Characterization, localization and functional analysis of Gpr1p, a protein affecting sensitivity to acetic acid in the yeast Yarrowia lipolytica

Antje Augstein1, Kathrin Barth1,{dagger}, Marcus Gentsch1, Sepp D. Kohlwein2 and Gerold Barth1

1 Institute of Microbiology, Dresden University of Technology, Mommsenstrasse 13, D-01062 Dresden, Germany
2 Department of Molecular Biology, Biochemistry and Microbiology, SFB Biomembrane Research Center, University Graz, Schubertstrasse 1, A-8010 Graz, Austria

Correspondence
Gerold Barth
gbarth{at}rcs.urz.tu-dresden.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Adaptation of cells to acetic acid requires a hitherto unknown number of proteins. Studies on the GPR1 gene and its encoded protein in the ascomycetous fungus Yarrowia lipolytica have revealed an involvement of this protein in the molecular processes of adaptation to acetic acid. Gpr1p belongs to a novel family of conserved proteins in prokaryotic and eukaryotic organisms that is characterized by the two motifs (A/G)NPAPLGL and SYG(X)FW (GPR1_FUN34_YaaH protein family). Analysis of four trans-dominant mutations and N-terminal deletion analysis of Gpr1p identified the amino acid sequence FGGTLN important for function of this protein in Y. lipolytica. Deletion of GPR1 slowed down adaptation to acetic acid, but had no effect on growth in the presence of acetic acid. Expression of GPR1 is induced by acetic acid and moderately repressed by glucose. It was shown by subcellular fractionation that Gpr1p is an integral membrane protein, which is also suggested by the presence of five to six putative transmembrane spanning regions. Fluorescence microscopy confirmed a localization to the plasma membrane. A model is presented describing a hypothetical function of Gpr1p during adaptation to acetic acid.


The EMBL accession number for the sequence reported in this paper is AJ313508.

{dagger}Present address: Institute of Anatomy, Dresden University of Technology, Fetscherstrasse 74, D-01307 Dresden, Germany.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Weak organic acids like acetic acid or lactic acid are widely used for preservation of food and beverages. However, several weak acid-tolerant micro-organisms can adapt to even higher concentrations of these compounds and can utilize them as sources for energy and carbon. The toxicity of weak acids is dependent on the pH of the environment. At low pH weak acids are mainly present in the undissociated form and will enter the cell by passive diffusion (Sikkema et al., 1995; van der Rest et al., 1995; Casal et al., 1996; Geros et al., 2000). Inside the cell the acids dissociate due to the more neutral pH and release protons, leading to a decrease of intracellular pH which interferes with several metabolic pathways (Krebs et al., 1983). Weak acids cause several strong changes in intracellular processes, for example in nutrient and ion transport, membrane structure, in fatty acid and phospholipid composition, as well as in protein synthesis (Sikkema et al., 1995). Although these intracellular effects are well documented, very little is known about the molecular mechanisms underlying the cellular response and the adaptation to these stress conditions (van der Rest et al., 1995). However, from these studies a model emerged that weak acids trigger a two-step process of adaptation and assimilation.

Piper et al. (1997, 1998) have shown that sorbic acid, ethanol and heat shock induce two plasma membrane proteins, Hsp30p and Pdr12p, in the yeast Saccharomyces cerevisiae. Hsp30p is involved in adaptation to weak acid by regulating the activity of the membrane H+-ATPase (Braley & Piper, 1997; Piper et al., 1997). Cells lacking Hsp30p are delayed in adaptation to sorbate. Pdr12p is a member of the ABC transporter protein family and is essential for the adaptation of S. cerevisiae cells to growth under weak acid stress, because pdr12-deleted cells are hypersensitive to sorbic, benzoic, propionic and acetic acids (Piper et al., 1998). It was shown that Pdr12p exports intracellularly accumulated anions of chain length from C1 to C7 out of the cell (Holyoak et al., 1999).

Tenreiro et al. (2000) identified the Azr1 protein, encoded by the ORF YGR224w in S. cerevisiae, which is required for adaptation to acetic acid, but which is not essential for utilization of this carbon source. Azr1p is a plasma membrane transporter of the major facilitator superfamily and is involved in multiple-drug resistance to azoles. The in vivo function of this protein in the puzzle of acetic acid resistance is still not clear. Azr1p does not export acetate anions out of the cell but may indirectly influence acetic acid uptake (Tenreiro et al., 2000).

In Yarrowia lipolytica, which frequently occurs in food and which is well adapted to low pH values, trans-dominant mutations were identified, which cause a sensitivity of cells to low concentrations of acetic acid. These mutations result in growth arrest and cell death after addition of acetic acid to glucose-growing cells (Tzschoppe et al., 1999). The gene affected in these mutants was originally termed GPR1 (glyoxylate pathway repressor), because of the lack of induction of enzymes of the glyoxylate cycle (Kujau et al., 1992; Schmid-Berger et al., 1994). Our current data suggest that Gpr1p is involved in a general response of cells to stress caused by acetic acid and that it is only indirectly involved in repression of the genes encoding glyoxylate cycle enzymes in Y. lipolytica (Tzschoppe et al., 1999).

