Curr Genet (1998) 34: 297–302
© Springer-Verlag 1998
O R I G I N A L PA P E R
Vladimíra Dzˇugasová · Margita Obernauerová Katarína Horváthová · Mariana Vachová Martina Zˇáková · Július Sˇubík
Phosphatidylglycerolphosphate synthase encoded by the PEL1/PGS1 gene in Saccharomyces cerevisiae is localized in mitochondria and its expression is regulated by phospholipid precursors Received: 15 June / 15 July 1998
Abstract The PEL1/PGS1 gene of the yeast Saccharomyces cerevisiae is essential for the viability of rho–/rho° mutants and the normal cardiolipin content of cells. The PEL1-GFP fusion gene has been found to complement the pel1/pgs1 mutation and its fluorescent protein was localized to mitochondria similarly to the β-galactosidase activity of a protein encoded by the PEL1-lacZ fusion gene. The expression of the PEL1-lacZ reporter gene was repressed in cells grown in the presence of inositol and choline, reduced in the ino2 and ino4 strains, but constitutive in the opi1 null-mutant strain. The results demonstrate that Pel1p, playing a vital role in cells impaired in the mitochondrial DNA, is localized in the mitochondria and expressed in response to inositol and choline. Key words Mitochondria · Transcription regulation · PEL1/PGS1 · Phospholipid
Introduction
Mitochondria are essential organelles of yeast cells. Asymmetrically distributed phospholipids and proteins in their membranes are synthesised in both mitochondria and cytoplasm, from where they have to be imported into mitochondria (Lithgow et al. 1995; Daum and Vance 1997). The contribution of mitochondria to cellular phospholipid biosynthesis is probably restricted to some steps of phosphatidylethanolamine and cardiolipin formation. Cardiolipin, a negatively charged phospholipid, is specifically localV. Dzˇugasová · M. Obernauerová · K. Horváthová · M. Vachová M. Zˇáková · J. Sˇubík (½) Department of Microbiology and Virology, Comenius University, Mlynská dolina B2, 842 15 Bratislava, Slovak Republic e-mail :
[email protected] Tel.: +421-7-60296 631 Fax: +421-7-65429 064 Communicated by A. Goffeau
ized in mitochondrial membranes. Its biosynthesis requires three sequential reactions catalysed by phosphatidylglycerolphosphate synthase (Minskoff and Greenberg 1997) phosphatidylglycerolphosphate phosphatase (Kelly and Greenberg 1990) and cardiolipin synthase (Schlame and Greenberg 1997). Recently, the gene for cardiolipin synthase was cloned and found not to be essential for the growth of yeast cells on non-fermentable carbon sources (Jiang et al. 1997; Tuller et al. 1998). The activities of a number of phospholipid biosynthetic enzymes in the yeast Saccharomyces cerevisiae are repressed in response to the availability of the precursors inositol and choline (Paltauf et al. 1992; Greenberg and Lopes 1995) . The structural genes encoding these enzymes are subject to co-ordinate transcriptional control that requires ICRE (inositol/choline-responsive element) motifs in their promoters and is mediated by a common set of regulatory factors, including the products of the INO2, INO4 and OPI1 genes. Transcription activators Ino2p a Ino4p are members of the basic helix-loop-helix (bHLH) family of DNA-binding proteins, whereas the negative regulator Opi1p contains the leucine-zipper motif and extensive polyglutamine stretches (Paltauf et al. 1992; Greenberg and Lopes 1995; Schwank et al. 1997). S. cerevisiae is a petite-positive yeast which can survive deletion (rho–) or loss (rhoo) of mitochondrial DNA. Although their cells tolerate the impairment of some mitochondrial functions they cannot entirely dispense with mitochondria. In fact, the prevention of mitochondrial biogenesis due to intra-mitochondrial ATP deficiency (Sˇubík et al. 1972a; Kovácˇ et al. 1977; Gbelská et al. 1983), or the deletion of some genes encoding the components of the mitochondrial protein import machinery (Baker and Schatz 1991), led to the arrest of cell proliferation. Mutations in at least three other nuclear genes, AAC2 (Kovácˇová et al. 1968), ADC1 (Ciriacy 1976) and PEL1 (allelic to PGS1) (Sˇubík 1974; Janitor and Sˇubík 1993; Chang et al. 1998), convert the petite-positive S. cerevisiae strains to petitenegative ones that cannot survive ethidium-bromide mutagenesis. The AAC2 gene is known to encode the main mitochondrial ATP/ADP translocator (Lawson and Doug-
