A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.

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.

Abstract. An efficient system for the import of newly synthesized proteins into highly purified rat liver peroxisomes was reconstituted in vitro. 35S-Labeled acyl-CoA oxidase (AOx) was incorporated into peroxisomes in a proteinase K-resistant fashion. This import was specific (did not occur with mitochondria) and was dependent on temperature, time, and peroxisome concentration. Under optimal conditions •30% of T hE biogenesis ofperoxisomes has unique features that distinguish it from the assembly of other organelles (21). In rat liver, for example, the peroxisomal matrix proteins, the core protein urate oxidase, and a major 22-kD integral membrane protein are all synthesized on free polyribosomes. Newly synthesized polypeptides are initially located in the cell cytosol and subsequently appear in peroxisomes, as shown by in vivo pulse-chase experiments. These findings imply the posttranslational import of newly synthesized polypeptides by preexisting peroxisomes. In this respect, peroxisome assembly resembles that of mitochondria and chloroplasts, although peroxisomes have only one membrane. In contrast to most other organelle proteins, peroxisomal proteins are generally synthesized at their final sizes and are not processed proteolytically upon import. Moreover, topogenic information is present in the carboxy-terminal region of the one peroxisomal protein so far studied (38).Practically no information is yet available on the details of the import mechanism, including the energy requirements (21). Bellion and Goodman have recently reported (2) that carbonylcyanide-m-chlorophenylhydrazone (CCCP) ~ prevents the import of alcohol oxidase into Candida boidinii peroxisomes. This effect is puzzling in view of the fact that peroxisomal membranes contain pores that allow the passage of molecules as large as 800 D (40). A requirement for ATP has emerged recently as a common factor in posttranslational Portions of this work have appeared in abstract form (1986. Eur. J. CellBiol.

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.

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 CITI 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.