Part I Peroxisome Biogenesis
©Copyright 2000 by Humana Press, Inc. All rights of any nature whatsoever reserved. 1085-9195/00/32/001–008/$12.00
In Silicio Search for Genes Encoding Peroxisomal Proteins in Saccharomyces cerevisiae Arnoud J. Kal,1,3 Ewald H. Hettema,1 Marlene van den Berg,1 Marian Groot Koerkamp,1 Lodewijk van Ijlst,2 Ben Distel,1 and Henk F. Tabak* ,1 1
Departments of Biochemistry and 2Clinical Chemistry, University of Amsterdam, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands; 3 Present address: Imperial Cancer Research Fund, Gene Expression Control Laboratory, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK; E-mail: H. F.
[email protected] ABSTRACT The biogenesis of peroxisomes involves the synthesis of new proteins that after, completion of translation, are targeted to the organelle by virtue of peroxisomal targeting signals (PTS). Two types of PTSs have been well characterized for import of matrix proteins (PTS1 and PTS2). Induction of the genes encoding these matrix proteins takes place in oleate-containing medium and is mediated via an oleate response element (ORE) present in the region preceding these genes. The authors have searched the yeast genome for OREs preceding open reading frames (ORFs), and for ORFs that contain either a PTS1 or PTS2. Of the ORFs containing an ORE, as well as either a PTS1 or a PTS2, many were known to encode bona fide peroxisomal matrix proteins. In addition, candidate genes were identified as encoding putative new peroxisomal proteins. For one case, subcellular location studies validated the in silicio prediction. This gene encodes a new peroxisomal thioesterase.
Index Entries: Thioesterase; Saccharomyces cerevisiae; peroxisome; oleate-response element (ORE).
INTRODUCTION
exhibit no homology with proteins of other organisms (1). Identification of their function is one of the challenges for future research. From a given gene sequence, several types of information can be extracted. First, amino acid sequence homology searches can identify proteins of known function in other organisms. Second, sequences upstream of open reading frames (ORFs) can be analyzed for the presence of regulatory elements. Some
The completion of the yeast genome sequence has provided a catalog of all (putative) genes present in this model eukaryote. Roughly 2000 of the approx 6200 genes encode proteins with unknown function, and *Author to whom all correspondence and reprint requests should be addressed. Cell Biochemistry and Biophysics
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2 regulatory elements are specific for proteins in a certain metabolic pathway or cellular function, e.g., the HAP-binding sites are found in genes required for mitochondrial function, and the stress response element is located in promoters of genes that respond to stress. Third, the amino acid sequence can be analyzed for presence of short motifs that are required for protein targeting (e.g., endoplasmic reticulum [ER] retention signal) or protein modification (e.g., myristylation, phosphorylation). Most important, gene function predictions become more accurate when two or more characteristics of a certain class of genes are present in one and the same gene. For example, a gene containing both an unfolded protein response element and an ER retention signal is likely to encode a folding enzyme of the ER. The authors are interested in the biogenesis and metabolic functions of peroxisomes. Peroxisomes are organelles that contain enzymes for a number of metabolic pathways, e.g., for β-oxidation of fatty acids. In Saccharomyces cerevisiae, the peroxisomal compartment and its content can be induced when cells are shifted from glucose-containing medium to a medium containing the fatty acid, oleate, as sole carbon source, rendering cells fully dependent on peroxisomal β-oxidation for energy and carbon components. The induction of genes encoding peroxisomal enzymes is mediated via the transcription factors, Pip2p and Oaf1p (2,3), which bind as a heterodimer to the oleate response element (ORE) (4,5). The ORE is the only known UAS that is specific for genes encoding peroxisomal matrix proteins. Genes encoding peroxisomal proteins are also regulated by trans-acting factors like Abf1p, Adr1p, and RP-A, but these also act on promoters of genes that are not related to peroxisome function (6). Peroxisomal proteins are imported after binding to cytosolic receptors that target the protein to the peroxisomes. Two receptors and two corresponding targeting signals have been identified. The peroxisomal targeting signal type 1 (PTS1) is defined by a C-terminal tripeptide with amino acid sequence SKL, or a variant thereof, and is recognized by Pex5p, the PTS1 receptor (7–9). The peroxisomal targeting signal type 2 (PTS2) is defined by an N-terminal Cell Biochemistry and Biophysics
Kal et al. amino acid sequence, and is recognized by Pex7p, the PTS2 (10,11). To identify genes encoding putative peroxisomal proteins, the authors searched the yeast genome sequence for the presence of OREs, PTS1, and PTS2 sequences, and combinations thereof.