Here we present the results of our studies on trans-dominant mutants (GPR1d) of Y. lipolytica. We show that the expression of the GPR1 gene is enhanced by acetic acid, but not required for the utilization of this carbon source for growth. The identification of trans-dominant mutation sites in the GPR1 gene, the functional analysis of the N-terminal part of Gpr1p and the detection of the intracellular localization of Gpr1p to the plasma membrane characterizes Gpr1p as a further piece in the puzzle of cellular adaptation to acetic acid. A model is presented to explain a putative function of Gpr1p suggested by our data.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Media, strains and vectors.
Y. lipolytica strains were grown in complete YPD medium or in the following minimal medium: 3·0 g (NH4)2SO4 l-1, 1·0 g KH2PO4 l-1, 0·16 g K2HPO4.3H2O l-1, 0·7 g MgSO4.7H2O l-1, 0·5 g NaCl l-1, 0·4 g Ca(NO3)2.4H2O l-1, 0·6 mg H3BO3 l-1, 0·48 mg CuSO4.5H2O l-1, 0·12 mg KI l-1, 0·48 mg MnSO4.4H2O l-1, 0·24 mg Na2MoO4.2H2O l-1, 0·48 mg ZnSO4.7H2O l-1, 2 mg FeCl3.6H2O l-1. The minimal medium was supplemented with 0·3 mg l-1 thiamine-hydrochloride and with 10 g glucose l-1, 10 ml ethanol l-1 or 4 g sodium acetate.3H2O l-1. If required, 20 mg uracil l-1, 60 mg leucine l-1 and/or 50 mg methionine l-1 were added.

For expression analysis Y. lipolytica cells were grown in 200 ml minimal medium with the desired carbon source after two precultivations in minimal medium with glucose. Cultures were inoculated at a cell density (OD600) of 1·0–1·5 and shaken at 250 r.p.m. at 28 °C. The pH of the medium was adjusted to 5·0–5·5 during the growth of the culture with 1 M NaOH or 1 M HCl.

Strains of Y. lipolytica used in this study are listed in Table 1.


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Table 1. Strains of Y. lipolytica used in this study

 
Transformation and amplification of recombinant plasmid DNA was done in Escherichia coli DH5{alpha}c (supE44 {Delta}lacU169 ({phi}80 lacZ {Delta}M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1). E. coli cells were grown at 37 °C in LB medium or in LB medium containing ampicillin (100 g ml-1) to maintain plasmids.

DNA manipulations.
All basic DNA manipulation procedures were performed according to Sambrook et al. (1989) and Ausubel et al. (1997). Plasmid preparations from E. coli were performed according to standard protocols (Ausubel et al., 1997). Plasmids and genomic DNA from Y. lipolytica were isolated according to the method of Hoffman & Winston (1987). Large amounts of genomic DNA were isolated after zymolyase/glusulase treatment and osmotic lysis of the produced protoplasts (Barth & Gaillardin, 1996). Labelling of probes and detection by Southern blot hybridization was performed with the Random Primed Labelling Module and the Gene Images CDP-Star Detection Module (Amersham Pharmacia Biotech). DNA sequencing was carried out according to Sanger et al. (1977) using the multiwell sequencing kit (Amersham Life Science). All PCR reactions were done with Combipol DNA polymerase (InViTek). Primers used in this study are listed in Table 2. Plasmids used in this study are summarized in Table 3 and Table 4.


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Table 2. List of primers used

 

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Table 3. Plasmids used in this work

 

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Table 4. N-terminal changes in Gpr1p and growth of strain PO1d transformed with plasmids, mutated in the GPR1 gene, on media containing glucose and acetic acid

 
Cloning strategies for isolation of GPR1 alleles.
At first, the mutant allele GPR1-1 from strain B204-12C-112 was isolated in connection with cloning of the retrotransposon Ylt1, which is inserted in the promoter of the GPR1 gene of Y. lipolytica strain B204-12C (Schmid-Berger et al., 1994). The strategy to clone the GPR1-1 allele was based on the trans-dominant action of this allele, which results in an Acu- (acetate non-utilization), Glc+ (glucose utilization) phenotype of wild-type cells transformed with the plasmid containing GPR1-1. A genomic DNA library from strain B204-12C-112 (GPR1-1) was created in the autonomously replicating vector pINA237 and used for transformation of strain B204-12A-213 (GPR1A). After transfer of leucine prototrophic transformants onto acetic-acid-containing plates, one Acu- Glc+ transformant was selected among 5000 colonies. The plasmid was isolated from this transformant, amplified in E. coli DH5{alpha}c and afterwards used for retransformation of strain B204-12A-213 to confirm the trans-dominant effect of the inserted gene.

