The carboxyl-terminal tripeptide serine-lysine-leucine of firefly luciferase is necessary but not sufficient for peroxisomal import in yeast.

Firefly luciferase is imported into peroxisomes in insects, mammals, plants, and yeast, which implies that the mechanism of protein translocation into peroxisomes has been conserved during eukaryotic evolution. The carboxyl-terminal tripeptide serine-lysine-leucine in luciferase acts as a peroxisomal import signal in mammalian cells. We have investigated whether this tripeptide is also involved in translocation of firefly luciferase into peroxisomes in yeast (Saccharomyces cerevisiae). We show by gene fusion experiments that the carboxyl-terminal 104 amino acids of luciferase can direct a heterologous protein to yeast peroxisomes. Luciferase mutant proteins were tested for their ability to be imported into yeast peroxisomes in vivo. We demonstrate that mutations in the carboxyl-terminal serine-lysine-leucine tripeptide abolish translocation of the protein into yeast peroxisomes. However, when a passenger protein was tagged at its carboxyl terminus with this tripeptide the fusion protein did not go to peroxisomes. These results indicate that, in yeast, the tripeptide is necessary but not sufficient for peroxisomal import.     

Peroxisomal localization and function of NADP-specific isocitrate dehydrogenases in yeast

Yeast peroxisomal NADP⁺-specific isocitrate dehydrogenase (IDP3) contains a canonical type I peroxisomal targeting sequence (a carboxyl-terminal Cys-Lys-Leu tripeptide), and provides the NADPH required for β-oxidation of some fatty acids in that organelle. Cytosolic yeast IDP2 carrying a PTS1 (IDP2^+CKL) was only partially localized to peroxisomes, and the enzyme was able to function in lieu of either peroxisomal IDP3 or cytosolic IDP2. The analogous isocitrate dehydrogenase enzyme (IDPA) from Aspergillus nidulans, irrespective of the presence or absence of a putative PTS1, was found to exhibit patterns of dual compartmental distribution and of dual function in yeast similar to those observed for IDP2^+CKL. To test a potential cellular limit on peroxisomal levels, authentic yeast IDP3, which is normally strictly peroxisomal, was over-expressed. This also resulted in dual distribution and function of the enzyme in both the cytosol and in peroxisomes, supporting the possibility of a restriction on organellar amounts of IDP.     

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.     

Peroxisomal protein import is conserved between yeast, plants, insects and mammals.

We have previously demonstrated that firefly luciferase can be imported into peroxisomes of both insect and mammalian cells. To determine whether the process of protein transport into the peroxisome is functionally similar in more widely divergent eukaryotes, the cDNA encoding firefly luciferase was expressed in both yeast and plant cells. Luciferase was translocated into peroxisomes in each type of organism. Experiments were also performed to determine whether a yeast peroxisomal protein could be transported to peroxisomes in mammalian cells. We observed that a C-terminal segment of the yeast (Candida boidinii) peroxisomal protein PMP20 could act as a peroxisomal targeting signal in mammalian cells. These results suggest that at least one mechanism of protein translocation into peroxisomes has been conserved throughout eukaryotic evolution.     

Import of human bifunctional enzyme into peroxisomes of human hepatoma cells in vitro.

A polypeptide containing the carboxyl-terminal fragment of human peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme was synthesized in vitro from its cDNA clone. This expression polypeptide was transported into purified rat liver peroxisomes. When the expression polypeptide was incubated with postnuclear supernatant fractions of human hepatoma cells and analyzed by Nycodenz gradient SDS-PAGE and fluorography, it was imported specifically into peroxisomes as indicated by its resistance to proteinase K degradation. A deletion of the last nine amino acid residues at the carboxyl-terminus of this polypeptide prevents its peroxisomal import. A tripeptide sequence, SKL, located at the carboxyl-terminus of human bifunctional enzyme appears to be the targeting signal for the peroxisomal importation of bifunctional enzyme in human cells.     