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las 1988; Kolarov et al. 1990) while ADC1 codes for a constitutive alcohol dehydrogenase (Bennetzen and Hall 1982). In order to gain more insight into the nucleo-mitochondrial interactions in eukaryotes we studied the biological role of the PEL1/PGS1 gene. The phenotype of the pel1/pgs1 mutations is pleiotropic. The mutant cells display a thermosensitive growth at 37°C, a reduced growth rate on fermentable and non-fermentable carbon sources, the absence of growth on minimal glycerol plus ethanol medium, a deficiency in cardiolipin content, and a synthetic-lethal phenotype after deletions either in the mitochondrial genome or in the CHO1 gene encoding phosphatidylserine synthase (Sˇubík 1974; Janitor and Sˇubík 1993; Janitor et al. 1995, 1996). The PEL1/PGS1 gene has been cloned and sequenced; its product was found to exhibit a significant homology with the phosphatidylserine synthase of Gram-negative bacteria (Janitor et al. 1995; Koonin 1996; Matsumoto 1997; Chang et al. 1998). Despite its structural homology with the phosphatidylserine synthases, the over-expression of the PEL1/PGS1 gene in the cho1 null mutant did not restore the wild-type properties of the transformed cells and failed to stimulate the incorporation of serine into total lipids of the intact cells. On the other hand, the cardiolipin deficiency of the pel1 mutant strain indicated that the PEL1/PGS1 gene could be involved in the biosynthesis of this mitochondrial phospholipid (Janitor et al. 1996). In fact, quite recently the PEL1/PGS1 gene was found to encode the phosphatidylglycerolphosphate synthase catalysing the committed and rate-limiting step in the biosynthesis of cardiolipin (Chang et al. 1998). In the present report we provide evidence to show that Pel1p is a mitochondrial protein which, like some other enzymes involved in phospholipid biosynthesis, is expressed in response to the availability of inositol and choline.
Materials and methods Yeast strains, media and growth conditions. The strains of S. cerevisiae used in this work are listed in Table 1. Strains were grown in semi-synthetic medium (0.5% peptone, 0.5% yeast extract, 0.5% or 2% glucose, 0.004% adenine and a mixture of inorganic salts) (Janitor and Sˇubík 1993). Synthetic medium contained 2% glucose, a mixture of inorganic salts, as well as vitamins (Janitor et al. 1996) and appropriate nutritional requirements. For inositol/choline repression, cells were grown with 0.2 mM inositol plus 2 mM choline. To achieve de-repressing conditions, cells were grown with 5 µM of inositol plus 5 µM of choline (Schwank et al. 1997). Media with glycerol contained 2% glycerol plus 1% ethanol instead of glucose. Solid media were prepared with 2% agar. The Escherichia coli strain XL1-Blue was used for transformation, plasmid amplification and propagation. Bacterial strains were grown in LB medium. When needed for plasmid maintenance, ampicillin was added at 50 µg/ml. Genetic transformation and ethidium-bromide mutagenesis. E. coli cells were transformed by the calcium-chloride procedure (Mandel and Higa 1970). For the transformation of S. cerevisiae cells, lithium acetate was used to induce competence (Ito et al. 1983). Respiratory deficient petite mutants from corresponding strains were prepared by ethidium-bromide (25 µM) mutagenesis (Slonimski et al. 1968).