The cloning of the GPR1 alleles from wild-type strains B204-12C (harbours the GPR1 gene with Ylt1 inserted in its promoter) and PO1d (harbours the authentic GPR1 gene), as well as from another mutant strain B204-12C-156 (GPR1-2) was done as follows. Enriched DNA libraries were constructed by digesting genomic DNA of these strains with SalI and HindIII, separation of the resulting fragments in agarose gels and analysis by Southern blotting, using a GPR1 probe from strain B204-12C-112. Fragments of about 2·1 kb (strains B204-12C and B204-12C-156) and fragments of about 4·1 kb (strain PO1d) were isolated and cloned into pUCBM20. Plasmids containing the respective GPR1 alleles were isolated after colony hybridization of E. coli transformants (Sambrook et al., 1989) using the XhoI–HindIII fragment of the GPR1 gene as a probe. The respective GPR1 alleles were sequenced and plasmids containing the correct inserts were named pGPD1 and pGPD2 (Table 3).

In a second approach the dominant GPR1 alleles (GPR1-3, GPR1-4) of mutant strains B204-12C-38 and B204-12C-124 were isolated by PCR-based amplification using genomic DNA of these strains and primers KT1 and KT1-Rev and Combipol polymerase. Three PCR fragments of each allele were sequenced to exclude potential mistakes introduced by PCR amplification.

Construction of GPR1-lacZ fusions.
For construction of lacZ fusions, the lacZ gene of E. coli was first cloned into the BamHI/SalI sites of pINA237. The resulting plasmid pINAlacZ was completed with the GPR1 terminator (yielding pINAlacZT), which was synthesized by PCR with the primers TNR and TSA and template plasmid pNS3. The resulting fragment was digested with SalI and cloned into the SalI/NruI-cut vector pINAlacZ. The promoter fragments for construction of plasmids pTSA1 (pTSB1) were synthesized by PCR with primers PBA1 and PBA2 (RP), and pNS3 (pGPD2) as the template. The PCR fragments were digested with BamHI and cloned into the BamHI-cut vector pINAlacZT. For construction of pTSC1 the GPR1 ORF was linked to the lacZ gene by attaching a BamHI site to the end of GPR1 (PCR with primers gprsc1 and GPRlacZ, template pGPD2). The synthesized fragment was digested with SalI and BamHI and ligated into pUCBM20 (cut with SalI and BamHI). The resulting plasmid, pUCGf, and plasmid pGPD2 were digested with SplI and SalI. The 3 kb vector fragment of pUCGf and the 3·7 kb fragment of pGPD2 were ligated and the resulting plasmid, pGPDf, was isolated. pGPDf was digested with BglII and BamHI, and the 2·7 kb GPR1 fragment was ligated into BamHI-opened pINAlacZT.

Construction of GPR1-GFP fusions.
The GFP gene, encoding the S65T variant of green fluorescent protein (Gfp; Cubitt et al., 1995), was fused to the 3' end of GPR1 with the authentic promoter. For this purpose plasmid pGPD2 was used as the template for PCR-based amplification of the GPR1 gene with primers GPR-3' and RP, creating a PacI site at the 3' end of GPR1. This fragment was cut with BamHI and PacI and inserted into the BamHI/PacI-opened plasmid pfaGFPkanMX6a (Wach et al., 1994), resulting in plasmid pGPR1-GFP. The SplI–ClaI fragment of pGPR1-GFP was inserted into the SplI/ClaI-opened plasmid pYLG3, resulting in plasmid pYLG3-GFP.

Construction of N-terminal mutations in Gpr1p.
Mutated GPR1 fragments were constructed by PCR with primers designed with the KpnI site or the BstEII site of the GPR1 ORF and the intended deletion or mutation (no. 9–27; Table 2). The PCRs were performed with primer pINA237-Tet and the respective mutation/deletion primer, and plasmid pYLG3 as the template. The resulting PCR fragments were digested with SalI and KpnI and cloned into the SalI/KpnI-digested vector pYLD303. For mutation of serine74 and tyrosine58, primer Nhe1s was used instead of primer pINA237-Tet. These PCR products were digested with NheI and KpnI or BstEII, respectively, and ligated into the vector pYLG3 digested with the same enzymes.

Construction of gene disruption plasmid pGPD2-dUra1 and disruption of the GPR1 gene.
A disruption of the GPR1 gene was done in strain PO1d, which contains only one copy of the GPR1 gene. For this purpose a disruption cassette was constructed by replacement of the 3' end of the GPR1 promoter, which contains the transcription start point, and the 5' end of the GPR1 ORF by the URA3 gene. pGPD2 was digested with NcoI and XhoI, treated with Klenow polymerase and ligated with the URA3 marker gene of Y. lipolytica (SalI fragment of pINA443, Klenow-polymerase-treated), resulting in plasmid pGPD2-dUra1. The disruption of the GPR1 gene was done by transforming strain PO1d with the gene disruption cassette isolated from plasmid pGPD2-dUra1 by cutting with BamHI and HindIII (Table 3). Transformants were selected by complementation of the uracil auxotrophy, and the correct GPR1-disrupted strain was isolated after Southern blot hybridization of Ura+ transformants.

Transformation of E. coli and Y. lipolytica.
Autonomously replicating plasmids were transformed into Y. lipolytica and E. coli by electroporation (Dower et al., 1988). Integrative transformations into the genomic DNA of Y. lipolytica for disrupting the GPR1 gene were done with lithium acetate according to Barth & Gaillardin (1996).

Microscopic techniques.
Cells were prepared for fluorescence microscopy as described by Kohlwein (2000). A sample (0·5 µl) of cell culture was placed on a coverslip and covered with a thin sheet of agarose, to immobilize cells under conditions of nutrient and oxygen supply. Cells were adapted to the microscopy conditions for up to 1 h, to minimize stress response. Microscopy was performed with a Leica TCS4D confocal microscope with GFP-optimized filter settings (488 nm laser excitation, 500–550 nm band pass detection).

Subcellular fractionation and Western blotting.
Cells (PO1d) transformed with either pYLG3-GFP or the control plasmid pINA237 were grown on minimal medium containing 1 % glucose to an OD600 of 7·5. Cells were collected by centrifugation, washed with minimal medium without carbon source, shifted into minimal medium containing 1 % ethanol and cultivated for an additional 2 h to induce the GPR1 promoter. All subsequent steps were performed on ice. A sample (200 ml) of cells (OD600=2) was harvested by centrifugation, resuspended in 3·5 ml TSP buffer (100 mM Tris/HCl, pH 7·4, 400 mM sorbitol, one tablet of Roche protease inhibitor cocktail complete per 50 ml) and homogenized using sterilized, chilled glass beads (0·45 mm). The resulting homogenate was centrifuged at 3000 g for 10 min to remove cell debris and intact cells. The supernatant was centrifuged at 10 000 g for 10 min. The pellet, containing membranes and some remaining cell debris, was resuspended in 400 µl TSP buffer. The supernatant was centrifuged for 1 h at 100 000 g. The resulting pellet contained membranes and bound proteins, the supernatant soluble cytoplasmic components. To distinguish between peripheral and integral membrane proteins the pellet was washed with 0·1 M Na2CO3, pH 11·5, for 1 h and centrifuged for 1 h at 220 000 g. Protein concentrations of these fractions were measured using the Lowry method. Samples (30 µg total protein) were separated on 12 % SDS-polyacrylamide gels and semi-dry-blotted onto PVDF membranes, according to standard protocols. Non-specific binding sites were blocked by incubating the membrane overnight in TBS-T (20 mM Tris/HCl, pH 7·6, 137 mM NaCl, 0·1 % Tween 20) containing 5 % non-fat dry milk. Gpr1-Gfp-fusion proteins were detected with the ECL-Plus Western blotting detection system (Amersham Pharmacia Biotech) using primary anti-Gfp antibodies (Boehringer Mannheim) and secondary anti-mouse antibodies (Amersham Pharmacia Biotech), diluted in TBS-T containing 5 % non-fat dry milk.

Determination of {beta}-galactosidase activity.
{beta}-Galactosidase activity was measured using the chloroform/SDS-permeabilized cell assay as described by Gaillardin & Ribet (1987) and Ausubel et al. (1997). In each case double measurements of samples from at least three independent cultivations of transformants were done. Activity was normalized to the OD600 of the culture (Miller units).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and sequencing of GPR1 alleles
It was previously shown that GPR1d alleles also have pleiotropic effects on life span, cell morphology, organelle structure and fatty acid/phospholipid composition (Tzschoppe et al., 1999). To understand the pleiotropic phenotype caused by trans-dominant mutations in the GPR1 gene we have cloned and sequenced the wild-type gene from strain B204-12C and four of its mutant alleles, as described in Methods. The identification of the mutation sites revealed that amino acid exchanges occurred in three cases in the N-terminal part of the protein (Gpr1-2p, -3p, -4p) (Fig. 1). These mutations cause the exchange of non-polar amino acids leucine65, glycine62 or glycine63 by the polar amino acids glutamine and serine and the acidic amino acid aspartic acid, respectively. The protein sequence around the three very closely localized mutations in the N-terminal protein part did not show any homologies to known functional protein motifs in the databases. However, these mutations are located between putative phosphorylation sites for a tyrosine kinase at tyrosine58 and for protein kinase C at serine74. This suggests that the exchanged amino acids could have some effects on binding or activities of kinases or phosphatases acting at these sites. However, the replacement of tyrosine58 by phenylalanine, which is the case in two other homologues of Gpr1p (ScFUN34p, SpSPAC5D6p), or by glutamic acid had no effect. Also, the replacements of serine74 by glycine, glutamine, histidine or aspartic acid had no effect on sensitivity of cells to acetic acid. Therefore, it appears unlikely that phosphorylation of tyrosine58 or serine74 is linked to the trans-dominant effects of the three mutations.



