Individual Peroxisomes Research Articles

A novel fluorescence-activated cell sorting (FACS)-based screening identified ATG14, the gene required for pexophagy in the methylotrophic yeast.

Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.

Quantitative analysis of peroxisome tracks using a Hidden Markov Model

Diffusion and mobility are essential for cellular functions, as molecules are usually distributed throughout the cell and have to meet to interact and perform their function. This also involves the cytosolic migration of cellular organelles. However, observing such diffusion and interaction dynamics is challenging due to the high spatial and temporal resolution required and the accurate analysis of the diffusional tracks. The latter is especially important when identifying anomalous diffusion events, such as directed motions, which are often rare. Here, we investigate the migration modes of peroxisome organelles in the cytosol of living cells. Peroxisomes predominantly migrate randomly, but occasionally they bind to the cell's microtubular network and perform directed migration, which is difficult to quantify, and so far, accurate analysis of switching between these migration modes is missing. We set out to solve this limitation by experiments and analysis with high statistical accuracy. Specifically, we collect temporal diffusion tracks of thousands of individual peroxisomes in the HEK 293 cell line using two-dimensional spinning disc fluorescence microscopy at a high acquisition rate of 10 frames/s. We use a Hidden Markov Model with two hidden states to (1) automatically identify directed migration segments of the tracks and (2) quantify the migration properties for comparison between states and between different experimental conditions. Comparing different cellular conditions, we show that the knockout of the peroxisomal membrane protein PEX14 leads to a decrease in the directed movement due to a lowered binding probability to the microtubule. However, it does not eradicate binding, highlighting further microtubule-binding mechanisms of peroxisomes than via PEX14. In contrast, structural changes of the microtubular network explain perceived eradication of directed movement by disassembly of microtubules by Nocodazole-treatment.

A rare case of proximal short limb dwarfism-rhizomelic chondrodysplasia punctata type-2

Rhizomelic chondrodysplasia punctata (RCDP) is one of the rare peroxisomal disorder (autosomal recessive inheritance) due to altered phytanic acid alpha oxidation and plasmalogen synthesis. RCDP 1 is the most frequent form of RCDP. It is a peroxisomal biosynthesis disorder. RCDP 2 and RCDP 3 are disorders of individual peroxisome enzyme. Authors described a case of RCDP type 2 in a 13 months old girl with characteristic features of typical chondrodysplastic facies, bilateral cataract, rhizomelic shortening of limbs, growth and global developmental delay; radiological features showed epiphyseal stippling. Genetic analysis showed apparent homozygous deletion of c.1848delC after full sequencing of her GNPAT genes.

Mechanism of impaired microtubule-dependent peroxisome trafficking and oxidative stress in SPAST-mutated cells from patients with Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) is an inherited neurological condition that leads to progressive spasticity and gait abnormalities. Adult-onset HSP is most commonly caused by mutations in SPAST, which encodes spastin a microtubule severing protein. In olfactory stem cell lines derived from patients carrying different SPAST mutations, we investigated microtubule-dependent peroxisome movement with time-lapse imaging and automated image analysis. The average speed of peroxisomes in patient-cells was slower, with fewer fast moving peroxisomes than in cells from healthy controls. This was not because of impairment of peroxisome-microtubule interactions because the time-dependent saltatory dynamics of movement of individual peroxisomes was unaffected in patient-cells. Our observations indicate that average peroxisome speeds are less in patient-cells because of the lower probability of individual peroxisome interactions with the reduced numbers of stable microtubules: peroxisome speeds in patient cells are restored by epothilone D, a tubulin-binding drug that increases the number of stable microtubules to control levels. Patient-cells were under increased oxidative stress and were more sensitive than control-cells to hydrogen peroxide, which is primarily metabolised by peroxisomal catalase. Epothilone D also ameliorated patient-cell sensitivity to hydrogen-peroxide. Our findings suggest a mechanism for neurodegeneration whereby SPAST mutations indirectly lead to impaired peroxisome transport and oxidative stress.