MATERIALS AND METHODS Pattern Searching For all searches, the Pattern Match program provided by the Saccharomyces Genome Database was applied (http://genome-www.stanford.edu/Sacch3D/patmatch.html). The PTS2 was searched within the first 70 amino acids of the coding regions. The PTS2 consensus was derived from Faber (12). For the PTS1, the three C-terminal amino acids were searched following the “two out of three ain’t bad” rule applied to SKL- and SKF-targeting signals (13). A consensus for the ORE was applied as reported from OREs found in promoters of well studied genes encoding peroxisomal proteins (2). OREs were searched within 500 bp upstream of each ORF.
Search strings PTS1: XKL>; XKF>; SXL>; SXF>; SKX> PTS2: indicates the Cterminus of the protein; [LVI] indicates an L, V, or I at that position; X indicates any amino acid, N indicates any nucleotide; {a,b} indicates repetition of the preceding character between a and b times.
Cloning, Expression, and Disruption of TES1 The TES1 gene was amplified via PCR on BJ1991 genomic DNA using primers P230 (5⬘GTC GAAT TCC AGC TAC AGG C-3⬘) and P232 (5⬘-TTT CTG CAG GAC CTT TTT CTA CTT AG-3⬘), digested with EcoRI and PstI, and cloned in EcoRI-PstI-digested pUC19, resulting Volume 32, 2000
In Silicio Gene Search in S. cerevisiae in pAK75. The TES1::LEU2 disruption construct was made by cloning the PvuII-BamHI fragment of pJJ252 (14) in MscI-BamHIdigested pAK75, resulting in pAK82. For generation of the tes1∆ knock-out strain, primers P230 and P232 were used to amplify the TES1::LEU2 fragment via PCR. The gel-purified fragment was transformed to BJ1991, and integrants were selected for growth on plates lacking leucine. Correct deletion was confirmed by Southern blotting (not shown). Constructs for expression of NH-tagged Tes1p were made as follows: The TES1 ORF was amplified via PCR on BJ1991 genomic DNA using primers P231 (5⬘-TTT TCT AGA ATG AGT GCT TCC AAA ATG GC-3⬘) and P232 (5⬘-TTT CTG CAG GAC CTT TTT CTA CTT AG-3⬘), digested with XbaI and PstI, and cloned behind the CTA1 promoter and NH-tag in XbaI-PstI digested pEW67 (CEN) and pEW68 (2µ) (E.H. Hettema), resulting in pAK80 and pAK81, respectively. The NH-tag has been described by Elgersma et al. (13).
Thioesterase Assay Thioesterase activity was measured in crude extracts from BJ1991, BJ1991tes1∆ and BJ1991 × pAK81, as described by Alexon and Nedergaard (15). Briefly, cell extracts were prepared in a buffer containing 200 mM Tris-HCl, pH 8.0, 10% (v/v) glycerol, 1 mM sodium-meta-bisulphite, and 1 mM ethylenediaminetetraacetic acid. Extracts were preincubated 3 min at room temperature in assay buffer containing 10 mM HEPES, pH 7.5, 50 mM KCl, 0.025% Triton X-100 and 0.3 mM 5,5⬘-dithiobis(nitrobenzoic acid). Subsequently, acyl-CoA (Sigma, St. Louis, MO) was added, to a final concentration of 50 mM, and thioesterase activity was followed spectrophotometrically as CoA production, measured as increase in absorbance at 412 nm.
