Paul B. Lazarow

Peroxisome biogenesis: advances and conundrums

Investigations of peroxisome biogenesis in diverse organisms reveal new details of this unique process and its evolutionary conservation. Interactions among soluble receptors and the membrane peroxins that catalyze protein translocation are being mapped. Ubiquitination is observed. A receptor enters the organelle carrying folded cargo and recycles back to the cytosol. Tiny peroxisome remnants - vesicles and tubules - are discovered in pex3 mutants that lack the organelle. When the mutant is transfected with a good PEX3 gene, these protoperoxisomes acquire additional membrane peroxins and then import the matrix enzymes to reform peroxisomes. Thus, de novo formation need not be postulated. Dynamic imaging of yeast reveals dynamin-dependent peroxisome division and regulated actin-dependent segregation of the organelle before cell division. These results are consistent with biogenesis by growth and division of pre-existing peroxisomes.

Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae

Pex7p is the soluble receptor responsible for importing into peroxisomes newly synthesized proteins bearing a type 2 peroxisomal targeting sequence. We observe that appending GFP to Pex7ps COOH terminus shifts Pex7ps intracellular distribution from predominantly cytosolic to predominantly peroxisomal in Saccharomyces cerevisiae. Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol. The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid. These results support the “extended shuttle” model of peroxisome import receptor function and open the way to future studies of receptor export.

Eci1p uses a PTS1 to enter peroxisomes: either its own or that of a partner, Dci1p.

Saccharomyces cerevisiae delta3,delta2-enoyl-CoA isomerase (Eci1p), encoded by ECI1, is an essential enzyme for the betaoxidation of unsaturated fatty acids. It has been reported, as well as confirmed in this study, to be a peroxisomal protein. Unlike many other peroxisomal proteins, Ecilp possesses both a peroxisome targeting signal type 1 (PTS1)-like signal at its carboxy-terminus (-HRL) and a PTS2-like signal at its amino-terminus (RIEGPFFIIHL). We have found that peroxisomal targeting of a fusion protein consisting of Eci1p in front of green fluorescent protein (GFP) is not dependent on Pex7p (the PTS2 receptor), ruling out a PTS2 mechanism, but is dependent on Pex5p (the PTS1 receptor). This Pex5p-dependence was unexpected, since the putative PTS1 of Ecilp is not at the C-terminus of the fusion protein; indeed, deletion of this signal (-HRL-) from the fusion did not affect the Pex5p-dependent targeting. Consistent with this, Pex5p interacted in two-hybrid assays with both Eci1p and Eci1PdeltaHRL. Ecilp-GFP targeting and Eci1pdeltaHRL interaction were abolished by replacement of Pex5p with Pex5p(N495K), a point-mutated Pex5p that specifically abolishes the PTS1 protein import pathway. Thus, Eci1p peroxisomal targeting does require the Pex5p-dependent PTS1 pathway, but does not require a PTS1 of its own. By disruption of ECI1 and DCI1, we found that Dci1p, a peroxisomal PTS1 protein that shares 50% identity with Eci1p, is necessary for Eci1p-GFP targeting. This suggests that the Pex5p-dependent import of Eci1p-GFP is due to interaction and co-import with Dci1p. Despite the dispensability of the C-terminal HRL for import in wild-type cells, we have also shown that this tripeptide can function as a PTS1, albeit rather weakly, and is essential for targeting in the absence of Dci1p. Thus, Eci1p can be targeted to peroxisomes by its own PTS1 or as a hetero-oligomer with Dcilp. These data demonstrate a novel, redundant targeting pathway for Eci1p.