PPAR gamma pro12Ala polymorphism and type 2 diabetes: a study in a spanish cohort

Type 2 diabetes (T2D) is a disease whose occurrence is increasing prevalent in westernized civilizations and is responsible for the proliferation in the morbidity and total mortality of patients with cardiovascular diseases, worldwide. However, the complexity in the treatment and prevention of T2D arises from the intricacy of the many physical and biological factors involved in its etiology. Impaired pathways for insulin signaling have been implicated as one the many factors in the development of T2D Individual peroxisome proliferator-activated receptors (PPARs) have previously exhibited associations with alterations of lipid profiles, fat tissue and T2D and displayed complications derived from high levels of glucose. However, PPARgamma has not yet been associated with the development or developmental pathways of T2D. We performed an observational study a Spanish cohort in order to better understand the association between the SNP PPARgamma polymorphism Pro12Ala in our patients and the incidence of T2D and other cardiovascular complications. We study did not find a statistically significant relationship between the Pro12Ala and T2D development in our cohort, future observations will help us to know the association with vascular disease in patients with T2D.

The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies

Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots™, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.

Myosin-driven peroxisome partitioning inS. cerevisiae

In Saccharomyces cerevisiae, the class V myosin motor Myo2p propels the movement of most organelles. We recently identified Inp2p as the peroxisome-specific receptor for Myo2p. In this study, we delineate the region of Myo2p devoted to binding peroxisomes. Using mutants of Myo2p specifically impaired in peroxisome binding, we dissect cell cycle–dependent and peroxisome partitioning–dependent mechanisms of Inp2p regulation. We find that although total Inp2p levels oscillate with the cell cycle, Inp2p levels on individual peroxisomes are controlled by peroxisome inheritance, as Inp2p aberrantly accumulates and decorates all peroxisomes in mother cells when peroxisome partitioning is abolished. We also find that Inp2p is a phosphoprotein whose level of phosphorylation is coupled to the cell cycle irrespective of peroxisome positioning in the cell. Our findings demonstrate that both organelle positioning and cell cycle progression control the levels of organelle-specific receptors for molecular motors to ultimately achieve an equidistribution of compartments between mother and daughter cells.

Peroxule extension over ER‐defined paths constitutes a rapid subcellular response to hydroxyl stress

Plants survive against myriad environmental odds while remaining rooted to a single spot. The time scale over which plant cells can respond to environmental cues is seldom appreciated. Fluorescent protein-assisted live imaging of peroxisomes reveals that they respond within seconds of exposure to hydrogen peroxide and hydroxyl radicals by producing dynamic extensions called peroxules. Observations of the Arabidopsis flu mutant and treatments with xenobiotics eliciting singlet oxygen and superoxide reactive oxygen species suggest that the observed responses are specific for hydroxyl radicals. Prolonged exposure to hydroxyl radicals inhibits peroxule extension, and instead causes motile and spherical peroxisomes in a cell to become immotile and elongate several-fold. Expression of photo-convertible EosFP-PTS1 demonstrates that vermiform peroxisomes result from rapid stretching of individual peroxisomes, while the subsequent 'beads-on-a-string' morphology results from differential protein distribution within an elongated tubule. Over time, the beads in elongated peroxisomes also extend peroxules randomly before undergoing asynchronous, asymmetrical fission. Peroxule extension does not appear to involve cytoskeletal elements directly, but is closely aligned with and reflects the dynamics of ER tubules. Peroxisomal responses reveal a rapidly invoked subcellular machinery that is involved in recognition of hydroxyl stress thresholds, and its possible remediation locally through extension of peroxules or globally by increasing peroxisome numbers. A matrix protein retro-flow mechanism that supports peroxisome-ER connectivity in plant cells is suggested.

Mitochondria use actin filaments as rails for fast translocation in Arabidopsis and tobacco cells

Although mitochondria are known to move actively in plant cells, little is known about how they move. In higher plants, actin filaments have been reported to be involved in the movement of organelles, such as chloroplasts, peroxisomes, endoplasmic reticulum and Golgi apparatus. Mitochondria were visualized in living Arabidopsis thaliana plants using fluorescent proteins fused to a mitochondria targeting signal. To compare the movement of mitochondria to the movements of another organelle, we also examined peroxisomes because of their similarity in size. Velocities of individual mitochondria and peroxisomes, as measured by time-lapse laser scanning microscopy, varied, although the average velocities of the two organelles were similar. Latrunculin B, an actin-depolymerizing drug, stopped movement of mitochondria and peroxisomes, demonstrating that movement of these organelles depends on actin filaments. On the other hand, propyzamide, a microtubule-depolymerizing drug, did not affect the movement of mitochondria and peroxisomes. In living Arabidopsis plants in which mitochondria or peroxisomes are visualized by red fluorescent protein and cytoskeletal elements are simultaneously visualized by green fluorescent protein, both organelles moved using actin filaments as rails. In contrast, their movement was not related to arrays of microtubules. We also examined mitochondrial movement in tobacco cultured cells visualizing mitochondria and cytoskeletal element simultaneously. Mitochondrial movement was often seen along actin filaments but not along microtubules. These findings suggest that mitochondria move along actin filaments rapidly and over long distances in higher plants.

In the yeastHansenula polymorpha, peroxisome formation from the ER is independent of Pex19p, but involves the function of p24 proteins

The peroxin Pex19p is important for the formation of functional peroxisomal membranes. Here we show that Hansenula polymorpha Pex19p is also required for peroxisome inheritance. Peroxisome inheritance is partly defective when Pex19p farnesylation is blocked, whereas deletion of PEX19 resulted in a severe defect in partitioning of peroxisomal structures. Time lapse imaging revealed that in newly formed buds, which had not inherited a peroxisome from the mother cell, new peroxisomes are formed that derive from the nuclear envelope/endoplasmic reticulum. This process was impaired upon deletion of EMP24 and ERP3, genes that encode p24 proteins. p24 Proteins are components of coated vesicles that mediate trafficking between the endoplasmic reticulum and Golgi apparatus. In an H. polymorpha wild-type background, deletion of EMP24 and ERP3 resulted in a strong reduction of organelle number in conjunction with an increase in the size of individual peroxisomes. This observation suggests that p24 proteins also play a role in peroxisome development in wild-type H. polymorpha cells.

Atg8 is Essential for Macropexophagy in Hansenula polymorpha

We have isolated a peroxisome-degradation-deficient (pdd) mutant of the methylotrophic yeast Hansenula polymorpha via gene tagging mutagenesis. Sequencing revealed that the mutant was affected in the HpATG8 gene. HpAtg8 is a protein with high sequence similarity to both Pichia pastoris and Saccharomyces cerevisiae Atg8 and appeared to be essential for selective peroxisome degradation (macropexophagy) and nitrogen-limitation induced microautophagy. Fluorescence microscopy revealed that a GFP.Atg8 fusion protein was located close to the vacuole. After induction of macropexophagy, the GFP.Atg8 containing spot extended to engulf an individual peroxisome. In cells of a constructed deletion strain, sequestration of individual organelles was never completed; analysis of series of serial sections revealed that invariably a minor diaphragm-like opening remained. We hypothesize that H. polymorpha Atg8 facilitates sealing of the sequestering membranes during selective peroxisome degradation.

Removal of Pex3p Is an Important Initial Stage in Selective Peroxisome Degradation in Hansenula polymorpha

Selective degradation of peroxisomes (macropexophagy) in Hansenula polymorpha involves the sequestration of individual organelles to be degraded by membranes prior to the fusion of this compartment with the vacuole and subsequent degradation of the whole organelle by vacuolar hydrolases. Here we show that Pex3p, a peroxisomal membrane protein essential for peroxisome biogenesis, escapes this autophagic process. Upon induction of macropexophagy, Pex3p is removed from the organelle tagged for degradation prior to its sequestration. Our data indicate that Pex3p degradation is essential to allow the initiation of the organellar degradation process. Also, in a specific peroxisome degradation-deficient (pdd) mutant in which sequestration still occurs but the vacuolar fusion event is disturbed, the turnover of Pex3p is still observed. Taken together, our data suggest that degradation of Pex3p is part of the initial degradation machinery of individual peroxisomes.

Stable and transient expression of chimeric peroxisomal membrane proteins induces an independent "zippering" of peroxisomes and an endoplasmic reticulum subdomain.

Peroxisomal ascorbate peroxidase (APX) (EC 1.11.1.11) was shown recently to sort through a subdomain of the ER (peroxisomal endoplasmic reticulum; pER), and in certain cases, alter the distribution and/or morphology of peroxisomes and pER when overexpressed transiently in Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells. Our goal was to gain insight into the dynamics of peroxisomal membrane protein sorting by characterizing the structure and formation of reorganized peroxisomes and pER. Specifically, we test directly the hypothesis that the observed phenomenon is due to the oligomerization of cytosol-facing, membrane-bound polypeptides. a process referred to as membrane "zippering". Results from differential detergent permeabilization experiments confirmed that peroxisomal APX is a C-terminal "tail-anchored" (Cmatrix-Ncytosol) membrane protein with a majority of the polypeptide facing the cytosol. Transient expression of several APX chimeras whose passenger polypeptides can form dimers or trimers resulted in the progressive formation of "globular" peroxisomes and circular pER membranes. Stable expression of the trimer-capable fusion protein yielded suspension cultures that reproducibly maintained a high degree of peroxisomal globules but relatively few detectable pER membranes. Electron micrographs revealed that the globules consisted of numerous individual peroxisomes, seemingly in direct contact with other peroxisomes and/or mitochondria. These peroxisomal clusters or aggregates were not observed in cells transiently expressing monomeric versions of APX. These findings indicate that the progressive, independent "zippering" of peroxisomes and pER is due to the post-sorting oligomerization of monomeric, cytosol-facing polypeptides that are integrally inserted into the membranes of "like" organelles. The dynamics of this process are discussed, especially with respect to the involvement of the microtubule cytoskeleton.

Glucose-induced and nitrogen-starvation-induced peroxisome degradation are distinct processes in that involve both common and unique genes

In the methylotrophic yeast Hansenula polymorpha non-selective autophagy, induced by nitrogen starvation, results in the turnover of cytoplasmic components, including peroxisomes. We show that the uptake of these components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast. A selective mode of autophagy in H. polymorpha, namely glucose-induced peroxisome degradation, involves sequestration of individual peroxisomes tagged for degradation by membrane layers that subsequently fuse with the vacuole where the organelle is digested. H. polymorpha pdd mutants are blocked in selective peroxisome degradation. We observed that pdd1-201 is also impaired in non-selective autophagy, whereas this process still normally functions in pdd2-4. These findings suggest that mechanistically distinct processes as selective and non-selective autophagy involve common but also unique genes.

Glucose-induced and nitrogen-starvation-induced peroxisome degradation are distinct processes in Hansenula polymorpha that involve both common and unique genes.

In the methylotrophic yeast Hansenula polymorpha non-selective autophagy, induced by nitrogen starvation, results in the turnover of cytoplasmic components, including peroxisomes. We show that the uptake of these components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast. A selective mode of autophagy in H. polymorpha, namely glucose-induced peroxisome degradation, involves sequestration of individual peroxisomes tagged for degradation by membrane layers that subsequently fuse with the vacuole where the organelle is digested. H. polymorpha pdd mutants are blocked in selective peroxisome degradation. We observed that pdd1-201 is also impaired in non-selective autophagy, whereas this process still normally functions in pdd2-4. These findings suggest that mechanistically distinct processes as selective and non-selective autophagy involve common but also unique genes.

Selective peroxisome degradation in Yarrowia lipolytica after a shift of cells from acetate/oleate/ethylamine into glucose/ammonium sulfate-containing media

We have shown that peroxisomes of the yeast Yarrowia lipolytica are subject to specific degradation after exposure of acetate/oleate-grown cells to glucose excess conditions. Electron microscopic analysis has revealed that the peroxisomes were degraded by uptake in the vacuole. Our results suggest that peroxisomes are taken up by macroautophagic processes, because sequestration of individual peroxisomes, which occurs typically at the beginning of microautophagy, was never observed. The observation that a peroxisomal amine oxidase activity is specifically induced by ethylamine was used for the development of a plate assay screening procedure to isolate peroxisome degradation-defective mutants.

The different effects of the peroxisome proliferators clofibric acid and bis(2-ethylhexyl)phthalate on the activities of peroxisomal oxidases in rat liver

Treatment with peroxisome proliferators induces increased numbers and alterations in the shape of peroxisomes in liver. It ultimately leads to hepatocellular carcinomas induced by the persistent production of high amounts of H2O2 as a result of a dramatical increase in acyl-CoA oxidase activity. The effects of peroxisome proliferators on other peroxisomal oxidase activities are less well documented. In the present study, the distribution patterns of the activity of SdD-amino acid oxidase, SlD-alpha-hydroxy acid oxidase, polyamine oxidase, urate oxidase and catalase activities were investigated in unfixed cryostat sections of liver, kidney and duodenum of rats treated with either clofibrate or bis(2-ethylhexyl)phthalate. The activities of xanthine oxidoreductase, which produces urate, a potent anti-oxidant, and xanthine oxidase, which produces oxygen radicals, were studied as well. The liver was the only organ that was affected by treatment. The number of peroxisomes increased considerably. SdD-Amino acid oxidase and polyamine oxidase activities were completely abolished by the treatment, whereas SlD-alpha-hydroxy acid oxidase activity decreased and urate oxidase activity increased periportally and decreased pericentrally. Total catalase activity increased because of the larger numbers of peroxisomes, but it decreased per individual peroxisome. Xanthine oxidoreductase activity decreased, whereas the percentage of xanthine oxidase remained constant. We conclude that oxidases in rat liver are affected differentially, indicating that the expression of activity of each oxidase is regulated individually. © 1998 Chapman & Hall

Molecular cloning and further characterization of rat peroxisomal trihydroxycoprostanoyl-CoA oxidase.

The composite trihydroxycoprostanoyl-CoA oxidase cDNA sequence, derived from overlapping clones isolated via screening of two different rat liver expression libraries, consisted of 2509 bases and contained an open reading frame of 2046 bases, encoding a protein of 681 amino acids with a calculated molecular mass of 76711 Da. The reading frame and identity of the trihydroxycoprostanoyl-CoA oxidase cDNA were confirmed by the location of various tryptic peptides, obtained from the purified enzyme, in the deduced amino acid sequence. The C-terminus (His-Lys-Met) of trihydroxycoprostanoyl-CoA oxidase did not seem to interact with the C-terminal peroxisomal targeting signal 1 (PTS1) import receptor, although the tripeptide fits the rule of conserved PTS1 variants for targeting of proteins to glycosomes of Trypanosomatidae. At the protein level, trihydroxycoprostanoyl-CoA oxidase showed 45% identical amino acids with rat palmitoyl-CoA oxidase, whereas the identity with pristanoyl-CoA oxidase was much lower (22%). Northern analysis of multiple rat tissues revealed a signal (approx. 2.6 kb) only in liver and (although much weaker) in kidney. Dot-blot analysis of total liver RNA revealed that the mRNA for trihydroxy-coprostanoyl-CoA oxidase is not induced after treatment of rats with structurally unrelated peroxisome proliferators and indicates that highly similar mRNAs are present in other mammals, including man. Immunocytochemistry showed a decrease in trihydroxycoprostanoyl-CoA oxidase protein in individual liver peroxisomes ('diluting-out effect') after treatment of rats with bezafibrate, whereas the palmitoyl-CoA oxidase labelling was significantly increased.

In situ heterogeneity of peroxisomal oxidase activities: an update.

Oxidases are a widespread group of enzymes. They are present in numerous organisms and organs and in various tissues, cells, and subcellular compartments, such as mitochondria. An important source of oxidases, which is investigated and discussed in this study, are the (micro)peroxisomes. Oxidases share the ability to reduce molecular oxygen during oxidation of their substrate, yielding an oxidized product and hydrogen peroxide. Besides the hydrogen peroxide-catabolizing enzyme catalase, peroxisomes contain one or more hydrogen peroxide-generating oxidases, which participate in different metabolic pathways. During the last four decades, various methods have been developed and elaborated for the histochemical localization of the activities of these oxidases. These methods are based either on the reduction of soluble electron acceptors by oxidase activity or on the capture of hydrogen peroxide. Both methods yield a coloured and/or electron dense precipitate. The most reliable technique in peroxisomal oxidase histochemistry is the cerium salt capture method. This method is based on the direct capture of hydrogen peroxide by cerium ions to form a fine crystalline, insoluble, electron dense reaction product, cerium perhydroxide, which can be visualized for light microscopy with diaminobenzidine. With the use of this technique, it became clear that oxidase activities not only vary between different organisms, organs, and tissues, but that heterogeneity also exists between different cells and within cells, i.e. between individual peroxisomes. A literature review, and recent studies performed in our laboratory, show that peroxisomes are highly differentiated organelles with respect to the presence of active enzymes. This study gives an overview of the in situ distribution and heterogeneity of peroxisomal enzyme activities as detected by histochemical assays of the activities of catalase, and the peroxisomal oxidases D-amino acid oxidase, L-alpha-hydroxy acid oxidase, polyamine oxidase and uric acid oxidase.

Identification of peroxisomal membrane ghosts with an epitope-tagged integral membrane protein in yeast mutants lacking peroxisomes.

Many yeast peroxisome biogenesis mutants have been isolated in which peroxisomes appear to be completely absent. Introduction of a wild-type copy of the defective gene causes the reappearance of peroxisomes, despite the fact that new peroxisomes are thought to form only from pre-existing peroxisomes. This apparent paradox has been explained for similar human mutant cell lines (from patients with Zellweger syndrome) by the discovery of peroxisomal membrane ghosts in the mutant cells (Santos, M. J., T. Imanaka, H. Shio, G. M. Small and P. B. Lazarow. 1988. Science 239, 1536-1538). Introduction of a wild-type gene is thought to restore to the ghosts the ability to import matrix proteins, and thus lead to the refilling of the peroxisomes. It is vitally important to our understanding of peroxisome biogenesis to determine whether the yeast mutants contain ghosts. We have solved this problem by introducing an epitope-tagged version of Pas3p, a peroxisome integral membrane protein (that is essential for peroxisome biogenesis). Nucleotides encoding a nine amino acid HA epitope were added to the PAS3 gene immediately before the stop codon. The tagged gene (PAS3HA) was inserted in the genome, replacing the wild-type gene at its normal locus. It was fully functional (the cells assembled peroxisomes normally and grew on oleic acid) but the expression level was too low to detect the protein with monoclonal antibody 12CA5. PAS3HA was expressed in greater quantity from an episomal plasmid with the CUP1 promoter. The gene product, Pas3pHA, was detected by immunogold labelling on the membranes of individual and clustered peroxisomes; the clusters appeared as large spots in immunofluorescence. PAS3HA was similarly expressed in peroxisome biogenesis mutants peb2 and peb4, which lack morphologically recognizable peroxisomes. Gold-labelled membranes were clearly visible in both mutants: in peb2 the labelled membrane vesicles were generally much smaller than those in peb4, which resembled normal peroxisomes in size.