A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase.

Several peroxisomal proteins do not contain the previously identified tripeptide peroxisomal targeting signal (PTS) at their carboxy-termini. One such protein is the peroxisomal 3-ketoacyl CoA thiolase, of which two types exist in rat [Hijikata et al. (1990) J. Biol. Chem., 265, 4600-4606]. Both rat peroxisomal thiolases are synthesized as larger precursors with an amino-terminal prepiece of either 36 (type A) or 26 (type B) amino acids, that is cleaved upon translocation of the enzyme into the peroxisome. The prepieces are necessary for import of the thiolases into peroxisomes because expression of an altered cDNA encoding only the mature thiolase, which lacks any prepiece, results in synthesis of a cytosolic enzyme. When appended to an otherwise cytosolic passenger protein, the bacterial chloramphenicol acetyltransferase (CAT), the prepieces direct the fusion proteins into peroxisomes, demonstrating that they encode sufficient information to act as peroxisomal targeting signals. Deletion analysis of the thiolase B prepiece shows that the first 11 amino acids are sufficient for peroxisomal targeting. We conclude that we have identified a novel PTS that functions at amino-terminal or internal locations and is distinct from the C-terminal PTS. These results imply the existence of two different routes for targeting proteins into the peroxisomal matrix.     

Peroxisomal acetoacetyl-CoA thiolase of an n-alkane-utilizing yeast, Candida tropicalis.

Two genes encoding acetoacetyl-CoA thiolase (thiolase I; EC 2.3.1.9), whose localization in peroxisomes was first found with an n-alkane-utilizing yeast, Candida tropicalis, were isolated from the lambda EMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N-terminus, which were named as thiolase IA and thiolase IB. The calculated molecular masses were 41,898 Da for thiolase IA and 41,930 Da for thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that thiolase IA and thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl-CoA thiolase (65% amino acids of thiolase IA and 64% of thiolase IB were identical with this thiolase). Considering the evolution of thiolases, the C. tropicalis thiolases and S. uvarum cytosolic acetoacetyl-CoA thiolase are supposed to have a common origin. It was noticeable that the carboxyl-terminal regions of thiolases IA and IB contained a putative peroxisomal targeting signal, -Ala-Lys-Leu-COOH, unlike those of other thiolases reported hitherto.     

The peroxin Pex17p of the yeast Yarrowia lipolytica is associated peripherally with the peroxisomal membrane and is required for the import of a subset of matrix proteins.

PEX genes encode peroxins, which are required for the biogenesis of peroxisomes. The Yarrowia lipolytica PEX17 gene encodes the peroxin Pex17p, which is 671 amino acids in length and has a predicted molecular mass of 75,588 Da. Pex17p is peripherally associated with the peroxisomal membrane. The carboxyl-terminal tripeptide, Gly-Thr-Leu, of Pex17p is not necessary for its targeting to peroxisomes. Synthesis of Pex17p is low in cells grown in glucose-containing medium and increases after the cells are shifted to oleic acid-containing medium. Cells of the pex17-1 mutant, the original mutant strain, and the pex17-KA mutant, a strain in which most of the PEX17 gene is deleted, fail to form normal peroxisomes but instead contain numerous large, multimembraned structures. The import of peroxisomal matrix proteins in these mutants is selectively impaired. This selective import is not a function of the nature of the peroxisomal targeting signal. We suggest a regulatory role for Pex17p in the import of a subset of matrix proteins into peroxisomes.     

Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins

As part of an effort to understand how proteins are imported into the peroxisome, we have sought to identify the peroxisomal targeting signals in four unrelated peroxisomal proteins: human catalase, rat hydratase:dehydrogenase, pig D-amino acid oxidase, and rat acyl-CoA oxidase. Using gene fusion experiments, we have identified a region of each protein that can direct heterologous proteins to peroxisomes. In each case, the peroxisomal targeting signal is contained at or near the carboxy terminus of the protein. For catalase, the peroxisomal targeting signal is located within the COOH-terminal 27 amino acids of the protein. For hydratase:dehydrogenase, D-amino acid oxidase, and acyl-CoA oxidase, the targeting signals are located within the carboxy-terminal 15, 14, and 15 amino acids, respectively. A tripeptide of the sequence Ser-Lys/His-Leu is present in each of these targeting signals as well as in the peroxisomal targeting signal identified in firefly luciferase (Gould, S.J., G.-A. Keller, and S. Subramani. 1987. J. Cell Biol. 105:2923-2931). When the peroxisomal targeting signal of the hydratase:dehydrogenase is mutated so that the Ser-Lys-Leu tripeptide is converted to Ser-Asn-Leu, it can no longer direct proteins to peroxisomes. We suggest that this tripeptide is an essential element of at least one class of peroxisomal targeting signals.     

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.     

Importance of sequences adjacent to the terminal tripeptide in the import of a peroxisomal Candida tropicalis protein in plant peroxisomes

 The peroxisome targeting signal (PTS) required for import of the rat acyl-CoA oxidase (AOX; EC 1.3.3.6) and the Candida tropicalis multifunctional protein (MFP) in plant peroxisomes was assessed in transgenic Arabidopsis thaliana (L.) Heynh. The native rat AOX accumulated in peroxisomes in A. thaliana cotyledons and targeting was dependent on the presence of the C-terminal tripeptide S-K-L. In contrast, the native C. tropicalis MFP, containing the consensus PTS sequence A-K-I was not targeted to plant peroxisomes. Modification of the carboxy terminus to the S-K-L tripeptide also failed to deliver the MFP to peroxisomes while addition of the last 34 amino acids of the Brassica napus isocitrate lyase, containing the terminal tripeptide S-R-M, enabled import of the fusion protein into peroxisomes. These results underline the influence of the amino acids adjacent to the terminal tripeptide of the C. tropicalis MFP on peroxisomal targeting, even in the context of a protein having a consensus PTS sequence S-K-L. Received: 19 July 1999 / Accepted: 19 February 2000     

Expression of the Cymbidium ringspot virus 33-kilodalton protein in Saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal

Open reading frame 1 in the viral genome of Cymbidium ringspot virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.     

Characterization of human and murine PMP20 peroxisomal proteins that exhibit antioxidant activity in vitro.

We have isolated the cDNAs encoding human and mouse homologues of a yeast protein, termed peroxisomal membrane protein 20 (PMP20). Comparison of the amino acid sequences of human (HsPMP20) and mouse (MmPMP20) PMP20 proteins revealed a high degree of identity (93%), whereas resemblance to the yeast Candida boidinii PMP20A and PMP20B (CbPMP20A and CbPMP20B) was less (30% identity). Both HsPMP20 and MmPMP20 lack transmembrane regions, as do CbPMP20A and CbPMP20B. HsPMP20 mRNA expression was low in human fetal tissues, especially in the brain. In adult tissues, HsPMP20 mRNA was expressed in the majority of tissues tested. HsPMP20 and MmPMP20 contained the C-terminal tripeptide sequence Ser-Gln-Leu (SQL), which is similar to the peroxisomal targeting signal 1 utilized for protein import into peroxisomes. HsPMP20 bound directly to the human peroxisomal targeting signal 1 receptor, HsPEX5. Mutagenesis analysis showed that the C-terminal tripeptide sequence, SQL, of HsPMP20 is necessary for its binding to HsPEX5. Subcellular fractionation of HeLa cells, expressing epitope-tagged PMP20, revealed that HsPMP20 is localized in the cytoplasm and in a particulate fraction containing peroxisomes. Double-staining immunofluorescence studies showed colocalization of HsPMP20 and thiolase, a bona fide peroxisomal protein. The amino acid sequence alignment of HsPMP20, MmPMP20, CbPMP20A, and CbPMP20B displayed high similarity to thiol-specific antioxidant proteins. HsPMP20 exerted an inhibitory effect on the inactivation of glutamine synthetase in the thiol metal-catalyzed oxidation system but not in the nonthiol metal-catalyzed oxidation system, suggesting that HsPMP20 possesses thiol-specific antioxidant activity. In addition, HsPMP20 removed hydrogen peroxide by its thiol-peroxidase activity. These results indicate that HsPMP20 is imported into the peroxisomal matrix via PEX5p and may work to protect peroxisomal proteins against oxidative stress. Because some portion of PMP20 might also be present in the cytosol, HsPMP20 may also have a protective effect in the cytoplasm.     

