Evidence for a novel pathway for the targeting of a Saccharomyces cerevisiae peroxisomal protein belonging to the isomerase/hydratase family.

We, and others, have identified a novel Saccharomyces cerevisiae peroxisomal protein that belongs to the isomerase/hydratase family. The protein, named Dci1p, shares 50% identity with Eci1p, a delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids. Both of these proteins are localized to peroxisomes, and both contain motifs at their amino- and carboxyl termini that resemble peroxisome targeting signals (PTS) 1 and 2. However, we demonstrate that the putative type 1 signaling motif is not required for the peroxisomal localization of either of these proteins. Furthermore, the correct targeting of Eci1p and Dci1p occurs in the absence of the receptors for the type 1 or type 2 peroxisome targeting pathway. Together, these data suggest a novel mechanism for the intracellular targeting of these peroxisomal proteins.

Global regulatory functions of Oaf1p and Pip2p (Oaf2p), transcription factors that regulate genes encoding peroxisomal proteins in Saccharomyces cerevisiae.

Two transcription factors, Oaf1p and Pip2p (Oaf2p), are key components in the pathway by which several Saccharomyces cerevisiae genes encoding peroxisomal proteins are activated in the presence of a fatty acid such as oleate. By searching the S. cerevisiae genomic database for the consensus sequence that acts as a target for these transcription factors, we identified 40 genes that contain a putative Oaf1p-Pip2p binding site in their promoter region. Quantitative Northern analysis confirmed that the expression of 22 of the genes identified is induced by oleate and that either one or both of these transcription factors are required for the activation. In addition to known peroxisomal proteins, the regulated genes encode novel peroxisomal proteins, a mitochondrial protein, and proteins of unknown location and function. We demonstrate that Oaf1p regulates certain genes in the absence of Pip2p and that both of these transcription factors play a role in maintaining the glucose-repressed state of one gene. Furthermore, we provide evidence that the defined consensus binding site is not required for the regulation of certain oleate-responsive genes.

A complex containing two transcription factors regulates peroxisome proliferation and the coordinate induction of beta-oxidation enzymes in Saccharomyces cerevisiae.

Expression of the POX1 gene, which encodes peroxisomal acyl coenzyme A oxidase in the yeast Saccharomyces cerevisiae, is tightly regulated and can be induced by fatty acids such as oleate. Previously we have shown that this regulation is brought about by interactions between trans-acting factor(s) and an upstream activating sequence (UAS1) in the POX1 promoter. We recently identified and isolated a transcription factor, Oaf1p, that binds to the UAS1 of POX1 and mediates its induction. A screening strategy has been developed and used to identify eight S. cerevisiae mutants, from three complementation groups, that are defective in the oleate induction of POX1. Characterization of one such mutant led to the identification of Oaf2p, a protein that is 39% identical to Oaf1p. Oaf1p and Oaf2p form a protein complex that is required for the activation of POX1 and FOX3 and for proliferation of peroxisomes. We propose a model in which these two transcription factors heterodimerize and mediate this activation process. The mutants that we have isolated, and further identification of the corresponding defective genes, provide us with an opportunity to characterize the mechanisms involved in the coordinate regulation of peroxisomal beta-oxidation enzymes.

Regulation of peroxisomal fatty acyl-CoA oxidase in the yeast. Saccharomyces cerevisiae.

Peroxisomes are specialized organelles found in most eukaryote cells, where their major functions are in cellular respiration and fatty acid oxidation. Proliferation of this organelle, and induction of peroxisomal enzymes, is a phenomenon that occurs in diverse species, and is stimulated by a number of physiological and pharmacological stimuli. A large number of chemically diverse compounds, including hypolipidemic drugs and industrial plasticizers, have been shown to cause peroxisome proliferation and the induction of peroxisomal enzymes in rodents. Chronic exposure to these compounds produces hepatocellular carcinomas, however, the mechanism by which this tumorigenic event occurs is unknown. In the yeast Saccharomyces cerevisiae peroxisomes are induced when a fatty acid such as oleate is supplied as a carbon source in the growth medium. In addition, many peroxisomal enzymes are induced by growth on oleate; these include enzymes of the peroxisomal beta-oxidation cycle. This regulation occurs at the transcription level, and is controlled by specific trans-acting factors. The research in our laboratory has focused on the mechanisms involved in this regulation, and on the identification and characterization of the proteins involved. Our recent results, and current research directions are summarized.

Evidence for the existence of a novel mechanism for the nuclear import of Hsc70.

Hsc70 is a multifunctional protein that is capable of shuttling between the nucleus and the cytoplasm. In this study we investigated the signal-mediated nuclear import step, using Xenopus oocytes as a model system. Purified rat hsc70, and hybrid proteins, which contained either the full-length or the mutated forms of rat hsc70 fused with the maltose binding protein, were labeled with 125I and used as transport substrates. In competition experiments, it was found that the nuclear import of neither purified hsc70 nor the full-length fusion protein was inhibited by an excess of SV40 large T or nucleoplasmin nuclear localization signals (NLSs) that were conjugated with BSA. Since hsc70 contains only a single basic domain (246KRKHKKDISENKRAVRR262), which has the characteristics of an NLS, we examined its role in nuclear import. It was determined, by conjugating this sequence with BSA, that it is capable of promoting nuclear import and, therefore, acts as a prototypical basic NLS. However, inactivation of this signal by deleting the first six amino acids (246KRKHKK251) had no effect on hsc70 import. Overall, the present results indicate that hsc70 utilizes a novel import signal and enters the nucleus by a different mechanism than that employed by simple and bipartite NLSs. The novel signal has not been identified, but we have obtained evidence that it is located in the amino terminus of hsc70.

