Peroxisomes of Saccharomyces cerevisiae are the exclusive site of fatty acid beta‐oxidation. We have found that fatty acids reach the peroxisomal matrix via two independent pathways. The subcellular site of fatty acid activation varies with chain length of the substrate and dictates the pathway of substrate entry into peroxisomes. Medium‐chain fatty acids are activated inside peroxisomes hby the acyl‐CoA synthetase Faa2p. On the other hand, long‐chain fatty acids are imported from the cytosolic pool of activated long‐chain fatty acids via Pat1p and Pat2p, peroxisomal membrane proteins belonging to the ATP binding cassette transporter superfamily. Pat1p and Pat2p are the first examples of membrane proteins involved in metabolite transport across the peroxisomal membrane.

Most peroxisomal matrix proteins contain a carboxyl-terminal tripeptide that directs them to peroxisomes. Within limits, these amino acids may be varied, without loss of function. The specificity of this peroxisomal targeting signal (PTS1) is remarkable considering its small size and its relaxed consensus sequence. Moreover, several peroxisomal proteins have a PTS1-like signal that does not fit the reported consensus sequence. Because many of these PTS1 variants seem to be functional in a species-dependent or protein context-dependent manner, we investigated the PTS1 requirements in a homologous context, using Saccharomyces cerevisiae and endogenous peroxisomal malate dehydrogenase (MDH3). Peroxisomal import of the MDH3-PTS1 variants was tested qualitatively by the ability to complement the Δmdh3 mutant and quantitatively by subcellular fractionation. We observed efficient import of MDH3 into peroxisomes with a large variety of PTS1 tripeptides. Many of these variants do not fit the observed PTS1 requirements for heterologously expressed proteins, which suggests that additional domains in the protein may be of decisive importance whether or not a certain PTS1 variant is recognized by the components of the peroxisomal import machinery. Because we show that dimerization of MDH3 precedes import into the organelle, these domains are most likely conformational domains.

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein.

In this paper we report on the subcellular localization of peroxisomal thiolase in rat liver using density-gradient centrifugation and immunoelectron microscopy. The results obtained show that peroxisomes display great biochemical heterogeneity and can not be regarded as one homogeneous population of particles. We conclude that rat liver contains at least three distinct populations of peroxisomes, which are present both in normal-fed rats as well in rats treated with a plasticizer, di-(2-ethylhexyl)phthalate, known to induce peroxisomes. The following types of peroxisomes could be discerned: (1) Low-density peroxisomal particles containing 69-kDa peroxisomal membrane protein (PMP), dihydroxyacetonephosphate acyltransferase (DHAPAT) and the precursor form of peroxisomal thiolase (44-kDa). (2) Intermediate-density peroxisomal particles containing 69-kDa peroxisomal membrane protein, dihydroxyacetonephosphate acyltransferase, both 41-kDa (mature) and 44-kDa (immature) peroxisomal thiolase, catalase and D-aminoacid oxidase. (3) High-density peroxisomes containing 69-kDa peroxisomal membrane protein, dihydroxyacetonephosphate acyltransferase, 41-kDa thiolase, catalase and D-aminoacid oxidase.

In the liver biopsy from an 8.5-year-old girl with the biochemical characteristics of rhizomelic chondrodysplasia punctata (RCDP), but with normal limbs, normal catalase-containing peroxisomes were absent. Light microscopy after diaminobenzidine staining for catalase activity (the peroxisomal marker enzyme) and immunostaining against catalase protein indicated a cytosolic localization of the enzyme. By electron microscopy, rare and extremely large, irregularly shaped vesicles were found in the parenchymal cells. The three peroxisomal beta-oxidation enzymes (acyl-CoA oxidase, bi(tri)functional enzyme, and 3-ketoacyl-CoA thiolase) and alanine-glyoxylate aminotransferase were immunolocalized in these organelles. However, a weak to negative label was obtained after staining against catalase. Diaminobenzidine staining demonstrated a minimal catalase reaction product in some vesicles only. Morphometry revealed a corrected mean d-circle of 1.44 microns and a maximum d-circle of 2.767 microns (controls: 0.635 microns and 1.027 microns, respectively). Numerical, volume, and surface densities were reduced to 3%, 41%, and 17% of control values, respectively. The large size, irregular shape, and rarity of the organelles are morphologic features of peroxisomal "ghosts." It seems that in this patient, apart from the known peroxisomal defects in RCDP, catalase incorporation into the peroxisomes is impaired together with a normal proliferation (division) of the organelles. In the cultured skin fibroblasts from the patient, however, immuno-electron microscopy showed normal catalase-containing peroxisomes in apparently normal numbers.