Carnitine-dependent transport of acetyl coenzyme A in Candida albicans is essential for growth on nonfermentable carbon sources and contributes to biofilm formation

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid beta-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C(2) carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid beta-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid beta-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.     

An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes

Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320- 322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences. Analysis of PMP47- dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes. Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles.     

Identification of peroxisomal acyl-CoA thioesterases in yeast and humans

A computer-based screen of the Saccharomyces cerevisiae genome identified YJR019C as a candidate oleate-induced gene. YJR019C mRNA levels were increased significantly during growth on fatty acids, suggesting that it may play a role in fatty acid metabolism. The YJR019C product is highly similar to tesB, a bacterial acyl-CoA thioesterase, and carries a tripeptide sequence, alanine-lysine-phenylalanineCOOH, that closely resembles the consensus sequence for type-1 peroxisomal targeting signals. YJR019C directed green fluorescence protein to peroxisomes, and biochemical studies revealed that YJR019C is an abundant component of purified yeast peroxisomes. Disruption of the YJR019C gene caused a significant decrease in total cellular thioesterase activity, and recombinant YJR019C was found to exhibit intrinsic acyl-CoA thioesterase activity of 6 units/mg. YJR019C also shared significant sequence similarity with hTE, a human thioesterase that was previously identified because of its interaction with human immunodeficiency virus-Nef in the yeast two-hybrid assay. We report here that hTE is also a peroxisomal protein, demonstrating that thioesterase activity is a conserved feature of peroxisomes. We propose that YJR019C and hTE be renamed as yeast and human PTE1 to reflect the fact that they encode peroxisomal thioesterases. The physical segregation of yeast and human PTE1 from the cytosolic fatty acid synthase suggests that these enzymes are unlikely to play a role in formation of fatty acids. Instead, the observation that PTE1 contributes to growth on fatty acids implicates this thioesterase in fatty acid oxidation.     

The helicase CaHmi1p is required for wild-type mitochondrial DNA organization in Candida albicans

The mechanistic details of mtDNA maintenance in petite-negative yeasts have remained largely unexplored. We report here that the DNA helicase Hmi1p plays a crucial role in mtDNA stability in Candida albicans. Like its counterpart in Saccharomyces cerevisiae, Hmi1p in C. albicans (CaHmi1p) contains a C-terminal mitochondrial targeting signal that is functional in both organisms. Biochemical analysis demonstrates that CaHmi1p is a protein possessing ATP-dependent 3'-5' DNA-unwinding activity. Deletion of both HMI1 alleles does not lead to complete loss of mtDNA in C. albicans; however, substantial fragmentation of the wild-type mitochondrial genome, reduction of mtDNA mass and loss of wild-type nucleoid distribution occur. Specific regions of the mitochondrial genome give rise to mtDNA molecule populations with altered characteristics upon CaHMI1 deletion. Fragmentation of the mitochondrial genome can be reversed by reintroduction of CaHmi1p. This is the first time that a gene required for wild-type mtDNA maintenance in S. cerevisiae has been demonstrated to be nonessential in a petite-negative yeast.