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Fig. 1. Genomic situation of GPR1 from strain B204-12C with the positions of nucleotide exchanges (underlined) in the mutant strains and the mutated amino acid sequences (bold) in Gpr1p. LTR, long terminal repeat of retrotransposon Ylt1.

 
The fourth mutation identified resulted in the exchange of glycine248 by aspartic acid in a region of the C-terminal part of the Gpr1 protein (Gpr1-1p), which, however, also lacks any homologies to sequence motifs in the databases.

Grp1p homologous proteins from other organisms
Sequence comparison of the Gpr1 protein with protein databases (SWISS-PROT, PIR) revealed that Gpr1p belongs to a group of highly conserved proteins occurring in archaebacteria and eubacteria as well as in cells of lower and higher eukaryotes. A common, highly conserved sequence feature of this class of proteins includes the sequences (A/G)NPAPLGL and SYG(X)FW (Fig. 2). Gpr1p harbours two putative tyrosine-specific phosphorylation sites, one of which (tyrosine145) is located in a conserved region. Similarly, one of three putative serine/threonine-specific phosphorylation sites (tyrosine194) is located in a conserved region of the protein; as shown previously, tyrosine58 and serine74 appear not to be phosphorylated.



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Fig. 2. Alignment and consensus sequence of the amino acid sequences of Gpr1p and some of its homologues: Y.lipol., Gpr1p of Y. lipolytica; S.cer1, Fun34p protein of S. cerevisiae; S.cer2, hypothetical protein Q12359p of S. cerevisiae; S.pombe, protein SPAC5D6p of Schizosaccharomyces pombe; C.albicans, hypothetical protein L04305p of C. albicans; Leishmania, hypothetical protein L5204.4p of Leishmania major; Methanob., hypothetical protein 000808p of Methanobacterium thermoautotrophicum; E.coli, hypothetical protein YaaHp of E. coli. Bold letters indicate amino acid residues which contribute to the consensus sequence; perfectly matching amino acid residues are bold and underlined. $, L or M; %, Y or F; !, I, L or V; #, N, D, Q or E.

 
Gpr1p contains a putative glycosylation site (asparagine219), which is also present in one of the homologues of S. cerevisiae (Fun34p). However, the size of the protein, estimated from HA- and Gfp fusion proteins, as well as treatment with glycosidase F demonstrated that glycosylation does not take place (data not shown).

Interestingly, all bacterial homologues do not contain the acidic N-terminal region, which is present in all eukaryotic homologues (see below). Gpr1p contains five to six putative membrane-spanning regions (Fig. 3; according to commonly used programs), which are well conserved in the prokaryotic as well as in the eukaryotic homologues. This suggests that these proteins are integral membrane proteins (see below).



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Fig. 3. Hydropathicity pattern of Gpr1p. Six putative {alpha}-helices are indicated by I–VI.

 
The exchanges of amino acids in the trans-dominant Gpr1 proteins took place in protein regions, which are not conserved among all eukaryotic and prokaryotic homologues. The four amino acids, exchanged in the Gpr1 mutant proteins, are conserved between the Y. lipolytica protein and two of the Gpr1p homologues of S. cerevisiae (glycine63 and glycine248 in Fun34p and Ycq10Cp) or in all of the S. cerevisiae homologues (glycine62). Two (glycine62, glycine248) out of these four mutations affect amino acid residues that are also conserved in the homologous protein of Schizosaccharomyces pombe. The other Gpr1p homologues have striking amino acid exchanges at these sites, suggesting that these regions may have different importance for the function of the various Gpr1p homologues.

Induction by acetic acid and by ethanol
We have shown previously (Tzschoppe et al., 1999) that GPR1d mutants are sensitive to low concentrations of acetic acid or ethanol, even in the presence of glucose, which represses the expression of genes of the gluconeogenetic pathway in Y. lipolytica (Barth, 1985). Therefore, we studied the regulation of expression of the GPR1 gene by the carbon sources acetate and ethanol, under control of the authentic GPR1 promoter (GPR1A) as well as of the Ylt1-disrupted promoter (GPR1B). The wild-type copy of the GPR1 gene from strain B204-12C and its four dominant alleles harbour the Ylt1 retrotransposon in their promoter regions (Schmid-Berger et al., 1994), implying that the regulation of GPR1 expression may be altered compared to other wild-type strains. Therefore, the authentic promoter GPR1B was also isolated from Y. lipolytica strain PO1d. Strain PO1d does not harbour any sequence of the retransposon Ylt1 (Juretzek et al., 2001). Restriction analysis revealed that the GPR1 gene is located on a 4·1 kb HindIII–SalI DNA fragment in strain PO1d. Due to the presence of a SalI site in the Ylt1 insertion, the GPR1 gene is present on a 2·1 kb HindIII–SalI DNA fragment in strain B204-12C. The 4·1 kb fragment of strain PO1d was cloned by colony hybridization from an enriched gene library and sequenced.

