Developmental analysis of a putative ATP/ADP carrier protein localized on glyoxysomal membranes during the peroxisome transition in pumpkin cotyledons

In order to clarify the peroxisomal membrane proteins (PMPs), we characterized one of the major PMPs, PMP38. The deduced amino acid sequence for its cDNA in Arabidopsis thaliana contained polypeptides with 331 amino acids and had high similarity with those of Homo sapiens PMP34 and Candida boidinii PMP47 known as homologues of mitochondrial ATP/ADP carrier protein. We expected PMP38 to be localized on peroxisomal membranes, because it had the membrane peroxisomal targeting signal. Cell fractionation and immunocytochemical analysis using pumpkin cotyledons revealed that PMP38 is localized on peroxisomal membranes as an integral membrane protein. The amount of PMP38 in pumpkin cotyledons increased and reached the maximum protein level after 6 d in the dark but decreased thereafter. Illumination of the seedlings caused a significant decrease in the amount of the protein. These results clearly showed that the membrane protein PMP38 in glyoxysomes changes dramatically during the transformation of glyoxysomes to leaf peroxisomes, as do the other glyoxysomal enzymes, especially enzymes of the fatty acid beta-oxidation cycle, that are localized in the matrix of glyoxysomes.     

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.     

Arabidopsis 22-kilodalton peroxisomal membrane protein. Nucleotide sequence analysis and biochemical characterization.

We sequenced and characterized PMP22 (22-kD peroxisomal membrane protein) from Arabidopsis, which shares 28% to 30% amino acid identity and 55% to 57% similarity to two related mammalian peroxisomal membrane proteins, PMP22 and Mpv17. Subcellular fractionation studies confirmed that the Arabidopsis PMP22 is a genuine peroxisomal membrane protein. Biochemical analyses established that the Arabidopsis PMP22 is an integral membrane protein that is completely embedded in the lipid bilayer. In vitro import assays demonstrated that the protein is inserted into the membrane posttranslationally in the absence of ATP, but that ATP stimulates the assembly into the native state. Arabidopsis PMP22 is expressed in all organs of the mature plant and in tissue-cultured cells. Expression of PMP22 is not associated with a specific peroxisome type, as it is detected in seeds and throughout postgerminative growth as cotyledon peroxisomes undergo conversion from glyoxysomes to leaf-type peroxisomes. Although PMP22 shows increased accumulation during the growth of young seedlings, its expression is not stimulated by light.     

NADPH is a specific inhibitor of protein import into glyoxysomes

We have studied the import of proteins into glyoxysomes in vitro and show that this process is specifically inhibited by NADPH. NADPH affects both binding and translocation of proteins into glyoxysomes, and inhibition is determined by the ratio of NADP⁺ to NADPH. The site of action of NADPH is most likely within the glyoxysome because (1) pretreatment of glyoxysomes with NADPH, followed by re-isolation of the organelles prior to the import assay, resulted in inhibition of import that could be restored by the addition of NADP⁺; (2) low concentrations of NADPH inhibited binding of proteins to broken glyoxysome membranes. The sensitivity of protein import to inhibition by NADPH declines as glyoxysomes are converted to leaf-type peroxisomes. A model is proposed that speculates on a possible role for NADPH in regulating protein import into plant peroxisomes.     

Saccharomyces cerevisiae pex3p and pex19p are required for proper localization and stability of peroxisomal membrane proteins

The mechanisms by which peroxisomal membrane proteins (PMPs) are targeted to and inserted into membranes are unknown, as are the required components. We show that among a collection of 16 Saccharomyces cerevisiae peroxisome biogenesis (pex) mutants, two mutants, pex3Delta and pex19Delta, completely lack detectable peroxisomal membrane structures and mislocalize their PMPs to the cytosol where they are rapidly degraded. The other pexDelta mutants contain membrane structures that are properly inherited during vegetative growth and that house multiple PMPs. Even Pex15p requires Pex3p and Pex19p for localization to peroxisomal membranes. This PMP was previously hypothesized to travel via the endoplasmic reticulum (ER) to peroxisomes. We provide evidence that ER-accumulated Pex15p is not a sorting intermediate on its way to peroxisomes. Our results show that Pex3p and Pex19p are required for the proper localization of all PMPs tested, including Pex15p, whereas the other Pex proteins might only be required for targeting/import of matrix proteins.     

Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae

A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.     

