptor for newly synthesized, class I peroxisomal membrane proteins (PMPs). PEX19 binds these PMPs in the cytoplasm and delivers them to the peroxisome for subsequent insertion into the peroxisome membrane, indicating that there may be a PEX19 docking factor in the peroxisome membrane. Here we show that PEX3 is required for PEX19 to dock at peroxisomes, interacts specifically with the docking domain of PEX19, and is required for recruitment of the PEX19 docking domain to peroxisomes. PEX3 is also sufficient to dock PEX19 at heterologous organelles and binds PEX19 via a conserved motif that is essential for this docking activity and for PEX3 function in general. Not surprisingly, transient inhibition of PEX3 abrogates class I PMP import but has no effect on class II PMP import or peroxisomal matrix protein import. Taken together, these results suggest that PEX3 plays a selective, essential, and direct role in PMP import as a docking factor for PEX19.

An Arabidopsis indole-3-butyric acid-response mutant defective in PEROXIN6, an apparent ATPase implicated in peroxisomal function.

Genetic evidence suggests that plant peroxisomes are the site of fatty acid beta-oxidation and conversion of the endogenous auxin indole-3-butyric acid (IBA) to the active hormone indole-3-acetic acid. Arabidopsis mutants that are IBA resistant and sucrose dependent during early development are likely to have defects in beta-oxidation of both IBA and fatty acids. Several of these mutants have lesions in peroxisomal protein genes. Here, we describe the Arabidopsis pex6 mutant, which is resistant to the inhibitory effects of IBA on root elongation and the stimulatory effects of IBA on lateral root formation. pex6 also is sucrose dependent during early seedling development and smaller and more pale green than WT throughout development. PEX6 encodes an apparent ATPase similar to yeast and human proteins required for peroxisomal biogenesis, and a human PEX6 cDNA can rescue the Arabidopsis pex6 mutant. The pex6 mutant has reduced levels of the peroxisomal matrix protein receptor PEX5, and pex6 defects can be partially rescued by PEX5 overexpression. These results suggest that PEX6 may facilitate PEX5 recycling and thereby promote peroxisomal matrix protein import.

Disturbed cholesterol homeostasis in a peroxisome-deficient PEX2 knockout mouse model.

We evaluated the major pathways of cholesterol regulation in the peroxisome-deficient PEX2(-/-) mouse, a model for Zellweger syndrome. Zellweger syndrome is a lethal inherited disorder characterized by severe defects in peroxisome biogenesis and peroxisomal protein import. Compared with wild-type mice, PEX2(-/-) mice have decreased total and high-density lipoprotein cholesterol levels in plasma. Hepatic expression of the SREBP-2 gene is increased 2.5-fold in PEX2(-/-) mice and is associated with increased activities and increased protein and expression levels of SREBP-2-regulated cholesterol biosynthetic enzymes. However, the upregulated cholesterogenic enzymes appear to function with altered efficiency, associated with the loss of peroxisomal compartmentalization. The rate of cholesterol biosynthesis in 7- to 9-day-old PEX2(-/-) mice is markedly increased in most tissues, except in the brain and kidneys, where it is reduced. While the cholesterol content of most tissues is normal in PEX2(-/-) mice, in the knockout mouse liver it is decreased by 40% relative to that in control mice. The classic pathway of bile acid biosynthesis is downregulated in PEX2(-/-) mice. However, expression of CYP27A1, the rate-determining enzyme in the alternate pathway of bile acid synthesis, is upregulated threefold in the PEX2(-/-) mouse liver. The expression of hepatic ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) involved in cholesterol efflux is not affected in PEX2(-/-) mice. These data illustrate the diversity in cholesterol regulatory responses among different organs in postnatal peroxisome-deficient mice and demonstrate that peroxisomes are critical for maintaining cholesterol homeostasis in the neonatal mouse.