To study the expression of the GPR1 gene by the two different promoters we constructed three different autonomously replicating plasmids based on the low-copy-number plasmid pINA237. Plasmid pTSA1 contains a lacZ fusion with the GPR1 promoter from strain B204-12C (GPR1A), which harbours the 9·4 kb retrotransposon Ylt1 (Schmid-Berger et al., 1994). Plasmid pTSB1 contains the lacZ gene fused to the authentic promoter GPR1B, and pTSC1 contains a GPR1-lacZ fusion under control of the authentic promoter GPR1B. Data of the expression analysis are summarized in Fig. 4(a). Analysis of {beta}-galactosidase activity expressed in these strains revealed that the insertion of Ylt1 in the promoter GPR1A reduced the expression dramatically to a very low level (about 2–5 Miller units of {beta}-galactosidase) on all tested carbon sources (Fig. 4b). This is important with respect to the GPRd alleles, which were isolated from strain B204-12C and contain the retrotransposon in their promoters, resulting in very low synthesis of the mutant proteins (see below). In contrast, under control of the authentic promoter GPR1B, moderate levels of expression of {beta}-galactosidase were observed on glucose (about 80–120 Miller units). Acetic acid and ethanol as sole carbon sources increased the expression of {beta}-galactosidase up to three times in comparison to glucose (Fig. 4). It is not clear whether ethanol acts directly or indirectly by intracellular conversion to acetate, as an inducer of GPR1 expression.



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Fig. 4. (a) Expression cassettes with lacZ from E. coli as a reporter gene fused to the promoter harbouring the retrotransposon Ylt1 (plasmid pTSA1), the authentic promoter of GPR1 (plasmid pTSB1) or the complete GPR1 gene (pTSC1). (b) {beta}-Galactosidase activities in transformants dependent on the expression cassette of these different plasmids. White bars, grown with glucose in medium; black bars, grown with acetate in medium; grey bars, grown with ethanol in medium.

 
The studies with plasmid pTSC1, which harbours GPR1 fused to lacZ under control of the GPR1B promoter, showed that GPR1 itself does not have any major effect on its expression on glucose or acetic acid as carbon sources. The slightly reduced expression observed with plasmid pTSC1 could be due to the localization of the fusion protein to the plasma membrane, thus decreasing the activity of {beta}-galactosidase (Fig. 4).

Effects of the GPR1 gene disruption and low expression of the GPR1d alleles
Disruption of the gpr1 gene showed that the Gpr1 protein did not affect utilization of glucose, acetate and ethanol, even at low pH. Furthermore, no reduction of growth or the number of cells at the stationary phase was observed. However, growth of the gpr1-disrupted strain was delayed on acetic-acid-containing medium, indicating that Gpr1p effects the adaptation of cell metabolism to the utilization of acetic acid (Fig. 5).



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Fig. 5. Growth of PO1d (triangles) and PO1d{Delta}gpr1 (diamonds) transformants (with pINA237) on minimal medium pH 4·0 with 30 mM acetic acid. After 57·5 h, 30 mM acetic acid was added again. PO1d{Delta}gpr1 displays an elongated lag phase to adapt to acetic acid.

 
We have previously shown that heterozygous GPR1d/GPR1 diploid strains constructed with the trans-dominant mutants derived from strain B204-12C together with strain B204-12A-213 are highly sensitive to acetic acid even in presence of glucose (Tzschoppe et al., 1999). Also, transformants of strain B204-12A-213 with plasmids harbouring a GPR1d allele were sensitive to acetic acid. Investigation of expression driven by the two different versions of the GPR1 promoter demonstrated that the insertion of Ylt1 into the GPR1A promoter in strains B204-12C and B204-12A-213 resulted in about 100 times lower expression rates, compared to the authentic promoter. However, PO1d, which harbours the GPR1 allele under control of the authentic promoter GPR1B responsible for a moderate expression of Gpr1 wild-type protein, became acetic-acid-sensitive when transformed with plasmids carrying GPR1d under control of the very weak promoter GPR1A.

These results demonstrate that Gpr1p functions in the adaptation of cells to acetic-acid-containing media in Y. lipolytica, but that it is not required for growth of cells already adapted to acetic acid. Furthermore, our data indicate that very low amounts of mutated Gpr1p reduce tolerance to acetic acid dramatically even in the presence of abundant wild-type levels of Gpr1p.