Cell Biology Techniques Immunolabeling of ultrathin cryosections with polyclonal antibodies against the NHepitope was performed on oleate-grown cells fixed with 2% paraformaldehyde and 0.5% glutaraldehyde, as described (16). Cell fractionations were performed as described (17). Cell Biochemistry and Biophysics
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Fig. 1. Pattern searches identified 216 PTS1-containing genes, 95 PTS2 containing genes, and 648 genes containing a putative ORE. The Venn diagram shows the numbers of genes contained in each class.
Miscellaneous For the nomenclature of genes coding for cytoplasmic ribosomal proteins, the guidelines proposed by Mager et al. (18) were followed (also available via htp://speedy.mips.biochem.mpg.de/mips/).
RESULTS AND DISCUSSION Pattern searches for PTS1-containing proteins resulted in 216 candidate genes, the search for PTS2 containing proteins revealed 95 genes, and finally 648 putative OREs were found (Fig. 1). The number of putative targeting signals and OREs was very high, and it seemed unlikely that over 10% of all 6000 yeast genes contained a functional ORE. However, finding these large numbers was not unexpected, because search strings for the pattern searches were not very stringent. In this way, false positives would be expected, but the chance of missing a gene with a true PTS1, PTS2, or ORE would be small. All data were combined in an Microsoft Excel database and it was found that 34 genes contained both a PTS1 and an ORE, 16 genes contained both a PTS2 and an ORE, four genes contained both a PTS1 and a PTS2, and one gene showed all three characteristics (Fig. 1). All these genes are listed in Table 1. Volume 32, 2000
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Kal et al. Table 1 Table of all Genes Identified with Pattern Searching that Contain Combinations of PTS1, PTS2, and ORE patternsa
aIn
bold are indicated genes that are known to encode peroxisomal proteins. In bold italics are indicated genes encoding putative peroxisomal proteins.
Besides eight known genes encoding peroxisomal proteins (CTA1, FAA2, PCS60/FAT2, FOX2, MDH3, MLS1, SPS19, and POT1/ FOX3), a number of other genes were found. Some of these genes are clearly not related to Cell Biochemistry and Biophysics
peroxisome function, e.g., the GAL4 gene and genes encoding ribosomal proteins. The list also contained genes for which no function has been described yet. To filter out genes encoding putative peroxisomal proteins, Volume 32, 2000
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Fig. 2. Pileup of acyl-CoA thioesterases from E. coli, H. influenzae, S. cerevisiae, and man. The Pileup program from GCG (Wisconsin Package Version 9.0, Genetics Computer Group [GCG], Madison, WI) was used with gap creation penalty set at 12, and gap extension penalty set at 4, to prepare the figure. Identities were calculated using the GCG Bestfit program, with gap creation penalty set at 6, and gap extension penalty set at 2. Identities were 31% between S. cerevisiae and E. coli, 32% between S. cerevisiae and H. influenzae, and 31% between S. cerevisiae and man. Amino acid residues in E. coli, H. influenzae, and H. sapiens, which are identical to S. cerevisiae, are boxed. two additional criteria were tested. First, it was determined whether the putative OREs also fitted the more stringent ORE consensus, 5⬘-CGGNNNTNAN(2–5)TNACCG-3⬘, allowing two mismatches in the TNA blocks. Second, protein sequences encoded by these genes Cell Biochemistry and Biophysics
were used to search Genbank for homologs in other organisms. Twenty genes fulfilled the more stringent ORE consensus, and, for two genes, the homology searches resulted in the finding of considerable homology to known proteins that are possibly involved in peroxisome Volume 32, 2000
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Fig. 3. Thioesterase activity in crude extracts from oleate-grown wild-type BJ1991, BJ1991 transformed with centromeric (CEN), or multi copy (2µ) plasmids expressing NHtagged Tes1p from the CTA1 promoter. The activity found for extracts containing NHTes1p expressed from multicopy plasmids with C12 acyl-CoA as a substrate was set at 100%.