In order to obtain information on the disputed nature of melanosomes a comparison was made between the localization of melanosomal markers with those of other well-defined subcellular organelles such as lysosomes and peroxisomes. The distribution of marker enzymes was studied using two different density gradient systems. i.e., Percoll and Nycodenz. Furthermore, the subcellular localization of various types of antigens was analyzed using indirect immunofluorescence and immuno-electron microscopy. All methods revealed the existence of partial co-localization of melanosomal and lysosomal proteins and different localization of peroxisomal markers. The results suggest that melanosomes may share a common origin with lysosomal structures.

To study peroxisome biogenesis, we developed a procedure to select for Saccharomyces cerevisiae mutants defective in peroxisomal protein import or peroxisome assembly. For this purpose, a chimeric gene was constructed encoding the bleomycin resistance protein linked to the peroxisomal protein luciferase. In wild-type cells this chimeric protein is imported into the peroxisome, which prevents the neutralizing interaction of the chimeric protein with its toxic phleomycin ligand. Peroxisomal import and peroxisome assembly mutants are unable to import this chimeric protein into their peroxisomes. This enables the bleomycin moiety of the chimeric protein to bind phleomycin, thereby preventing its toxicity. The selection is very efficient: upon mutagenesis, 84 (10%) of 800 phleomycin resistant colonies tested were unable to grow on oleic acid. This rate could be increased to 25% using more stringent selection conditions. The selection procedure is very specific; all oleic acid non utilizing (onu) mutants tested were disturbed in peroxisomal import and/or peroxisome assembly. The pas (peroxisome assembly) mutants that have been used for complementation analysis represent 12 complementation groups including three novel ones, designated pas20, pas21 and pas22.

Degradation of the peroxisomal enzymes fatty acyl-CoA oxidase and catalase was studied in hepatocytes isolated from rats treated with clofibrate and from control rats. Hepatocytes were incubated in the absence of amino acids in order to ensure maximal flux through the autophagic pathway and in the presence of cycloheximide to inhibit protein synthesis. (1) Degradation of the two peroxisomal enzymes in hepatocytes from clofibrate-fed rats, but not in hepatocytes from control rats, was much faster than that of other intracellular enzymes. This increased degradation of the peroxisomal enzymes was almost completely prevented by 3-methyladenine, an inhibitor of macroautophagic sequestration. (2) The increased degradation of the peroxisomal enzymes was also inhibited by a long-chain (C16:0) and a very-long-chain (C26:0) fatty acid, but not by C12:0, a medium-chain fatty acid, or by C8:0, a short-chain fatty acid. These results provide direct evidence for the proposal that autophagic sequestration can be highly selective [(1987) Exp. Mol. Pathol. 46, 114-122]. It is concluded that preferential autophagy of peroxisomes is prevented when these organelles are supplied with their fatty acid substrates.

Previous studies have shown that in fibroblasts from patients with the Zellweger syndrome (ZS) aberrant membrane structures are present which contain peroxisomal membrane proteins (Santos, M. J. et al., Science 239, 1536-1538 (1988)). In order to characterize these structures we have performed double labeling immunoelectron microscopy experiments using antisera directed against the 69 kDa peroxisomal integral membrane protein (PMP) and lysosomal hydrolases. The results indicate that at least 80% of the structures earlier referred to as 'peroxisomal ghosts' contain lysosomal hydrolases. In addition, we have studied the effect of culture of ZS fibroblasts in the presence of 3-methyladenine, an inhibitor of autophagy, on the intracellular distribution of the 69 kDa PMP. Immunofluorescence experiments showed that in the presence of 3-methyladenine there is an increase in fluorescent spots and a change in the distribution of the spots from mainly perinuclear to randomly distributed throughout the cytoplasm. Double labeling immunoelectron microscopy revealed that after culture in the presence of 3-methyladenine the 69 kDa PMP also accumulates mainly in compartments containing lysosomal hydrolases. In one ZS cell line we found that after culture in the presence of 3-methyladenine there was also an accumulation of structures which were as small as normal microperoxisomes. We conclude that in ZS fibroblasts the 69 kDa PMP is mainly present in lysosomal compartments, presumably degradative autophagic vacuoles. Furthermore, in ZS fibroblasts peroxisomes of apparently normal morphology may be synthesized, but they are degraded by autophagic proteolysis.