Immunoelectron microscopic evidence for organ differences in the composition of peroxisome-specific membrane polypeptides among three rat organs: liver, kidney, and small intestine

We examined the distribution of peroxisome-specific membrane polypeptides (PMPs) among peroxisomes of the liver, renal cortex, and jejunal mucosa, using antibodies for 70 KD, 26 KD and 22 KD PMPs. Immunoblot analysis showed signals for 70 KD polypeptide in all three kinds of tissue, but for the other two only in the liver and renal cortex, with neither being detected in jejunal mucosa. The total amounts of PMPs increased in all three organs with DEHP (di-(2-ethylhexyl)phthalate) administration. By immunoelectron microscopic analysis using protein A-gold, the three PMPs were localized along the peroxisomal membrane. Quantitation of the gold particles associated with the peroxisomal membrane showed an increase in the density of 70 KD and 26 KD PMPs but a decrease in 22 KD PMP with the administration of DEHP. The presence of tissue-specific localizations of PMPs suggest the 70 KD PMP is a common constituent of peroxisomes of these three tissues, whereas 26 KD and 22 KD PMPs are absent in microperoxisomes of jejunal mucosal epithelium.     

Light-quality effects on Arabidopsis development. Red, blue and far-red regulation of flowering and morphology

Arabidopsis thaliana (L.) Heynh. race Columbia plants were grown in red. blue, red + far-red, blue + far-red and various light mixtures of red + blue + far-red light under 14 h light/10 h dark photoperiods. Each single light source and light mixture maintained a constant irradiance (50 μmol m⁻² s⁻¹) and the mixtures of red + blue + far-red maintained a constant ratio of red/far-red light, but varied in the ratio of blue to red + far-red light. Depending on the method used for calculation, values of the fraction of phytochrome in the far-red absorbing form (P_fr/P_tot) for these light mixtures were either constant or decreased slightly with increasing percentage of blue light in the mixtures. Arabidopsis flowered early (20 days) in blue, blue + far-red and red + far-red light and late (55 days) in red light. In mixtures of red + blue + far-red light, each of which established a nearly constant P_fr/P_tot flowering was in direct relation to time and irradiance level of blue light. Leaf area and petiole length were also correlated with blue light irradiance levels.     

Presence of peroxisomal membrane proteins in liver and fibroblasts from patients with the Zellweger syndrome and related disorders: evidence for the existence of peroxisomal ghosts

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.     

Cytochemical localization of glycolate oxidase in microbodies (glyoxysomes and peroxisomes) of higher plant tissues with the CeCl3 technique

Summary Alpha hydroxy acid oxidase activity (using glycolate as substrate) was demonstrated cytochemically in leaf-type peroxisomes, glyoxysomes, and unspecialized peroxisomes of higher plant tissues with the CeCl₃ technique in which cerous ions react with enzyme-generated H₂O₂ to form insoluble, electron-dense cerium perhydroxide. In all peroxisomes examined, reaction product was deposited throughout the matrices. None of the three types of microbody inclusions (crystals, amorphous nucleoids, or fibrillar, threadlike structures) observed in leaftype peroxisomes showed cytochemical reactivity. However, results with crystal-containing peroxisomes of guayule and tobacco leaves indicate an intimate association of glycolate oxidase with the crystals; reaction product was deposited in the spaces between the structural units of the crystal.Prolonged (18- versus 3-hour) incubation with glycolate and CeCl₃ were required for reliable cytochemical reactivity in glyoxysomes of castor bean endosperm and unspecialized peroxisomes of barley coleoptile, both of which contain relatively low enzyme activity. The CeCl₃ procedure may prove useful for helping identify microbodies observed with the electron microscope as peroxisomes. The lack of significant background deposits, and resolution of reaction product within crystals, illustrate qualities of the CeCl₃ procedure superior to those of the ferricyanide-reduction method, which was previously used to localize glycolate oxidase in higher plant microbodies.     

Lecithin synthesis during microbody biogenesis in watermelon cotyledons

During the normal development of watermelon seedlings, leaf peroxisomes succeed glyoxysomes as the major microbody component in the cotyledons. The possibility has thus been raised that the two organelles are ontogenetically related; that leaf peroxisomes are derived from glyoxysomes. The behavior of lecithin, an important constituent of the membranes of both kinds of organelle was examined in this study. Using labeled choline as a precursor of lecithin, its incorporation into various membrane fractions was followed during the period when glyoxysomal activity was declining and that of leaf peroxisomes increasing after exposure to light. The results showed that glyoxysomal membrane was selectively destroyed during this period. Furthermore, from double-labeling experiments using [¹⁴C]- and [³H]choline it was shown that newly synthesized lecithin was incorporated into the membranes of the developing leaf peroxisomes. These results support the thesis that leaf peroxisomes are not derived from glyoxysomes and instead represent two distinct microbody populations.     