Fig. 4. TES1 mRNA induction is dependent on Pip2p. 7.5 µg of total RNA isolated from BJ1991 wild-type (WT) or BJ1991∆pip2 (∆pip2) was run on a 1% agarose gel (left panel, ethidium bromide staining), blotted to nitrocellulose, and probed with a 32P-labeled TES probe (right panel).
function: YJR019C and YNL009W. BLAST searches (19) demonstrated that the protein encoded by YJR019C had significant homology to acyl-CoA thioesterases from Escheridria coli and H. influenzae, and recently a human homolog was reported (20; Fig. 2). The yeast amino acid sequence ends in AKF, which conforms to the consensus PTS1 sequence. To study the function of the encoded gene product, the authors overexpressed the YJR019c gene in S. cerevisiae, and were able to show that increased thioesterase activity was present in the overexpression strain. The highest activity was toward medium-chain fatty acylCoA (C12–C14) (Fig. 3). The authors therefore propose TES1 as the gene name for YJR019C. Northern blotting showed that induction of TES1 depends on transcription factor Pip2p, as expected, on the basis of the ORE preceding the TES1 gene (Fig. 4). To study its subcellular location, Tes1p was tagged with a N-terminal epitope (NH-tag) (13), and it was observed that NH-tagged Tes1p is present in peroxisomes via immunoelectron microscopy (Fig. 5). Import of Tes1p was dependent on the PTS1 receptor, Pex5p, as demonstrated by
cell fractionation studies (not shown). A tes1∆ knock-out strain was able to grow on oleate and showed no obvious phenotype. Thus, the role of Tes1p in peroxisome metabolism remained unclear. The other ORF that was identified, YNL009w, showed high homology to nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate dehydrogenases from various organisms. For S. cerevisiae, two NADP-dependent isocitrate dehydrogenases were already known: a mitochondrial Idp1p and a cytoplasmic Idp2p. The authors showed that this enzyme is located in the peroxisomal matrix (22), and therefore propose to call the gene IDP3. IDP3 expression is oleate inducible and dependent for its expression on the transcription factor Pip2p. Idp3p is required for the regeneration of NADPH, which is consumed by the action of 2,4-dienoyl-CoA reductase when yeast cells grow on polyunsaturated fatty acids with double bonds at even positions (22). Although this in silicio search, combined with biochemical validation tests, was useful to identify new genes such as TES1 and IDP3,
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Fig. 5. Immunoelectron microscopy of oleate-grown wild-type BJ1991 cells transformed with a centromeric plasmid expressing NH-tagged Tes1p from the CTA1 promoter. Labeling was performed with a polyclonal anti-NH antiserum and gold-conjugated protein A. Bar = 0.5 µm. P, peroxisome; M, mitochondrion; N, nucleus; V, vacuole; F, fat droplet. some matrix proteins were missed because of the lack of PTS1 or PTS2, or because of the lack of an ORE. Examples are the gene encoding the acyl-CoA oxidase (FOX1/POX1), which lacks a PTS1 and PTS2, and the gene encoding peroxisomal citrate synthase 2 (CIT2), which lacks an ORE. These genes were present in the uncombined datasets, however. When new protein motifs are identified, e.g., specific targeting signals for peroxisomal membrane proteins, this type of search can be repeated to give new leads to identification of gene functions.
ACKNOWLEDGMENTS The authors thank Saccharomyces Genome Database curators Steve Chervitz and Catherine Ball for support in pattern searching, and Fred Wittkampf for advice in database programming. This work was financially supported by the Netherlands Foundation for Chemical Research/Netherlands Foundation for Scientific Research.
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