Results Toxicity of HzOz Produced during Peroxisomal Oleate Metabolism in Catalase-lacking Yeast

The goal of this research is to identify and characterize the protein machinery that functions in the intracellular translocation and assembly of peroxisomal proteins in Saccharomyces cerevisiae. Several genes encoding proteins that are essential for this process have been identified previously by Kunau and collaborators, but the mutant collection was incomplete. We have devised a positive selection procedure that identifies new mutants lacking peroxisomes or peroxisomal function. Immunofluorescence procedures for yeast were simplified so that these mutants could be rapidly and efficiently screened for those in which peroxisome biogenesis is impaired. With these tools, we have identified four complementation groups of peroxisome biogenesis mutants, and one group that appears to express reduced amounts of peroxisomal proteins. Two of our mutants lack recognizable peroxisomes, although they might contain peroxisomal membrane ghosts like those found in Zellweger syndrome. Two are selectively defective in packaging peroxisomal proteins and moreover show striking intracellular clustering of the peroxisomes. The distribution of mutants among complementation groups implies that the collection of peroxisome biogenesis mutants is still incomplete. With the procedures described, it should prove straightforward to isolate mutants from additional complementation groups. p IEROXlSOME biogenesis occurs by the import of proteins posttranslationally, following synthesis on cytosolic free polyribosomes (19, 41). The membrane of the peroxisome grows by the incorporation of newly synthesized membrane proteins and, presumably, lipids. The peroxisome is capable of division by fission to form new peroxisomes, and segregation upon cell division, even when the membrane is nearly empty of the proteins that are normally packaged within it. This conclusion is based on studies of cells from human patients with Zellweger syndrome, in which peroxisomal membranes are present, but the import of proteins inside the peroxisomes is defective (27, 28). Three types of targeting sequences have been discovered thus far, each of which functions to direct some proteins to peroxisomes. A noncleaved carboxy-terminal SKL tripeptide is sufficient to target proteins to peroxisomes in several species (10, 11), and is present on many peroxisomal proteins (12). Variants of the SKL tripeptide function in several yeasts (1). Rat thiolase contains an amino-terminal, cleavable peroxisome targeting oligopeptide (24, 38). Candida tropicalis acyl-CoA oxidase has two internal, redundant topogenic peptides (35). Catalase A of Saccharomyces cerevisiae has both a noncleaved COOH-terminal SSNSKF and an internal topogenic sequence (17). To fully understand the import process, it is critical to Address all correspondence to Dr. Paul B. Lazarow, Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, Fifth Avenue and 100th Street, Box 1007, New York, NY 10029-6574. identify and characterize the proteins that make up the machinery for the translocation of newly synthesized proteins into peroxisomes. Several such proteins have been identified recently (7, 15, 31, 47). This progress has been the result of genetic experiments in which mutant cells that were defective in peroxisome biogenesis were isolated. The genes were then cloned by functional complementation and sequenced. The mutants were made in CHO cells (40, 50) and in several species of yeast (4-6, 13, 20, 42). Peroxisomes may be induced in S. cerevisiae by growth in the presence of fatty acids, such as oleate, and repressed by growth in glucose (45). Yeast peroxisomes can be isolated by cell fractionation (21, 39), and an in vitro import assay has been established to study the translocation of newly synthesized proteins into the organelle (39). These features make S. cerevisiae an attractive model organism for analysis of peroxisome biogenesis. Some peroxisome assembly mutants in S. cerevisiae were identified by screening for cells unable to use oleic acid as the sole carbon source (6). Several of these mutants had a phenotype resembling Zellweger syndrome, but there were far fewer than the nine complementation groups that have been demonstrated thus far among human patients (30, 48). Therefore, we set out to devise a positive selection procedure to find additional mutants. Strategy for Isolation o f Peroxisome Biogenesis Mutants In S. cerevisiae, fatty acid /3-oxidation occurs in peroxi© The Rockefeller University Press, 0021-9525/93/12/1133/15 $2.00 The Journal of Cell Biology, Volume 123, Number 5, December 1993 1133-11,'7 1133 on M ay 9, 2017 D ow nladed fom Published December 1, 1993