Antibodies against pex14p block ATP-independent binding of matrix proteins to peroxisomes in vitro.

The membrane protein Pex14p is a key component of the protein import machinery of peroxisomes. Antibodies raised against human Pex14p recognise a 66 kDa protein in sunflower glyoxysomes (HaPex14p) and immunoprecipitate in vitro-translated Arabidopsis Pex14p (AtPex14p). These antibodies inhibit the ATP-independent binding to sunflower peroxisome membranes of peroxisome targeting signal type (PTS) 1- and PTS2-targeted matrix proteins, but not an integral membrane protein. These results suggest that Pex14p functions before the ATP-dependent step of peroxisome assembly.     

Activated oxygen‐mediated metabolic functions of leaf peroxisomes

Peroxisomes are subcellular organelles with an essentially oxidative type of metabolism. The presence in these organelles of superoxide dismutases and the generation of superoxide radicals (O₂^??) was first demonstrated in plant tissues and in recent years different experimental evidence has suggested the existence of cellular functions related to activated oxygen species. Some of these functions are analyzed in this work. In purified intact peroxisomes from pea (Pisum sativum L.) leaves, xanthine oxidase and urate oxidase were found to be present. The occurrence and the level of the metabolites xanthine, hypoxanthine, uric acid, and allantoin were studied in extracts of pea leaf peroxisomes by HPLC. Xanthine, uric acid, and allantoin were detected in peroxisomes. These results suggest a cellular role for leaf peroxisomes in the catabolism of purines. In peroxisomal membranes, 3 polypeptides (PMPs) with molecular masses of 18, 29 and 32 kDa, respectively, have been shown to generate superoxide radicals. These PMPs were purified from pea leaf peroxisomal membranes and characterized. While the 18- and 32-kDa PMPs use NADH as electron donor for O₂^?? production, the 29-kDa PMP was clearly dependent on NADPH. Very recently, the occurrence in pea leaf peroxisomes of all the enzymes of the ascorbate-glutathione cycle has been demonstrated. NADPH is required for the glutathione reductase activity of the cycle and this implies the reduction of NADP⁺ to NADPH. This recycling function could be carried out by the NADP-dependent glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and isocitrate dehydrogenase (ICDH). These 3 dehydrogenases have been demonstrated to be present in the matrix of pea leaf peroxisomes. The catabolism of purines, the superoxide-generating PMPs, the ascorbate-glutathione cycle, and the dehydrogenase-mediated recycling of NADPH, are activated oxygen roles of leaf peroxisomes that add to other functions previously known for peroxisomes from eukaryotic cells.     

Targeting signals in peroxisomal membrane proteins

Peroxisomal membrane proteins (PMPs) are encoded by the nuclear genome and translated on cytoplasmic ribosomes. Newly synthesized PMPs can be targeted directly from the cytoplasm to peroxisomes or travel to peroxisomes via the endoplasmic reticulum (ER). The mechanisms responsible for the targeting of these proteins to the peroxisomal membrane are still rather poorly understood. However, it is clear that the trafficking of PMPs to peroxisomes depends on the presence of cis-acting targeting signals, called mPTSs. These mPTSs show great variability both in the identity and number of requisite residues. An emerging view is that mPTSs consist of at least two functionally distinct domains: a targeting element, which directs the newly synthesized PMP from the cytoplasm to its target membrane, and a membrane-anchoring sequence, which is required for the permanent insertion of the protein into the peroxisomal membrane. In this review, we summarize our knowledge of the mPTSs currently identified.     

Evaluation of the role of the endoplasmic reticulum-Golgi transit in the biogenesis of peroxisomal membrane proteins in wild type and peroxisome biogenesis mutant CHO cells

Peroxisomes are thought to be formed by division of pre-existing peroxisomes after the import of newly synthesized proteins. However, it has been recently suggested that the endoplasmic reticulum (ER) provides an alternative de novo mechanism for peroxisome biogenesis in some cells. To test a possible role of the ER-Golgi transit in peroxisome biogenesis in mammalian cells, we evaluated the biogenesis of three peroxisomal membrane proteins (PMPs): ALDRP (adrenoleukodystrophy related protein), PMP70 and Pex3p in CHO cells. We constructed chimeric genes encoding these PMPs and green fluorescent protein (GFP), and transiently transfected them to wild type and mutant CHO cells, in which normal peroxisomes were replaced by peroxisomal membrane ghosts. The expressed proteins were targeted to peroxisomes and peroxisomal ghosts correctly in the presence or absence of Brefeldin A (BFA), a drug known to block the ER-Golgi transit. Furthermore, low temperature did not disturb the targeting of Pex3p-GFP to peroxisomes. We also constructed two chimeric proteins of PMPs containing an ER retention signal "DEKKMP": GFP-ALDRP-DEKKMP and myc- Pex3p-DEKKMP. These proteins were mostly targeted to peroxisomes. No colocalization with an ER maker was found. These results suggest that the classical ER-Golgi pathway does not play a major role in the biogenesis of mammalian PMPs.     