Rhizomelic Chondrodysplasia Punctata, a Peroxisomal Biogenesis Disorder Caused by Defects in Pex7p, a Peroxisomal Protein Import Receptor: A Minireview

Rhizomelic chondrodysplasia punctata (RCDP) is a lethal autosomal recessive disease correspondingto complementation group 11 (CG 11), the second most common of the thirteen CGs of peroxisomalbiogenesis disorders (PBDs). RCDP is characterized by proximal limb shortening, severely disturbedendochondrial bone formation, and mental retardation, but there is an absence of the neuronal migrationdefect found in the other PBDs. Plasmalogen biosynthesis and phytanic acid oxidation are deficient, butvery long chain fatty acid (VLCFA) oxidation is normal. At the cellular level, RCDP is unique in thatthe biogenesis of most peroxisomal proteins is normal, but a specific subset of at least four, and maybemore, peroxisomal matrix proteins fail to be imported from the cytosol. In this review, we discuss recentadvances in understanding RCDP, most prominently the cloning of the affected gene, PEX7,and identification of PEX7 mutations in RCDP patients. Human PEX7 wasidentified by virtue of its sequence similarity to its Saccharomyces cerevisiae ortholog, whichhad previously been shown to encode Pex7p, an import receptor for type 2 peroxisomal targetingsequences (PTS2). Normal human PEX7 expression rescues the cellular defects in culturedRCDP cells, and cDNA sequence analysis has identified a variety of PEX7 mutations in RCDP patients,including a deletion of 100 nucleotides, probably due to a splice site mutation, and a prevalent nonsensemutation which results in loss of the carboxyterminal 32 amino acids. Identification of RCDP as a PTS2import disorder explains the observation that several, but not all, peroxisomal matrix proteins aremistargeted in this disease; three of the four proteins deficient in RCDP have now been shown to bePTS2-targeted.

Peroxisome Structure, Function, and Biogenesis—Human Patients and Yeast Mutants Show Strikingly Similar Defects in Peroxisome Biogenesis

Peroxisomes are found in almost all eukaryotic cells. Two major functions of the organelle are in lipid metabolism: peroxisomes catalyze the initial steps in the biosynthesis of plasmalogens, which are phospholipids that are present in large amounts in myelin. Peroxisomes also catalyze the beta-oxidation of fatty acids; this pathway is essential for the catabolism of a variety of substrates that are not oxidized by mitochondria. A third important function is in cellular respiration, involving the metabolism of H2O2, for which the peroxisome is named. Peroxisomes increase in size by the post-translational import of newly synthesized proteins from the cytosol; these pre-existing peroxisomes divide to form new peroxisomes. Proteins are targeted to peroxisomes by three different types of topogenic sequences, and it is hypothesized that a receptor exists for each type. The newly made proteins are translocated through the peroxisomal membrane into the interior by a machinery that is energized by ATP hydrolysis. Human patients and yeast mutants have remarkably similar defects in peroxisome biogenesis. Some such mutants are defective in the import of a subset of peroxisomal proteins that share a topogenic sequence type; other mutants fail to import all newly made proteins into peroxisomes, regardless of the type of targeting sequence they possess. These mutants might be defective in receptors and in translocation machinery components, respectively. Cloned genes that are essential for peroxisome biogenesis encode diverse proteins: some likely receptors, some transmembrane proteins possibly involved in translocation, and others hydrophilic proteins that may play other roles in peroxisome assembly.

Genetic approaches to studying peroxisome biogenesis

How proteins are imported into peroxisomes is a question attracting considerable interest at present. Peroxisomal proteins, including the integral membrane proteins of the membrane bounding the peroxisome, are synthesized on free cytoplasmic ribosomes. They assemble post-translationally into pre-existing peroxisomes. New peroxisomes are believed to form exclusively by division of old ones. Few molecular details of this process have been elucidated so far, but genetic approaches are now beginning to identify the proteins catalysing peroxisome assembly.

Protein import into peroxisomes in vitro.