Table 1 Strains of S. cerevisiae used in this work Strain Genotype DW4-2A GA74D/3A GA74D/3C SH302 SH303 SH304 SH307
MATα pel1 ade2-1 ura3-1 his3-11 trp1-1 MATα ade8 ura3 his3 trp1 leu2 MATa ade8 ura3 his3 trp1 leu2 pel1::HIS3 MATa trp1 his3 ura3 leu2 MATa trp1 his3 ura3 leu2 ino2::TRP1 MATa trp1 his3 ura3 leu2 opi1::LEU2 MATα trp1 his3 ura3 leu2 ino4::LEU2
Construction of the PEL1-GFP expression plasmid and fluorescence microscopy. Plasmid pBKC/GFP (Stratagene) containing the gene for the green fluorescent protein (GFP) was digested with PstI and HindIII. The resulting 783-bp fragment was inserted into pTZ19R to yield the plasmid pTZ19R/GFP. From this the 771-bp XbaI-HindIII fragment containing the gene encoding GFP was inserted into the XbaI and HindIII sites of the plasmid YEp352/PEL1 bearing the PEL1/GFP1 gene to yield YEp352/PEL1-GFP (Fig. 1A). The expressed Pel1-GFP fusion protein encoded by this plasmid contains a truncated Pel1p (deleted for 25 amino acids) linked at its C-terminus to GFP via an eight amino-acid linker (Fig. 1B). Epifluorescence micrographs were recorded on Kodak transparency film with an OLYMPUS B201 microscope using a suitable filter set (420–480 excitation, DM 500 dichroic reflector, and 515 emission filters). Preparation and analysis of DNA. Plasmid DNA from E. coli cells was prepared by the alkaline-lysis method. For restriction analysis, gel-electrophoresis and the purification of DNA fragments, standard protocols were followed (Sambrook et al. 1989). Isolation and proteinase treatment of mitochondria. Yeast mitochondria were isolated from spheroplasts (Daum et al. 1982) and their structural integrity was demonstrated by the efficiency of oxidative phosphorylation determined polarographically (Sˇubik et al. 1972b). In the presence of NADH, isolated mitochondria exhibited the respiratory control ratio 2.3 and their ADP/O ratio was 1.7. For proteinase treatment, mitochondria at a concentration of 1 mg/ml were incubated on ice in 0.6 M sorbitol, 10 mM Tris-Cl, pH 7.4, in the presence of 0.1 mg/ml of proteinase K and 0.5% Triton X-100 (Adrian et al. 1986; Jarosch et al. 1996). After incubation for 30 min, phenylmethylsulphonyl fluoride was added to 1 mM and β-galactosidase activities were immediately determined in triplicate samples.
β-galactosidase assays. For the analysis of β-galactosidase expression, transformants were grown overnight in synthetic medium to mid-log phase. Cells were broken by vortexing with glass beads (0.5mm diameter). Specific β-galactosidase activity was assayed in the crude cell extracts, in mitochondria, or in the post-mitochondrial supernatant as described by Rose and Botstein (1983). The proteins were determined by the method of Bradford (1976). For each host strain and growth condition, at least three independent yeast transformants were assayed in duplicate and each experiment was repeated three to five times. Specific β-galactosidase activities are given in nmol of o-nitrophenyl-β-D-galactoside hydrolysed per min per mg of protein (U/mg).
Results
The PEL1/PGS1 gene encodes a mitochondrial protein containing a functional pre-sequence Intracellular localization of Pel1p was determined using the expressed fusion genes. The PEL1/PGS1 gene deleted for the sequence encoding 25 C-terminal amino acids
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A
B
Fig. 1 Construction of the YEp352/PEL1-GFP reporter plasmid (A) and the structure of the Pel1-GFP fusion protein (B). Numbers correspond to amino-acid residues. The position of the protease cleavage site for removal of the mitochondrial targeting sequence (shaded region) is indicated by a vertical arrow
(Chang et al. 1998) was fused in-frame with the gene for the green fluorescent protein (GFP) of Aequorea victoria through an eight amino-acid linker (Fig. 1). After transformation the PEL1-GFP fusion gene was able to fully complement the pel1/pgs1 null mutation in the host strain GA74D/3C (Fig. 2). In contrast to the mutant strain transformed with the vector, the transformants bearing the PEL1GFP fusion gene like those with the intact PEL1/PGS1 gene were able to grow at 37 °C (Fig. 2B), grew on minimal medium containing glycerol plus ethanol (Fig. 2D) and survived ethidium-bromide mutagenesis (Fig. 2F). These re-
Fig. 2 A–F Complementation of the pel1 null mutation with the PEL1-GFP fusion gene in the GA74D/3C strain. Serial dilutions of transformants bearing the vector YEp352 (first line), centromere plasmid YCp50/PEL1 carrying the PEL1 gene (second line), and episomal plasmid YEp352/PEL1-GFP carrying the fusion reporter gene (third line) were spotted on the indicated media and growth was scored after 5 days. Semi-synthetic glucose medium at 30°C (A ) and 37°C (B). Semi-synthetic (C) and synthetic glycerol plus ethanol medium at 30°C (D). Growth on semi-synthetic glucose medium after ethidium-bromide mutagenesis of cells transformed with YEp352 (E) and YEp352/PEL1-GFP (F)