Purification, identification, and properties of a Saccharomyces cerevisiae oleate-activated upstream activating sequence-binding protein that is involved in the activation of POX1.

Peroxisomes have a central function in lipid metabolism, and it is well established that these organelles are inducible by many compounds including fatty acids. Peroxisomes are the sole site for the beta-oxidation of fatty acids in yeast. The first and rate-limiting enzyme of this cycle is fatty acyl-CoA oxidase. The gene encoding this enzyme in Saccharomyces cerevisiae (POX1) undergoes a complex regulation that is dependent on the growth environment. When this yeast is grown in medium containing oleic acid as the main carbon source, peroxisomes are induced and POX1 expression is activated. When cells are grown in the presence of glucose, the expression of POX1 mRNA is repressed, whereas growth on a carbon source such as glycerol or raffinose causes derepression. This rigorous regulation is brought about by the complex interactions between trans-acting factors and cis-elements in the POX1 promoter. Previously, we characterized regulatory elements in the promoter region of POX1 that are involved in the repression and activation of this gene (Wang, T., Luo, Y., and Small, G. M. (1994) J. Biol. Chem. 269, 24480-24485). In this study we have purified and identified an oleate-activated transcription factor (Oaf1p) that binds to the activating sequence (UAS1) in the POX1 gene. The protein has a predicted molecular mass of approximately 118 kDa.

The POX1 gene encoding peroxisomal acyl-CoA oxidase in Saccharomyces cerevisiae is under the control of multiple regulatory elements.

Transcription of POX1, the gene encoding peroxisomal acyl-CoA oxidase in the yeast Saccharomyces cerevisiae, is controlled by the carbon source given for cell growth. The gene is repressed in glucose, derepressed in glycerol, and induced in oleate. This regulation is controlled by cis-acting elements in the 5' region of the gene, which bind regulatory proteins. By deletion analysis and DNA band shift assays, we have characterized three such elements in the POX1 upstream sequence, two upstream repression sequences and an upstream activating sequence. Each was able to regulate the transcription of a heterologous gene construct iso-1-cytochrome-c (CYC1)/lacZ.

Localization of glucocorticoid receptors and glucocorticoid receptor mRNAs in the rat cochlea.

The distribution of glucocorticoid (GR) receptor messenger RNAs (mRNAs) and GR receptors was studied by in situ hybridization histochemistry and immunocytochemistry, respectively. In situ hybridization histochemistry was performed with a biotin-labeled riboprobe complementary to rat GR receptor mRNA. GR receptor mRNAs were demonstrated in spiral ligament cells, spiral limbus cells, and spiral ganglion cells. GR receptor mRNAs were demonstrated neither in cells of the stria vascularis nor in cells of the organ of Corti region. With the use of a monoclonal and a polyclonal antibody, GR receptors were observed in the spiral ligament cells, stria vascularis cells, spiral limbus cells, and spiral ganglion cells by immunocytochemistry. Binding of anti-GR-receptor antibodies to a lesser extent was observed in the organ of Corti region; however, cellular distribution of the GR receptors could not be resolved with the applied techniques. These results suggest that the GR receptor is expressed differently in the heterogeneous cochlear tissues.

Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae.

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.

A negative regulating element controlling transcription of the gene encoding acyl-CoA oxidase in Saccharomyces cerevisiae.

Peroxisomes are induced in Saccharomyces cerevisiae when this yeast is grown in the presence of oleate, and are repressed when glucose is supplied as the carbon source. Concomitant with this is an induction/repression of peroxisomal beta-oxidation enzymes. We are investigating the transcriptional control of acyl-CoA oxidase, the first and rate-limiting enzyme in the peroxisomal beta-oxidation cycle. The promoter region of POX1 from S. cerevisiae has been analyzed in POX1/lacZ fusions. Expression of the POX1/lacZ fusion protein underwent glucose repression and oleate induction. By deletion, DNA band shift and DNase I footprinting analyses we have identified a region that is involved in transcriptional repression of POX1. Elimination of this DNA sequence results in constitutive expression of POX1 when S. cerevisiae is grown on a fermentable carbon source or glycerol.

Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.

The nucleotide sequence of POX18, a gene encoding a small oleate-inducible peroxisomal protein from Candida tropicalis.