N-terminal deletions and their effects on growth
Sequence comparisons showed that the acidic N-terminal part of the protein is present in all eukaryotic Gpr1p homologues, but absent in bacteria (Fig. 2), and that three of the trans-dominant mutations of Gpr1p are localized in this region (Fig. 1). Computer analysis did not reveal any motif indicative of a signal peptide or other known functional sequences in the N-terminal part of the protein. Thus, we analysed by deletion analysis and amino acid replacements, whether the entire N terminus or a part of it were essential for Gpr1p function. This study was done with plasmid pYLG3, harbouring the GPR1 gene on a 4·1 kb DNA fragment of strain PO1d. There was only a slightly increased acetic acid sensitivity associated with deletion of the N-terminal part up to residue alanine60 of Gpr1p (pYLG3-d1B, Table 4). Deletion of the N terminus, including phenylalanine61 (pYLG3-d1A) and glycine62 (pYLG3-d1), fully inhibited growth on acetic acid (Table 4). Partial deletions of the N-terminal part, including phenylalanine61, resulted in the same trans-dominant effect (pYLG3-d1 to pYLG3-d6, pYLG3-d2A). Furthermore, we found that cells did not grow on acetic acid if only phenylalanine61 was deleted (pYLG3-d7) or replaced by glutamic acid (pYLG3-m61B), but grew like wild-type if phenylalanine61 was exchanged with tyrosine (pYLG3-m61A). Phenylalanine61 is part of the motif ‘FGGTLN’ that is also present in two homologues of S. cerevisiae. Interestingly, three out of the four trans-dominant mutations in Y. lipolytica are located in this motif. Transformation of the gpr1-disrupted strain PO1d{Delta}gpr1 with these N-terminal deletions resulted in the same behaviour as for the transformed wild-type. This result demonstrates that none of the created N-terminal deletions caused recessive phenotypes. All these transformants also exhibited the same behaviour on media containing acetic acid together with glucose as on acetic acid alone (data not shown).

Intracellular localization
For the intracellular localization of Gpr1p a fusion protein of Gpr1p with green fluorescent protein (Gfp) was constructed and expressed under the control of the authentic GPR1B promoter (plasmid pYLG3-GFP). Subcellular fractionation and immunolocalization using Gfp-directed antibodies was done with cells of strain PO1d transformed with plasmid pYLG3-GFP. Cell fractionation and subsequent SDS-PAGE and Western blotting confirmed a membrane localization of Gpr1p (Fig. 6). A band of about 51 kDa, corresponding to Gpr1p-Gfp, was detected in all membrane-containing fractions (Fig. 6, lanes 1, 3, 5 and 7). Gpr1p is most likely an integral membrane protein as alkaline treatment with 0·1 M Na2CO3 (pH 11·5) of the 100 000 g pellet fraction did not result in solubilization of Gpr1p from the membrane (Fig. 6, lanes 6 and 7).



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Fig. 6. Subcellular localization of Gpr1p. Cells (PO1d) containing the plasmid pYLG3-GFP were harvested, homogenized and fractionated. The samples were analysed by Western blotting using antibodies against Gfp. Lanes: 1, cell extract of PO1d with pYLG3-GFP; 2, cell extract of PO1d with control plasmid pINA237; 3, 10 000 g pellet; 4, 100 000 g supernatant; 5, 100 000 g pellet. The 100 000 g pellet was washed with 0·1 M Na2CO3, pH 11·5 and separated by centrifugation for 1 h at 220 000 g: lane 6, supernatant; lane 7 pellet. Molecular mass standards are indicated.

 
Confocal laser scanning microscopy of Y. lipolytica transformants expressing a Gpr1-Gfp fusion protein revealed the presence of the fusion protein in the cytoplasmic membrane (Fig. 7a). Four hours after transfer of cells into acetic-acid-containing medium fluorescence was also found in intracellular vesicles (Fig. 7b), which are most probably endocytotic vesicles, and subsequently (24 h) in the vacuole (Fig. 7c). The same localization pattern was observed with transformed cells overexpressing GPR1-GFP under control of the strong heterologous POT1 promoter (Juretzek et al., 2000) and in cells expressing GPR1-GFP under a weak promoter (data not shown).



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Fig. 7. Gpr1p is localized to the cell surface (a) and later shifted into endocytotic vesicles (b) and into the vacuole (c). Y. lipolytica B204-12A-213 cells were transformed with plasmid pYLG3-GFP and analysed after growth on acetate-containing medium. Samples were taken after 2 (a), 4 (b) and 24 h (c). I, micrographs of differential interference contrast (DIC) microscopy; II, GFP-fluorescence micrographs (confocal laser scanning microscopy).

 
In summary, the subcellular fractionation data and fluorescence microscopy confirm a membrane localization of Gpr1p.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The genetics and molecular biology of the yeast Yarrowia lipolytica have been well established and data suggest that this yeast has diverged considerably from other yeasts, displaying high similarities to higher eukaryotic cells in many respects (Barth & Gaillardin, 1997). Commonly used genetic techniques are well adapted to this yeast, which represents a suitable model organism for studies on a number of cellular processes, including secretion and mechanisms of cellular resistance (Barth & Gaillardin, 1996).