sults show that the C-terminal 25 amino acids are apparently not essential for the function of Pel1p, which can also tolerate a substantial addition at its C-terminus. When cells of the pel1/pgs1 mutant strains DW4-2A or GA74D/3C, transformed with plasmid YEp352/PEL1GFP and expressing the fusion protein, were examined by fluorescence microscopy a strong green fluorescence was co-localized with mitochondrial DNA visualised by DAPI (4,6-diamidino-2-phenylindole) (Fig. 3). The cytoplasm, nucleus and vacuoles were virtually free of green fluorescence. This pattern of fluorescence was found only in living cells and not in cells fixed in 50% ethanol. The ability of the Pel1p pre-sequence to target the protein into mitochondria was analysed using the PEL1-lacZ fusion gene. The gene for β-galactosidase in plasmid YEp357 was fused in-frame with the promoter and part of the coding region of PEL1 specifying the first 63 N-terminal amino acids (Janitor et al. 1996). The fusion gene allowed the generation of hybrid protein whose subcellular localization was followed by the determination of β-galactosidase activity. When examined in different subcellular fractions of the transformed cells the highest total and specific β-galactosidase activity was observed in the mitochondria. About a 10-times lower specific β-galactosidase
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Fig. 3 A, B Detection of the Pel1p-GFP fusion protein in living yeast cells. Yeast strain DW4-2A was transformed with YEp352/PEL1GFP and examined by fluorescence microscopy. A DNA in fixed cells stained with DAPI. B green fluorescence co-localized with the mitochondria
activity was found in total yeast cell extracts and the postmitochondrial supernatant. In intact isolated mitochondria the Pel1-β-galactosidase fusion protein was not accessible to proteinase K (Table 2). However, more than 87% of the β-galactosidase activity was inactivated by protease when the mitochondria were solubilized with 0.5% Triton-X100. Similar results were observed with the mitochondrially targeted hybrid protein containing the N-terminal part derived either from the yeast mitochondrial F1-ATPase β-subunit or from the ATP/ADP translocator encoded by the AAC2 gene (Adrian et al. 1986). PEL1/PGS1 gene expression responds to inositol and choline We analysed the 327-bp PEL1/PGS1 promoter region by a comparative search for binding sites of known transcripTable 2 Expression and localization of the Pel1-β-galactosidase fusion protein in the strain SH302 transformed with YEp352/PEL1-lacZ. Total units of β-galactosidase activity in the cell extract were 3609 U
Cell fractions
Total cell extract Mitochondria Post-mitochondrial supernatant
Fig. 4 Inositol/choline-dependent expression of the PEL1-lacZ fusion gene in wild-type and in ino2, ino4 or opi1 regulatory mutants. Strains SH302 (wild-type), SH303 (∆ino2), SH304 (∆opi1) and SH307 (∆ino4) were transformed with an episomal plasmid carrying the PEL1 promoter (–327 to +194 bp) fused to lacZ in the vector YEp357. Transformants were grown under inositol/choline-repressing (+IC) or de-repressing conditions (–IC). Error bars represent the standard deviation of the mean values
tion regulators encoded by the INO2 and INO4 genes. Interestingly, we identified two sequences, 5′-CATGTGTGAA3′ and 5′-CAAGTGAATG-3′, starting at positions –159 and –284 relative to the putative translation start site. A third sequence, 5-CATGTGCTAC-3, was found in the complementary strand at position –116 but in the opposite orientation. All three elements exhibit a significant similarity to the ICRE motif (consensus: 5′-CATGTGAAAT3′) previously described as the UAS of structural genes required for phospholipid biosynthesis (Paltauf et al. 1992; Greenberg and Lopes 1995; Schwank et al. 1997). Taking into account the ICRE-like motifs in the PEL1/PGS1 promoter and the cardiolipin deficiency in the pel1 mutant (Janitor et al. 1996) we investigated the expression of the PEL1/PGS1 gene in response to the soluble phospholipid precursors, inositol and choline. The PEL1-lacZ fusion gene was transformed into wild-type strain SH302 and its isogenic derivatives containing disrupted INO2, INO4 or OPI1 genes, whose specific β-galactosidase activities expressed under different growth conditions were determined. Transformants of the wildtype strain grown in log-phase in the presence of repress-
β-Galactosidase activity (U/mg protein)
% Total activity
41.8 ±3.1
100.0
397.1 ±28.9
75.3
28.8 ±2.5
24.7
Accessibility to proteinase K (% activity remaining) –Triton X-100
+Triton X-100
86.2
12.3
301
ing concentrations of inositol and choline (Schwank et al. 1997) displayed only 46% of the β-galactosidase activity found in the same strain grown under de-repressed conditions (Fig. 4). PEL1-lacZ activation in the ino2 and ino4 null mutants grown under either repressing or de-repressing conditions was reduced to 38% and 45% of the de-repressed wild-type level. On the other hand, a constitutive expression of the reporter gene was observed in the opi1 null-mutant strain, defective for a negative regulator of INO1 transcription (White et al. 1991). Thus, functional regulatory INO2 and INO4 genes are required for maximal expression of the PEL1 gene both under de-repressing conditions and in the absence of a functional OPI1 gene.