We report the molecular cloning and nucleotide sequence of the nuclear gene, POX18, encoding an oleate-inducible peroxisomal protein from the yeast Candida tropicalis. POX18 has a single open reading frame of 381 nucleotides (nt), which encodes a protein of 127 amino acids. The predicted Mr of this protein is 13,792. Codon usage in the expression of POX18 is non-random, and shows a pattern similar to that used for other peroxisomal genes from C. tropicalis and highly expressed genes from Saccharomyces cerevisiae. Northern analysis of total RNA from oleate-grown cells determined that POX18 mRNA is approximately 750 nt in length. The POX18 gene was expressed in vitro, which resulted in a single translation product that co-migrated in denaturing polyacrylamide gels with an abundant peroxisomal protein (apparent mass of 16 kDa) and was immunoprecipitated by an antiserum against peroxisomal protein.

Zellweger syndrome amniocytes: morphological appearance and a simple sedimentation method for prenatal diagnosis.

Zellweger syndrome is the prototype of a growing group of genetic diseases caused by an absence or deficiency of peroxisomes. The defect causes the enzyme catalase to remain in the cytosol instead of being packaged into peroxisomes. This mislocalization can be easily detected by sedimentation analysis. Amniocytes were homogenized and then centrifuged to pellet organelles. Catalase was found to sediment with the peroxisomes in the homogenates of normal cells, but to remain in the supernatant with Zellweger syndrome amniocyte homogenates. This striking difference is unambiguous and reproducible, and provides a simple method for prenatal diagnosis. Moreover, it allows one to differentiate diseases in which peroxisomes are deficient from other peroxisomal diseases in which the organelle is intact, but one enzyme is defective. Electron microscopic observations support the biochemical determinations. Normal amniocytes contain small peroxisomes in which a weak cytochemical reaction for catalase may be demonstrated. Zellweger amniocytes appear to lack these organelles, although some cells have rare structures that might be residual or abnormal peroxisomes.

Acyl-CoA oxidase contains two targeting sequences each of which can mediate protein import into peroxisomes.

Acyl-CoA oxidase is a major induced enzyme in peroxisomes of Candida tropicalis grown on fatty acids. The gene, POX4, encoding acyl-CoA oxidase was expressed in vitro, and the resulting polypeptide was imported into purified peroxisomes in a temperature-dependent fashion. Plasmids containing fragments of POX4 were prepared, expressed and the polypeptides tested for import into peroxisomes. We identified two regions of acyl-CoA oxidase (amino acids 1-118 and 309-427) that contained information that specifically targeted fragments of acyl-CoA oxidase to peroxisomes. The corresponding regions of the gene were fused to cDNA encoding the cytosolic enzyme dihydrofolate reductase (DHFR), and the expressed fusion proteins were likewise imported into peroxisomes. DHFR itself neither bound to, nor was imported into peroxisomes. Thus, there are at least two regions of peroxisomal targeting information in the acyl-CoA oxidase gene.

Peroxisomal membrane ghosts in Zellweger syndrome--aberrant organelle assembly.

Peroxisomes are apparently missing in Zellweger syndrome; nevertheless, some of the integral membrane proteins of the organelle are present. Their distribution was studied by immunofluorescence microscopy. In control fibroblasts, peroxisomes appeared as small dots. In Zellweger fibroblasts, the peroxisomal membrane proteins were located in unusual empty membrane structures of larger size. These results suggest that the primary defect in this disease may be in the mechanism for import of matrix proteins.

Immunoblotting of hydrophobic integral membrane proteins.

For diagnosis and research purposes it is frequently desirable to measure by immunoblotting small amounts of proteins in complex mixtures such as tissue biopsy homogenates. Standard immunoblot procedures that give excellent results for soluble proteins unexpectedly gave low and irreproducible signals with some hydrophobic membrane proteins. We found that this was due to inefficient electrophoretic transfer to nitrocellulose, which could be corrected by modification of the transblot buffer. Hydrophobic integral membrane proteins of peroxisomes as well as other rat and human liver proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. The nitrocellulose-bound proteins were detected both by staining and by immunoblotting with an antiserum against the 22-kDa integral membrane protein of peroxisomes plus 125I-labeled protein A. A modified transblot buffer with 0.7 M glycine and 25 mM Tris (pH 7.7) but no methanol allowed use of a much shorter transfer time and strikingly improved the electrophoretic transfer of membrane proteins such that a peroxisomal integral membrane protein could be easily detected in human liver biopsy homogenates.

Peroxisomal integral membrane proteins in livers of patients with Zellweger syndrome, infantile Refsum's disease and X-linked adrenoleukodystrophy.

Livers from seven patients with peroxisome disorders, three with Zellweger syndrome, one with infantile Refsum's syndrome and three with X-linked adrenoleukodystrophy, were analysed by immunoblotting. The bifunctional protein catalysing two peroxisomal beta-oxidation reactions was deficient in all Zellweger livers and in the infantile Refsum's liver, consistent with the absence of morphologically recognizable peroxisomes. Three peroxisomal integral membrane proteins (IMPs) (69, 53 and 22 kDa) were present in normal amounts in all the Zellweger and adrenoleukodystrophy samples and they sedimented in a membrane fraction. These membrane proteins were also present in the infantile Refsum's liver. We suggest, on the basis of these results, that aberrant peroxisomal membranes may be present in Zellweger syndrome and that the defect is in the transport of matrix proteins into the organelle.