Peroxisomal Catalase in the Methylotrophic Yeast Candida boidinii : Transport Efficiency and Metabolic Significance

Autor: Nobuo Kato, Hirofumi Horiguchi, Tomoyuki Nakagawa, Yasuyoshi Sakai, Toh-Kheng Goh, Hiroya Yurimoto
Rok vydání: 2001
Předmět:
Zdroj: Journal of Bacteriology. 183:6372-6383
ISSN: 1098-5530
0021-9193
DOI: 10.1128/jb.183.21.6372-6383.2001
Popis: Catalase, which degrades H2O2 to oxygen and H2O, is present along with various types of H2O2-generating oxidases in the matrix of the peroxisome, an organelle found in virtually all eukaryotic cells. Because of this, catalase has been used for a long time as a marker enzyme for peroxisomes (6, 24, 52). A lack of catalase in peroxisomes is thought to result in accumulation of toxic H2O2 and/or other reactive oxygen species derived from H2O2 (7, 18, 44, 53, 60), and the physiological importance of catalase activity is shown by the existence of a genetic disease, known as acatalasemia (52). In addition, a defect in catalase import into the peroxisome has been reported to lead to a severe neurological disorder (44). The peroxiredoxine Pmp20 family is another peroxisomal antioxidant system capable of degrading H2O2 that has recently been identified in both mammalian and yeast cells (20, 57). The physiological significance of catalase is reevaluated in this report and compared with the function of members of the Pmp20 family of proteins. Peroxisomal proteins are encoded by nuclear genes, are synthesized on free polyribosomes, and, following translation, are imported into the peroxisome (24). The targeting of proteins to peroxisomes is mediated by cis-acting peroxisomal targeting signals (PTSs) and their corresponding receptors. PTSs are necessary and sufficient for peroxisomal targeting. At least three types of PTSs are known, including a C-terminal tripeptide, PTS1 (16); an NH2-terminal peptide, PTS2 (12, 13, 50); and mPTS, which is specific for an integral peroxisomal membrane protein (8, 27, 28). The PTS1 receptor, Pex5p, and the PTS2 receptor, Pex7p, have been shown to bind and recruit PTS1 and PTS2 proteins to peroxisomes, respectively (46). Another feature of peroxisomal protein import is the presence of some proteins that can be imported into peroxisomes in a folded oligomeric state. Such oligomeric transport has been demonstrated with artificial PTS1-tagged chloramphenicol acetyltransferase in yeast and human cells (29) and also with some native PTS1 and PTS2 proteins, including Saccharomyces cerevisiae thiolase (13), S. cerevisiae malate dehydrogenase 3 (9), human alanine/glyoxylate amino transferase 1 (26), and plant isocitrate lyase (25). In these studies, a reporter protein from which PTS had been deleted was shown to be transported into peroxisomes only when the same reporter protein harboring a PTS was coexpressed in the same cell. This finding was supported by the results of microinjection studies of human cell lines in which colloidal gold particles (diameter, 9 nm) that were coated with a human serum albumin-PTS1 conjugate were shown to be imported into the peroxisome (54). In contrast, two major methanol-induced peroxisomal proteins, dihydroxyacetone synthase and alcohol oxidase (AOD), seemed to differ in that these two proteins fold within peroxisomes after they are imported into these organelles (40, 56). There is no direct evidence that oligomeric transport of catalase occurs, but there is indirect evidence which suggests that oligomeric transport of catalase could occur. For example, in cells of patients with Zellweger syndrome, in which catalase and other peroxisomal matrix proteins are synthesized on ribosomes normally but are not transported across the peroxisomal membrane (52), catalase assembles into catalytically active tetramers in the cytosol (55). These cells can be divided into distinct complementation groups so that fusion of cells from different groups results in the appearance of catalase-containing peroxisomes (4, 45). However, studies performed with monoclonal antibodies specific for tetrameric or dimeric-monomeric catalase subunits showed that in contrast to what happens in rodent liver, human skin fibroblasts assemble cytosolic tetrameric catalase in the cytosol (within 1 h of synthesis), and this catalase is then targeted to peroxisomes for disassembly and import (30). We have used the methylotrophic yeast Candida boidinii extensively as a model organism to study peroxisomal metabolism and protein import. C. boidinii can grow on three metabolically distinct peroxisome-inducing carbon sources, methanol, fatty acids, and d-alanine (15, 38). When this organism is used in combination with a gene manipulation system (37, 41), the metabolic significance of a specific protein can be evaluated by examining the growth defect with cells grown on a peroxisome-inducing carbon source. For example, in a previous study we showed that the function of CbPmp20 is specific for methanol metabolism (20). In the present study we sought to evaluate the physiological contributions of peroxisomal catalase in various types of peroxisome metabolism. In addition, the following two aspects of peroxisomal import of catalase were studied: the efficiency of transport; and oligomeric transport in which green fluorescent protein (GFP) was used, which enabled us to visualize the localization of GFP-Cta1p fusion protein in living cells. We were able to show that the efficiency of catalase import into peroxisomes depends on the growth conditions (i.e., the carbon source). Furthermore, our findings suggest that the variations in PTSs in different proteins are related to the metabolic significance of each protein. In this report we show that not only peroxisomal metabolism but also the efficiency of peroxisomal protein transport can change depending on the environmental conditions.
Databáze: OpenAIRE
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