Publisher Summary The principle of the in vitro reconstitution of peroxisomal protein assembly is simple: newly synthesized peroxisomal proteins (labeled with [ 35 S]methionine) are mixed with purified peroxisomes and the proteins enter the organelle in a time- and temperature-dependent fashion. Import is analyzed by digesting the nonimported proteins with a protease. Those proteins that have been imported into the peroxisomes are protected from proteolysis by the membrane of the organelle. This in vitro reconstitution of import requires a good supply of stable, purified peroxisomes, sufficient synthesis of radiolabeled peroxisomal proteins, appropriate incubation conditions for import, the selection of protease(s) that can digest nonimported proteins without destroying the organelle membrane, and careful controls. Each of these issues is discussed in detail in the chapter. To adapt the import assays for the study of another peroxisomal protein, it is necessary to verify that the protease protection assay functions for the new protein, and, if not, to find suitable conditions. The selection of the protease is based on two criteria: (1) the 35 S-labeled protein has to be digestible by the protease and (2) the peroxisomes have to remain intact during the protease treatment.

A novel 57 kDa peroxisomal membrane polypeptide detected by monoclonal antibody (PXM1a/207B)

BALB/c mice were immunized with peroxisomal membranes prepared from rat liver. Spleen cells were fused with myeloma cells (P3/U1) and the hybridomas were selected using peroxisomal membranes. A monoclonal antibody (PXM1a/207B) which recognized peroxisomal membranes was selected. Using the antibody, a novel 57 kDa polypeptide was identified in the peroxisomal membrane fraction. Immunoblot analysis of the subcellular fractions demonstrated that the 57 kDa polypeptide was present exclusively in peroxisomal membranes. The 57 kDa polypeptide was partially digested by trypsin and chymotrypsin under conditions where peroxisomal particles remained intact, indicating that the polypeptide is exposed to the cytosolic face of the peroxisomal membrane. The amount of 57 kDa polypeptide increased in parallel with proliferation of peroxisomes by administration of clofibrate.

Peroxisomal ghosts are intracellular structures distinct from lysosomal compartments in Zellweger syndrome: a confocal laser scanning microscopy study.

Summary— Peroxisome ghosts are aberrant peroxisomal structures found in cultured skin fibroblasts from patients affected by Zellweger Syndrome (ZS), a genetic disorder of peroxisomal assembly. They contain peroxisomal integral membrane proteins (PxIMPs) and they lack most of the matrix enzymes that should be inside the organelle (Santos et al., Science 239 (1988) 1536–1538). Considerable evidence indicates that these ghosts result from genetic defects in the cellular machinery for importing newly‐synthesized peroxisomal proteins into the organelle. In contrast to these observations, (Heikoop et al., Eur. J. Cell Biol. 57 (1992) 165–171) report that in Zellweger Syndrome, peroxisomal membranes are located within lysosomes and/or contain lysosomal enzymes. We have undertaken a more detailed and systematic investigation of this matter, employing confocal laser scanning microscopy (CLSM). In fibroblasts derived from ZS patients belonging to different complementation groups, peroxisomes were labeled with antibodies against PxIMPs and lysosomes were labeled with an antibody against a lysosome associated membrane protein (LAMP‐2) or with LysoTracker. The results unambiguously demonstrated no appreciable colocalization of PxIMPs and LAMPs (or LysoTracker), indicating that peroxisomal ghosts are distinct subcellular structures, occupying separate subcellular locations.

Analysis of peroxisomes in lymphoblasts: Zellweger syndrome and a patient with a deletion in chromosome 7.

ABSTRACT: Lymphoblasts are useful cells for the diagnosis and basic studies of several human genetic disorders. Peroxisomal disorders are usually diagnosed by using fibroblasts or blood samples. Here, we report the characterization of peroxisomes in lymphoblasts. We demonstrated that lymphoblasts from a patient with Zellweger syndrome, the prototypical disorder of peroxisome biogenesis, contained peroxisomal ghosts like those described previously in Zellweger fibroblasts. We also found that lymphoblasts that carry a deletion on chromosome 7 (q11.23q22.1), a region thought to contain one Zellweger syndrome gene, contained normal peroxisomes.