We have developed a positive selection system for the isolation of Saccharomyces cerevisiae mutants with disturbed peroxisomal functions. The selection is based on the lethality of hydrogen peroxide (H2O2) that is produced in wild type cells during the peroxisomal beta-oxidation of fatty acids. In total, 17 mutants having a general impairment of peroxisome biogenesis were isolated, as revealed by their inability to grow on oleic acid as the sole carbon source and their aberrant cell fractionation pattern of peroxisomal enzymes. The mutants were shown to have monogenetic defects and to fall into 12 complementation groups. Representative members of each complementation group were morphologically examined by immunocytochemistry using EM. In one mutant the induction and morphology of peroxisomes is normal but import of thiolase is abrogated, while in another the morphology differs from the wild type: stacked peroxisomal membranes are present that are able to import thiolase but not catalase. These mutants suggest the existence of multiple components involved in peroxisomal protein import. Some mutants show the phenotype characteristic of glucose-repressed cells, an indication for the interruption of a signal transduction pathway resulting in organelle proliferation. In the remaining mutants morphologically detectable peroxisomes are absent: this phenotype is also known from fibroblasts of patients suffering from Zellweger syndrome, a disorder resulting from impairment of peroxisomes.

Rhizomelic Chondrodysplasia Punctata (RCDP) is an autosomal recessive disorder in which plasmalogen biosynthesis and phytanate catabolism are impaired. Peroxisomal structure and the intracellular localization of catalase, the 69 kDa peroxisomal integral membrane protein (PMP), and 3-oxoacyl-CoA thiolase were studied in cultured skin fibroblasts from control subjects and patients with RCDP. A punctate fluorescence pattern characteristic for peroxisomes was seen in control cells incubated with either anti-(catalase), anti-(69 kDa PMP) or anti-(3-oxoacyl-CoA thiolase). Incubation of mutant cells with anti-(catalase) or anti-(69 kDa PMP) resulted in the same pattern. However, when RCDP fibroblasts were incubated with a monoclonal anti-(3-oxoacyl-CoA thiolase) antibody no punctate fluorescence could be observed. Cryosections from control and RCDP cells were examined by electron microscopy using double immunogold labelling. RCDP fibroblasts contained structures indistinguishable from control peroxisomes, the membranes reacting with anti-(69 kDa PMP) and the matrix with anti-(catalase). However, the matrix of RCDP peroxisomes, unlike control peroxisomes, did not react with anti-(3-oxoacyl-CoA thiolase). We conclude that RCDP fibroblasts contain regularly shaped peroxisomes, comparable to control peroxisomes in number as well as in content of catalase and 69 kDa PMP. However, in RCDP peroxisomes the amount of 3-oxoacyl-CoA thiolase protein proved to be below the limit of detection.

The presence and intracellular localization of peroxisomal integral membrane proteins (PMP) were investigated in liver and cultured skin fibroblasts from control subjects and patients with the Zellweger syndrome and related disorders in which peroxisomes are virtually absent. Immunoblotting experiments showed that 22, 36 and 69 kDa PMPs were present and were confined to the membranous fraction both in the control liver and in the livers from the Zellweger patients. The 22 and 36 kDa PMPs were present in significantly lower amounts in the patients' livers than in the control liver. A reduced amount of the 69 kDa PMP was found in liver from one Zellweger but not in liver from another. The subcellular localization in fibroblasts of catalase and the 69 kDa PMP was studied by indirect immunofluorescence. A characteristic punctate fluorescence was seen in control cells incubated with either anti-(catalase) or with anti-(69 kDa PMP). Incubation of mutant cells with anti-(catalase) resulted in a diffuse fluorescence, whereas with anti-(69 kDa PMP) fluorescent particles were visualized which, in some cell lines, were larger and fewer in number than in control cells. Cryosections of control and mutant cells were examined by electron microscopy using immunogold labeling. Control cells contained small structures consisting of a single membrane enclosing a homogeneous matrix; the membranes reacted with anti-(69 kDa PMP) and the matrix with anti-(catalase). The mutant cell lines contained spherical or ellipsoidal structures whose membranes reacted with anti-(69 kDa PMP); no labeling was observed with anti-(catalase). We conclude that peroxisomal ghosts, the membranes of which contain the 69 kDa PMP, are present in peroxisome-deficient cell lines from all complementation groups studied so far.