Discussion
The results reported here demonstrate that the phosphatidylglycerolphosphate synthase encoded by the PEL1/PGS1 gene (Chang et al. 1998) is a mitochondrial enzyme. Two lines of evidence are presented. First, the Pel1-GFP fusion protein expressed from a multicopy plasmid was shown to be co-localized with mitochondria in vivo. Second, Pel1p was found to possess a functional amino-terminal pre-sequence that was responsible for mitochondrial targeting of the β-galactosidase expressed by the Pel1-lacZ fusion protein. These results are corroborated by a high specific activity of phosphatidylglycerolphosphate synthase detected in mitochondrial extracts (Gaynor et al. 1991) as well as by purification of this enzyme from the mitochondrial membrane solubilized with Triton X-100 (Jiang et al. 1998). Like other genes specifying enzymes of fatty acid and phospholipid biosynthesis, the promoter of the PEL1/ PGS1 gene was also found to contain DNA sequences that match well with the ICRE motif (Nikoloff and Henry 1991; Paltauf et al. 1992). Apparently, at least some of them are functional since the expression of the PEL1-lacZ fusion gene was found to be regulated in response to the availability of inositol and choline, and required intact Ino2p and Ino4p transcription regulators. Individual genes of this family follow the same overall pattern of transcription regulation, but they show widely disparate repression ratios ranging from 2, as found also for the PEL1/PGS1 gene, to 30, as demonstrated for INO1 (Paltauf et al. 1992). Thus, in the yeast S. cerevisiae phosphatidyglycerolphosphate synthase expression is co-ordinately regulated with general phospholipid synthesis at the level of transcription and repressed when cells are grown in the presence of the phospholipid precursors inositol and choline. In addition, inositol is able to reduce the specific activity of phosphatidylglycerolphosphate synthase within minutes of its addition and this rapid effect is most likely the result of inactivation and/or degradation of the enzyme (Greenberg et al. 1988). Despite cardiolipin (Janitor et al. 1996) and phosphatidylglycerol (Chang et al. 1998) deficiency, the pel1/pgs1 mutant strain is able to grow on a semi-synthetic medium with non-fermentable carbon sources (Sˇubík 1974; Janitor
and Sˇubík 1993). Non-essentiality of cardiolipin for aerobic growth was also demonstrated in a mutant strain disrupted in the gene encoding cardiolipin synthase (Jiang et al. 1997; Tuller et al. 1998). Since at the same time the pel1/pgs1 mutant cells are not able to survive ethidiumbromide mutagenesis one can conclude that anionic cardiolipin or its precursor, phosphatidylglycerol, is essential for the viability of rho–/rho° mutants. Mitochondrial inner-membrane structure and mitochondrial biogenesis are probably impaired in the pel1rho– double mutant to such a degree that the integrity of mitochondria, or of some mitochondrial functions, will become limiting for normal cell proliferation, as has been previously observed with respiratory deficient yeast cells starved for intra-mitochondrial ATP (Sˇubík et al. 1972; Kovácˇ et al. 1977; Gbelská et al. 1983). In conclusion, the PEL1 gene is expressed in response to phospholipid precursors and encodes a mitochondrial enzyme that is essential both for the biogenesis of mitochondria and the viability of yeast cells impaired in mitochondrial DNA. Acknowledgements We are grateful to J. Nosek for help with fluorescence microscopy. We thank S. A. Henry and P. Griacˇ for the yeast strains containing disrupted INO2, INO4 and OPI1 genes. Thanks are also due to S. D. Kohlwein for useful discussions. This work was supported by grants from Comenius University, from the Scientific Grant Agency (VEGA) and from the Slovak Ministry of Education. The research of J. S. was also supported by an International Research Scholars Grant from the Howard Hughes Medical Institute.
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