We have previously demonstrated that trans-dominant-acting mutations in the GPR1 gene of Y. lipolytica have several effects on sensitivity to acetic acid, cell and colony morphology, yeast-to-hyphae transition and life span of the cell (Tzschoppe et al., 1999). Here, we report the identification of functional regions of the Gpr1 protein which cause, when mutated or deleted, these pleiotropic effects. Noticeably, very low expression levels of GPR1d alleles act trans-dominantly over the highly expressed wild-type gene. The GPR1-disrupted strain exhibited no striking effects on the utilization of acetic acid/acetate. However, the gpr1-deleted strain needed longer to adapt to acetic acid in the growth medium, similar to the azr1-deleted strain of S. cerevisiae (Tenreiro et al., 2000).

Our studies identify Gpr1p as an integral membrane protein of the plasma membrane. The transcription of the GPR1 gene is induced/derepressed threefold in acetic acid- or ethanol-containing medium, compared to growth in glucose-containing medium.

Gpr1p is the first described member (Kujau et al., 1992) of a family of highly conserved proteins present in archaebacteria and eubacteria, as well as in lower and higher eukaryotic cells, harbouring five to six putative membrane-spanning regions (Figs 2 and 3). These proteins contain several conserved sequence motifs (Fig. 2), among them the sequences (A/G)NPAPLGL and SYG(X)FW. A search with the INTERPRO program revealed several proteins in which the sequences (A/G)NPAPLGL and SYG(X)FW are fully conserved, forming the GPR1_FUN34_YaaH protein family. Further proteins were detected, which have the consensus sequences NP(A/V/G)P(L/F/V)GL and (Y/F)G(X)FW in common. The function of all of these proteins is unknown; however, most of them are predicted to be plasma-membrane proteins.

The first motif harbours the sequence NPA that is characteristic for members of the MIP (major intrinsic protein) family, which transport water (aquaporins) and other solutes. The Gpr1-like proteins have a similar size and also contain five to six transmembrane domains like MIP-family proteins. The characteristic NPA motif is present twice in MIP proteins, between the second and the third and between the fifth and sixth transmembrane domains (Agre et al., 1995; Reizer et al., 1993). In contrast, the NPA motif is present only once in the N-terminal part preceding the first transmembrane domain in GPR1_FUN34_YaaH proteins, and which makes them clearly different from proteins of the MIP family. Whether Gpr1p can specifically effect the transport of acetate or acetic acid out of the cell is unknown and subject to current studies.

The sensitivity of the trans-dominant mutants is only detectable at low pH values (Tzschoppe et al., 1999). This observation fits with the model of Piper et al. (1998) on the action of weak acids on the cells. The trans-dominant effects of the four identified mutant alleles and the minor effect of the deletion of GRP1 in strain PO1d{Delta}gpr1 on growth suggest that Gpr1p may act as an inhibitor in the adaptation processes to acetic acid (Fig. 8). In the absence of acetic acid, Gpr1p is suggested to be active and may inhibit an anion-transporting ATPase (functional homologue of ScPdr12p) and affect the plasma membrane H+-ATPase (functional homologue of ScPma1p) (Fig. 8a). In the presence of acetic acid, Gpr1p becomes inactive, resulting in activation of the Pdr12p and Pma1p homologues (Fig. 8b), which inhibit intracellular acidification by pumping out protons and anions (Piper et al., 1998). In the gpr1-deleted strain, Pma1p and Pdr12p homologues are not expected to be inhibited, independent of carbon source, resulting in no strong phenotypic effects. Mutated Gpr1p is suggested to be permanently active and could thus cause inhibition of Pma1p and Pdr12p homologues, leading to intracellular acidification in the presence of acetic acid and ultimately, to cell death.



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Fig. 8. Hypothetical model of Gpr1p function. (a) In the absence of acetic acid Gpr1p is in its active form and inhibits a weak acid efflux pump (Pdr12p homologue) and/or the plasma membrane H+-ATPase (Pma1p homologue). (b) In the presence of acetic acid, the protonated form of the acid freely diffuses through the plasma membrane, Gpr1p becomes inactivated and the weak acid efflux pump as well as the plasma membrane ATPase are activated.

 
Summarizing our results we conclude that Gpr1p is an intrinsic plasma membrane protein, which helps in adaptation to acetic acid, and therefore affects the sensitivity/resistance of Y. lipolytica cells to acetic acid. Synthesis of Gpr1p is induced by acetic acid and moderately repressed by glucose. Further studies are in progress to elucidate the molecular function of the members of the GPR1_FUN34_YaaH protein family. It will be interesting to see whether these proteins are directly involved in the extrusion of acetic acid from the cells and whether they interact with other proteins like homologues of Azr1p, Pdr12p or Hsp30p of S. cerevisiae, which are directly or indirectly involved in the mechanism of adaptation to acetic acid.


   ACKNOWLEDGEMENTS
 
We thank S. Mauersberger for helpful discussion and K. Ruecknagel for excellent technical assistance. This work was supported by grants from EU-INTAS (0788-99) to G. B. and a grant from the Austrian Science Fund, FWF (F706) to S. D. K.


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DISCUSSION
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Received 31 July 2002; revised 16 October 2002; accepted 3 December 2002.



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