PHOTOMORPHOGENESIS OF THE GAMETOPHYTES OF LYGODIUM JAPONICUM

Protonemata of Lygodium japonicum turn biplanar in both red and blue light regimes and remain filamentous in far-red light. Biplanar gametophytes formed in red light are longer than broad with long, rectangular cells, whereas in blue light they appear broader than long with short, isodiametric cells. Transfer of protonemata of all ages from far-red regime to red or blue light induces a morphological type of growth characteristic of the new light regime. However, only relatively young biplanar forms transferred from red or blue light are able to resume filamentous type of growth in a subsequent regime of far-red light.     

Characterization of the targeting signal of the Arabidopsis 22-kD integral peroxisomal membrane protein

Using a combination of in vivo and in vitro assays, we characterized the sorting pathway and molecular targeting signal for the Arabidopsis 22-kD peroxisome membrane protein (PMP22), an integral component of the membrane of all peroxisomes in the mature plant. We show that nascent PMP22 is sorted directly from the cytosol to peroxisomes and that it is inserted into the peroxisomal boundary membrane with its N- and C-termini facing the cytosol. This direct sorting of PMP22 to peroxisomes contrasts with the indirect sorting reported previously for cottonseed (Gossypium hirsutum) ascorbate peroxidase, an integral PMP that sorts to peroxisomes via a subdomain of the endoplasmic reticulum. Thus, at least two different sorting pathways for PMPs exist in plant cells. At least four distinct regions within the N-terminal one-half of PMP22, including a positively charged domain present in most peroxisomal integral membrane-destined proteins, functions in a cooperative manner in efficient peroxisomal targeting and insertion. In addition, targeting with high fidelity to peroxisomes requires all four membrane-spanning domains in PMP22. Together, these results illustrate that the PMP22 membrane peroxisomal targeting signal is complex and that different elements within the signal may be responsible for mediating unique aspects of PMP22 biogenesis, including maintaining the solubility before membrane insertion, targeting to peroxisomes, and ensuring proper assembly in the peroxisomal boundary membrane.     

Multiple Domains in PEX16 Mediate Its Trafficking and Recruitment of Peroxisomal Proteins to the ER

Peroxisomes rely on a diverse array of mechanisms to ensure the specific targeting of their protein constituents. Peroxisomal membrane proteins (PMPs), for instance, are targeted by at least two distinct pathways: directly to peroxisomes from their sites of synthesis in the cytosol or indirectly via the endoplasmic reticulum (ER). However, the extent to which each PMP targeting pathway is involved in the maintenance of pre‐existing peroxisomes is unclear. Recently, we showed that human PEX16 plays a critical role in the ER‐dependent targeting of PMPs by mediating the recruitment of two other PMPs, PEX3 and PMP34, to the ER. Here, we extend these results by carrying out a comprehensive mutational analysis of PEX16 aimed at gaining insights into the molecular targeting signals responsible for its ER‐to‐peroxisome trafficking and the domain(s) involved in PMP recruitment function at the ER. We also show that the recruitment of PMPs to the ER by PEX16 is conserved in plants. The implications of these results in terms of the function of PEX16 and the role of the ER in peroxisome maintenance in general are discussed.      

PEX19 binds multiple peroxisomal membrane proteins, is predominantly cytoplasmic, and is required for peroxisome membrane synthesis

Peroxisomes are components of virtually all eukaryotic cells. While much is known about peroxisomal matrix protein import, our understanding of how peroxisomal membrane proteins (PMPs) are targeted and inserted into the peroxisome membrane is extremely limited. Here, we show that PEX19 binds a broad spectrum of PMPs, displays saturable PMP binding, and interacts with regions of PMPs required for their targeting to peroxisomes. Furthermore, mislocalization of PEX19 to the nucleus leads to nuclear accumulation of newly synthesized PMPs. At steady state, PEX19 is bimodally distributed between the cytoplasm and peroxisome, with most of the protein in the cytoplasm. We propose that PEX19 may bind newly synthesized PMPs and facilitate their insertion into the peroxisome membrane. This hypothesis is supported by the observation that the loss of PEX19 results in degradation of PMPs and/or mislocalization of PMPs to the mitochondrion.