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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Institute of Anatomy, Dresden University of Technology, Fetscherstrasse 74, D-01307 Dresden, Germany.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1·01·5 and shaken at 250 r.p.m. at 28 °C. The pH of the medium was adjusted to 5·05·5 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.
|
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
.
|
|
|
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 XhoIHindIII 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 SplIClaI 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. 927; 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, 500550 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 -galactosidase activity.
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.
|
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).
|
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 HindIIISalI 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 HindIIISalI 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
-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 25 Miller units of
-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
-galactosidase were observed on glucose (about 80120 Miller units). Acetic acid and ethanol as sole carbon sources increased the expression of
-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.
|
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).
|
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
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).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (editors) (1997). Current Protocols in Molecular Biology. New York: Wiley.
Barth, G. (1985). Genetic regulation of isocitrate lyase in the yeast Yarrowia lipolytica. Curr Genet 10, 119124.
Barth, G. & Gaillardin, C. (1996). Yarrowia lipolytica. In Nonconventional Yeasts in Biotechnology, pp. 313388. Edited by W. K. Berlin. Heidelberg, New York: Springer.
Barth, G. & Gaillardin, C. (1997). Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol Rev 19, 219237.[CrossRef][Medline]
Braley, R. & Piper, P. W. (1997). The C-terminus of yeast plasma membrane H+-ATPase is essential for the regulation of this enzyme by heat shock protein Hsp30, but not for stress activation. FEBS Lett 418, 123126.[CrossRef][Medline]
Casal, M., Cardoso, H. & Leao, C. (1996). Mechanisms regulating the transport of acetic acid in Saccharomyces cerevisiae. Microbiology 142, 13851390.[Abstract]
Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A. & Tsien, R. Y. (1995). Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20, 448455.[CrossRef][Medline]
Dower, W. J., Miller, J. F. & Ragsdale, C. W. (1988). High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16, 61276145.[Abstract]
Fournier, P., Abbas, A., Chasles, M. & 9 other authors (1993). Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeast Yarrowia lipolytica. Proc Natl Acad Sci U S A 90, 49124916.[Abstract]
Gaillardin, C. & Ribet, A. M. (1987). LEU2 directed expression of beta-galactosidase activity and phleomycin resistance in Yarrowia lipolytica. Curr Genet 11, 369375.[Medline]
Geros, H., Cassio, F. & Leao, C. (2000). Utilization and transport of acetic acid in Dekkera anomala and their implications on the survival of the yeast in acidic environments. J Food Prot 63, 96101.[Medline]
Hoffman, C. S. & Winston, F. (1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267272.[CrossRef][Medline]
Holyoak, C. D., Bracey, D., Piper, P. W., Kuchler, K. & Coote, P. J. (1999). The Saccharomyces cerevisiae weak-acid-inducible ABC transporter Pdr12 transports fluorescein and preservative anions from the cytosol by an energy-dependent mechanism. J Bacteriol 181, 46444652.
Juretzek, T., Wang, H.-J., Nicaud, J.-M., Mauersberger, S. & Barth, G. (2000). Comparison of promoters suitable for regulated overexpression of heterologous genes in the alkane-utilizing yeast Yarrowia lipolytica. Biotech Bioprocess Eng 5, 320326.
Juretzek, T., Le Dall, M. T., Mauersberger, S., Gaillardin, C., Barth, G. & Nicaud, J. M. (2001). Vectors for gene expression and amplification in the yeast Yarrowia lipolytica. Yeast 18, 97113.[CrossRef][Medline]
Kohlwein, S. D. (2000). The beauty of the yeast: Live Cell Microscopy at the limits of optical resolution. Microsc Res Tech 51, 511529.[CrossRef][Medline]
Krebs, H. A., Wiggins, D., Stubbs, M., Sols, A. & Bedoya, F. (1983). Studies on the mechanism of the antifungal action of benzoate. Biochem J 214, 657663.[Medline]
Kujau, M., Weber, H. & Barth, G. (1992). Characterization of mutants of the yeast Yarrowia lipolytica defective in acetyl-coenzyme A synthetase. Yeast 8, 193203.[Medline]
Le Dall, M. T., Nicaud, J. M. & Gaillardin, C. (1994). Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26, 3844.[Medline]
Piper, P., Mahe, Y., Thompson, S., Pandjaitan, R., Holyoak, C., Egner, R., Muhlbauer, M., Coote, P. & Kuchler, K. (1998). The Pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast. EMBO J 17, 42574265.
Piper, P. W., Ortiz-Calderon, C., Holyoak, C., Coote, P. & Cole, M. (1997). Hsp30, the integral plasma membrane heat shock protein of Saccharomyces cerevisiae, is a stress-inducible regulator of plasma membrane H(+)-ATPase. Cell Stress Chaperones 2, 1224.[Medline]
Reizer, J., Reizer, A. & Saier, M. H., Jr (1993). The MIP family of integral membrane channel proteins: sequence comparisons, evolutionary relationships, reconstructed pathway of evolution, and proposed functional differentiation of the two repeated halves of the proteins. Crit Rev Biochem Mol Biol 28, 235257.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular cloning. A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 54635467.[Abstract]
Schmid-Berger, N., Schmid, B. & Barth, G. (1994). Ylt1, a highly repetitive retrotransposon in the genome of the dimorphic fungus Yarrowia lipolytica. J Bacteriol 176, 24772482.[Abstract]
Sikkema, J., de Bont, J. A. & Poolman, B. (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 59, 201222.[Abstract]
Tenreiro, S., Rosa, P. C., Viegas, C. A. & Sa-Correia, I. (2000). Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae. Yeast 16, 146981.[CrossRef][Medline]
Tzschoppe, K., Augstein, A., Bauer, R., Kohlwein, S. D. & Barth, G. (1999). Trans-dominant mutations in the GPR1 gene cause high sensitivity to acetic acid and ethanol in the yeast Yarrowia lipolytica. Yeast 15, 16451656.[CrossRef][Medline]
van der Rest, M. E., Kamminga, A. H., Nakano, A., Anraku, Y., Poolman, B. & Konings, W. N. (1995). The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis. Microbiol Rev 59, 304322.[Abstract]
Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. (1994). New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 17931808.[Medline]
Received 31 July 2002;
revised 16 October 2002;
accepted 3 December 2002.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |