Angewandte Tumorvirologie, Abteilung F0100 and Virologie Appliquée à lOncologie (Unité INSERM 375), Deutsches Krebsforschungszentrum, P. 1011949, D-69009 Heidelberg, Germany1
Laboratoire de Génétique et Microbiologie, UPRES-A 7010 ULP/CNRS, Institut de Botanique, 28 rue Goethe, F-67083 Strasbourg cedex, France2
Author for correspondence: Jean-Claude Jauniaux. Tel: +49 6221 42 49 71. Fax: +49 6221 42 52 49 71. e-mail: j.jauniaux{at}dkfz.de
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ABSTRACT |
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Keywords: gene disruption, membrane protein, gene tandem repeats, two-hybrid system, yeast
Abbreviations: GFP, green fluorescent protein
a Present address: Centro de Biologia Molecular Severo Ochoa, Universidad Autonoma, Canto Blanco, 28049, Madrid, Spain.
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INTRODUCTION |
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Saccharomyces cerevisiae S288C potentially encodes 5651 ORFs (Malpertuy et al., 2000 ). Of these, 914 (16·2%) belong to two-gene families and 1544 (27·3%) belong to multigene families with between three and over 20 members (Blandin et al., 2000
). In some cases, there are physiological reasons for the presence of multigene families. For example the cytoplasmic and mitochondrial methionyl-tRNA synthetases are encoded by two related but different nuclear genes, resulting in different localizations (Schneller et al., 1978
). The genes encoding alcohol dehydrogenase (ADHI and ADHII) are very similar, but ADHI catalyses the formation of ethanol from acetaldehyde whereas ADHII catalyses the formation of acetaldehyde from ethanol (Johnston & Carlson, 1992
). Another well-studied case is the CUP1 locus, which encodes a copper- or cadmium-chelating metallothionein expressed after induction with metals. The reference strain, S288C, contains only two copies of CUP1, but copper- or cadmium-resistant strains can contain up to 15 copies (Karin et al., 1984
). The expression of most hexose transporters is tightly regulated by glucose concentration, starvation, osmotic pressure and the physiological state of the cells. In fact, to abolish glucose consumption and transport activity completely, all 18 members of the hexose transporter family, HXT117, GAL2 and three members of the maltose transporter family (AGT1, YDL247w and YJR160c) have to be deleted (Wieczorke et al., 1999
).
In comparison with these established examples and with the exception of the PAU and OSBP families (Rachidi et al., 2000 ; Beh et al., 2001
), the large gene families identified by sequencing programmes remain mostly unstudied. This is the case for the DUP240 gene family. This family is particularly interesting because of its unusually high copy number (10), the high level of nucleotide identity between some of its members and the specific chromosomal organization of the members (Fig. 1
). The YAR027w, YAR028w, YAR029w, YAR031w and YAR033w ORFs are arranged as tandem repeats on chromosome I and the YGL051w and YGL053w ORFs are arranged as tandem repeats on chromosome VII. The nucleotide sequences of YAR033w and YAR031w are 98% identical to those of YGL051w and YGL053w (Feuermann et al., 1997
). Most of the corresponding proteins are approximately 240 aa long. Moreover, these ORFs appear to be specific for the Saccharomyces sensu stricto group (Bon et al., 2000
).
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METHODS |
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Cloning of a PCR product in a linearized plasmid by homologous recombination in yeast.
Plasmids were linearized by two different endonucleases and dephosphorylated with calf intestine phosphatase (Boehringer). The sequences to be cloned were amplified by PCR (Saiki et al., 1985 ) from genomic DNA of yeast strain FY1679 with oligonucleotide primers having 20 additional nucleotides at the 5' ends. These ends were homologous to the ends of the linearized plasmid and used for cloning by homologous recombination in this plasmid (Muhlrad et al., 1992
). The PCR conditions were: 94 °C for 1·5 min, (94 °C for 30 s, 48 °C for 2 min, 70 °C for 2·5 min) x10 cycles, (94 °C for 30 s, 60 °C for 2 min, 70 °C for 2·5 min) x35 cycles, 70 °C for 10 min. The PCR product and the linearized plasmid were treated with phenol/chloroform prior to ethanol precipitation. High-efficiency transformations were performed according to the method described by Gietz et al. (1995)
. For cloning in pGBT9 (Bartel et al., 1993a
), pSOS (Stratagene) and pGRU1 (see below), the vector was linearized by EcoRI and SalI, NcoI and MluI, and EcoRI and SalI, respectively. The PCR product and the corresponding linearized plasmid were introduced into the yeast cells simultaneously. The oligonucleotide sequences are given in Table 2
. We ensured that there were no errors in the junctions between the plasmid and the inserts for pGBT9-YGL051w and pGBT9-YGL053w by sequencing.
Determination of Dup240GFP localization in vivo.
The PCR-amplified DUP240 ORFs were cloned in-frame at the C-terminal end of the green fluorescent protein-S65T (GFP-S65T) gene of pGRU1 (Michel Aigle, IBGC, Bordeaux, France) and of pGRU1-Padh1. Transformed yeast cells were selected in SD medium without uracil because URA3 was used as the marker gene for selection of pGRU1. Dup240GFP and control GFP were excited with a 488 nm laser and viewed under a Leica confocal microscope equipped with a x63 objective.
Activation of the RAS signalling pathway through hSos1pDup240 fusion.
We modified the cytoplasmic two-hybrid system (Aronheim et al., 1997 ) marketed by Stratagene (CytoTrap) so that it would test for the presence of a hybrid protein at the plasma membrane. This system uses the S. cerevisiae mutant CDC25H, containing a thermosensitive mutation of the CDC25 gene product (Petitjean et al., 1990
). This gene is homologous to the human hSOS1 gene (Chardin et al., 1993
) and encodes a GDP/GTP exchange factor. By stimulating the exchange of GDP associated with the Ras1 and Ras2 proteins for GTP, Cdc25p stimulates the signalling pathway involving these proteins. hSos1p is cytoplasmic in the system. The cdc25H mutation prevents cell growth at 37 °C while growth is normal at 25 °C. Cell growth can be restored by targeting hSos1p to the plasma membrane (Aronheim et al., 1994
). To test whether Dup240 proteins can target hSos1p to the plasma membrane we constructed hSos1pDup240 fusion proteins by introducing PCR-amplified DUP240 ORFs into the pSOS vector (CytoTrap) in-frame at the 5' end of the hSOS1 gene. We then tested the ability of the resulting constructions to restore the growth of the CDC25H yeast strain (CytoTrap) at 37 °C. Drops of liquid control and transformed yeast cultures were deposited onto YPD plates and incubated for 23 days at 37 °C to evaluate growth.
Two-hybrid screen strategy.
Two-hybrid screens were carried out using a system based on that described by Fields & Song (1989) . We used the Gal4pAD-yeast genomic library (mechanically sheared genomic DNA fragments with a mean size of 800 bp inserted into the pACTII vector which bears the marker gene LEU2; A. Ramne & P. Sunnerhagen, http://www.mips.biochem.mpg.de/proj/eurofan/eurofan_1/b5/index.html). This library was introduced (Georgakopoulos et al., 2001
) into the haploid yeast PJ69-4a (Table 1
). This strain contains three markers (HIS3, ADE2 and lacZ) controlled by three different GAL promoters (GAL1, GAL2 and GAL7, respectively), which can each be activated in the two-hybrid system (James et al., 1996
). This library was composed of 2·1x107 independent transformed yeast cells. It was amplified in liquid culture and aliquot vials containing 6·5x108 yeast cells were stored at -80 °C. A mating strategy inspired by Bendixen et al. (1994)
and modified by Fromont-Racine et al. (1997)
was used to obtain a wide range of diploids containing both bait and target plasmids. High mating efficiency enabled us to test over 5x107 interactions per experiment. The haploid yeast PJ69-4
(Table 1
) was transformed with the bait cloned by homologous recombination and fused with the Gal4pBD (Gal4p-DNA binding domain) of the pGBT9 vector bearing the marker gene TRP1. For each screen, one vial containing 6·5x108 transformed PJ69-4a was mixed with 109 PJ69-4
cells. Cells were concentrated onto filters and incubated on rich medium for 4·5 h at 30 °C prior to collection. The cells were diluted and spread onto SC-Leu, SC-Trp and SC-Leu-Trp plates to count the number of parental cells and the number of diploids. The rest of the cell suspension was spread onto 24 SC-Leu-Trp-His plates (24 cmx24 cm) containing 2 mM 3-aminotriazole. The plates were incubated at 30 °C for 4 days. His-positive clones were subjected to a second selection on SC-Leu-Trp-adenine plates. Plasmids were rescued in E. coli HB101. Insert junctions with Gal4pAD were sequenced and precisely identified in the yeast genome using the MIPS (Munich Information Centre for protein sequences; http://mips.gsf.de) Yeast Genome Database (MYGD) or the Saccharomyces Genome Database from Stanford University (http://genome-www.stanford.edu/Saccharomyces).
ß-Galactosidase filter assays to test the two-hybrid interactions in another genetic background.
Yeast strain SFY526 was transformed by the LiAc method (Gietz et al., 1995 ) simultaneously with two plasmids which gave a two-hybrid interaction in PJ69-4 and spread onto SD-Leu-Trp agar in 150 mm diameter Petri plates. After 2 days at 30 °C these plates were replicated onto 125 mm diameter filters, which were incubated on YPD medium plates for 1 day at 30 °C. The filters were frozen quickly in liquid nitrogen and put onto plates containing, in a total of 3·5 ml: 58·5 µl 2% X-Gal in dimethylformamide, 9·5 µl ß-mercaptoethanol, 16·1 g Na2HPO4.7H2O l-1, 5·5 g NaH2PO4.H2O l-1, 0·75 g KCl l-1 and 0·246 g MgSO4 . 7H2O l-1 at pH 7·0. The time required for yeasts to stain blue was measured at room temperature (22 °C).
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RESULTS |
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Subcellular localization of different Dup240GFP fusion proteins
The ORFs YGL051w and YGL053w under the control of their own promoters were each individually cloned in-frame with the gene encoding GFP-S65T. The fluorescent signal yielded by the subsequent constructions on the multicopy plasmids (pGRU1-P-YGL051w and pGRU1-P-YGL053w, respectively) were hardly detectable. This result could be related to a low expression level as previously reported by Northern blot (Barton et al., 1997 ) and DNA array data (Jelinsky & Samson, 1999
). To enhance transcription, the yeast ADH1 promoter was introduced into pGRU1, such that the GFP-S65T gene was under the control of this strong promoter; the derivative plasmid was named pGRU1-Padh1. The ORFs YGL051w, YGL053w, YAR029w, YAR033w, YAR031w and YHL044w were cloned in-frame at the 5' end of GFP-S65T ORF into this pGRU1-Padh1, and each one was individually introduced into the reference strain FY1679.
The control experiment with the vector without the insert revealed the expected homogeneous distribution of GFP within the cytoplasm (Fig. 6). Ygl051p, Ygl053p, Yar033p, Yar031p and Yhl044pGFP fusion proteins were localized at the plasma membrane. The Yar031p and Yhl044pGFP fusion proteins appeared to concentrate as specific spots corresponding to a higher focal concentration at the plasma membrane. In addition Ygl051p, Ygl053p and Yar033pGFP fusion proteins surrounded the nucleus, a situation typical of an endoplasmic reticulum localization. Moreover, Ygl053p and Yar033pGFP fusion proteins appeared to accumulate into additional membranes that could correspond to the Golgi apparatus. Conversely, the Yar029pGFP fusion protein showed a similar distribution to GFP alone, but with additional spots corresponding to a higher focal concentration not linked to the plasma membrane. This could be because Yar029p is the shortest member of the Dup240 family and the only one that lacks the predicted transmembrane domain. The localization of the Dup240 protein at the plasma membrane was further addressed by studying the hSos1pDup240 fusion RAS signalling pathway activation system.
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This result can be explained by at least two mechanisms. Firstly, these bait proteins could interact physically with endogenous membrane protein targets, allowing the co-localization of the hSos1p protein at the plasma membrane. hSos1p could then fulfil its deoxyriboguanosine exchange function. Alternatively, the bait proteins Yar033p, Ygl051p and Ygl053p may themselves be membrane proteins. This would localize the fused hSos1p protein at the plasma membrane, allowing it to fulfil its role as an enzyme.
The fusion proteins hSos1pYar029p and hSos1pYar031p were unable to restore the growth of the yeast CDC25H strain. These results were expected for Yar029p (see preceding section on GFP fusions) but were surprising for Yar031p because the Yar031pGFP fusion product appeared to be located at the plasma membrane.
Partners that interact with the YGL051w and YGL053w gene products as identified by two-hybrid screens
Proteinprotein interactions are essential for many biological processes. Therefore, the identification of protein partners of proteins of unknown function may help to determine the role played by these proteins in the cell. We used the two-hybrid system to screen for the protein partners of Ygl051p and Ygl053p. The entire sequences of YGL051w and YGL053w were cloned in-frame with the nucleotide sequence of the Gal4p-binding domain in pGBT9 and introduced into the yeast strain PJ69-4. The resulting vectors were named pGBT9-YGL051w and pGBT9-YGL053w, respectively.
For YGL053w, the two-hybrid screen was performed on 6·5x107 diploid yeast cells obtained by mating. Selection for histidine yielded 384 clones and 103 clones were obtained after selection for adenine. Seven of these clones corresponded to antisense constructs or intergenic regions, and 46 clones to the OAF1 (=YAF1) gene. As OAF1 encodes a transcription factor involved in the regulation of peroxisome proliferation and inserts of this gene were found in all the two-hybrid screenings performed in the laboratory with this library, these clones were considered to be false-positives. The two-hybrid interactions with the other 50 putative positives, corresponding to 15 different inserts, were verified in another genetic context: yeast strain SFY526 (Bartel et al., 1993b ) bearing the lacZ gene under the control of the GAL1 promoter. For this purpose pGBT9-empty or pGBT9-YGL053w were introduced into SFY526 simultaneously with each of the 15 plasmids encoding a putative positive partner. After ß-galactosidase filter assays for all these constructs, 45 clones corresponded to 11 different inserts but only two different genes were retained as positives. Five of the 11 inserts corresponded to a part of the FIR1 gene (ORF of 2775 nt) which encodes a protein that is thought to be involved in 3'-mRNA processing. The five inserts contained a common sequence of 320 nt corresponding to a region ending at 200 nt upstream from the 3' end of the FIR1 ORF (Russnak et al., 1996
). The six other inserts corresponded to YNL078w, a 1221 nt ORF of unknown function, and all of them contained the last 236 nt of the ORF (Fig. 7
).
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DISCUSSION |
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To learn more about the function of this multigene family we studied the subcellular localizations of the gene products and searched for interacting partners. Fluorescent staining of proteins Ygl051p, Ygl053p, Yar031p and Yar033p fused to GFP revealed that they were localized on the yeast plasma membrane and that Ygl051p, Ygl053p and Yar033p were also located in the endoplasmic reticulum. Takahashi et al. (2000) reported that YAR027p fused to GFP was localized at the nuclear envelope and plasma membrane regions. The membrane localization was further supported by the in silico analysis, which suggested that all of the proteins coded by the DUP240 ORFs (except Yar029p, which has no predicted transmembrane domain) possess two 17 aa hydrophobic predicted transmembrane domains. The activation of the RAS signalling pathway by hSos1pDup240 fusions confirmed the plasma membrane localization, showing that Ygl051p, Ygl053p and Yar033p were able to target activation in this system, either directly because they are integral membrane proteins, or indirectly by interacting with associated membrane proteins. Yar029p is much shorter than the other Dup240 proteins and Yar029pGFP was located throughout the cytoplasm, as was GFP alone. This finding is consistent with the inability of Yar029p to target hSos1p to the RAS signalling pathway. The Yar031p protein contains 62 extra amino acids at its N-terminus compared to Ygl053p. The C-terminal parts of these two ORFs are nevertheless highly conserved: 100% identity for the first 93 C-terminal amino acids, and only five differences among the 132 C-terminal amino acids. The identity of the N-terminal domain of Ygl053p and the central region of Yar031p is just 41·7% (48 out of 115 amino acids). When fused to hSos1p, Yar031p was unable to activate the RAS signalling pathway despite its apparent localization as indicated by GFP fusion at the plasma membrane and the presence of two predicted transmembrane domains. This discrepancy might result from the fact that GFP-fused Yar031p always seems to form aggregates at the membrane, and may consequently be unable to target hSos1p correctly for RAS activation.
The Ygl051p and Ygl053p partners identified by screening of the two-hybrid yeast library were in different classes. The product of the ORF YGL051w fused to the binding domain of Gal4p interacted with many integral membrane proteins and many proteins of unknown function predicted to have six or more transmembrane domains. This finding further suggests that Ygl051p is located at the plasma membrane. The two-hybrid partners of Ygl053p, Fir1p and Ynl078p do not appear to be plasma membrane proteins, as they lack predicted transmembrane domains; however, this finding does not exclude a plasma membrane localization for Ygl053p. The common sequence from the different inserts encoding parts of Fir1p or Ynl078p should encode the domains from Fir1p and Ynl078p required for interaction with the Ygl053p protein. Recently, high-throughput two-hybrid screens revealed that Yar031p and Apg12p interact, and that Ygl051p and Yar033p interact (Uetz et al., 2000 ). We were unable to replicate this finding (data not shown). Apg12p is involved in autophagy and the targeting of proteins to the vacuole. Most free Apg12p seems to be associated with the endoplasmic reticulum (Mizushima et al., 1998
). Two-hybrid interactions have also been reported between Yar027p and Cks1p, an essential, physically associated, component that interacts with the protein kinase Cdc28p (Hadwiger et al., 1989
), between Yar027p and Yar030p, between Ygl053p and Ylr065p, and between Yhl044p and Ykr035p (Ito et al., 2000
). The Yar030p, Ylr065p and Ykr035p proteins have unknown functions but possess two and five putative transmembrane domains, respectively, so these proteins are themselves predicted to be membrane associated. The proteins coded by YGL051w, YGL053w, YAR031w and YAR033w are predicted to have structural and/or functional roles at membranes.
Our data suggest that we are dealing with a gene family specific to the genus Saccharomyces sensu stricto. This gene family belongs to the set of ascomycete-specific genes, a class of genes that tend to be more sensitive to evolutionary divergence than the average (Malpertuy et al., 2000 ). Even without specific information on the precise function of the proteins encoded by this gene family, we have shown that when amino acid divergence is detectable, the changes often affect a limited number of amino acids localized within a specific domain of the protein. The proteins encoded by this gene family, with the exception of Yar029p, appear to be located at membranes. We have evidence that they have specialized functions because when they were fused to GFP they did not show identical fluorescence patterns and when fused to hSos1p they did not show identical RAS activation properties. In addition, Ygl051p and Ygl053p both appear to interact with a specific set of non-redundant proteins. Thus, our data suggest that the 10 Dup240 members do not have identical functions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Aronheim, A., Engelberg, D., Li, N., al-Alawi, N., Schlessinger, J. & Karin, M. (1994). Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell 78, 949-961.[Medline]
Aronheim, A., Zandi, E., Hennemann, H., Elledge, S. J. & Karin, M. (1997). Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol Cell Biol 17, 3094-3102.[Abstract]
Bartel, P. L., Chien, C. T., Sternglanz, R. & Fields, S. (1993a). Using the two-hybrid system to detect protein-protein interactions. In Cellular Interactions in Development: a Practical Approach , pp. 153-179. Edited by D. A. Hartley. Oxford: Oxford University Press.
Bartel, P. L., Chien, C. T., Sternglanz, R. & Fields, S. (1993b). Elimination of false positives that arise in using the two-hybrid system. Biotechniques 14, 920-924.[Medline]
Barton, A. B., Bussey, H., Storms, R. K. & Kaback, D. B. (1997). Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: characterization of the 54 kb right terminal CDC15-FLO1-PHO11 region. Yeast 13, 1251-1263.[Medline]
Beh, C. T., Cool, L., Phillips, J. & Rine, J. (2001). Overlapping functions of the yeast oxysterol-binding protein homologues. Genetics 157, 1117-1140.
Bendixen, C., Gangloff, S. & Rothstein, R. (1994). A yeast mating-selection scheme for detection of protein-protein interactions. Nucleic Acids Res 22, 1778-1779.[Medline]
Blandin, G., Durrens, P., Tekaia, F. & 19 other authors (2000). Genomic exploration of the hemiascomycetous yeasts. 4. The genome of Saccharomyces cerevisiae revisited. FEBS Lett 487, 3136.[Medline]
Bon, E., Neuveglise, C., Casaregola, S., Artiguenave, F., Wincker, P., Aigle, M. & Durrens, P. (2000). Genomic exploration of the hemiascomycetous yeasts. 5. Saccharomyces bayanus var. uvarum. FEBS Lett 487, 37-41.[Medline]
Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P. & Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115-132.[Medline]
Chardin, P., Camonis, J. H., Gale, N. W., van Aelst, L., Schlessinger, J., Wigler, M. H. & Bar-Sagi, D. (1993). Human Sos1: a guanine nucleotide exchange factor for Ras that binds to Grb2. Science 260, 1338-1343.[Medline]
Delneri, D., Gardner, D. C., Bruschi, C. V. & Oliver, S. G. (1999). Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain. Yeast 15, 1681-1689.[Medline]
Dujon, B. (1998). European Functional Analysis Network (EUROFAN) and the functional analysis of the Saccharomyces cerevisiae genome. Electrophoresis 19, 617-624.[Medline]
Eng, W. K., Faucette, L., McLaughlin, M. M., Cafferkey, R., Koltin, Y., Morris, R. A., Young, P. R., Johnson, R. K. & Livi, G. P. (1994). The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth. Gene 151, 61-71.[Medline]
Fairhead, C., Thierry, A., Denis, F., Eck, M. & Dujon, B. (1998). Mass-murder of ORFs from three regions of chromosome XI from Saccharomyces cerevisiae. Gene 223, 33-46.[Medline]
Feuermann, M., de Montigny, J., Potier, S. & Souciet, J. L. (1997). The characterization of two new clusters of duplicated genes suggests a Lego organization of the yeast Saccharomyces cerevisiae chromosomes. Yeast 13, 861-869.[Medline]
Fields, S. & Song, O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340, 245-246.[Medline]
Fromont-Racine, M., Rain, J. C. & Legrain, P. (1997). Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nat Genet 16, 277-282.[Medline]
Georgakopoulos, T., Koutroubas, G., Vakonakis, I., Tzermia, M., Prokova, V., Voutsina, A. & Alexandraki, D. (2001). Functional analysis of the Saccharomyces cerevisiae YFR021w/YGR223c/YPL100w ORF family suggests relations to mitochondrial/peroxisomal functions and amino acid signalling pathways. Yeast 18, 1155-1171.[Medline]
Gietz, R. D., Schiestl, R. H., Willems, A. R. & Woods, R. A. (1995). Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355-360.[Medline]
Goffeau, A., Park, J., Paulsen, I. T., Jonniaux, J. L., Dinh, T., Mordant, P. & Saier, M. H. (1997). Multidrug-resistant transport proteins in yeast: complete inventory and phylogenetic characterization of yeast open reading frames with the major facilitator superfamily. Yeast 13, 43-54.[Medline]
Hadwiger, J. A., Wittenberg, C., Mendenhall, M. D. & Reed, S. I. (1989). The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex. Mol Cell Biol 9, 2034-2041.[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, 267-272.[Medline]
Ito, T., Tashiro, K., Muta, S., Ozawa, R., Chiba, T., Nishizawa, M., Yamamoto, K., Kuhara, S. & Sakaki, Y. (2000). Toward a protein-protein interaction map of the budding yeast: a comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins. Proc Natl Acad Sci USA 97, 1143-1147.
James, P., Halladay, J. & Craig, E. A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425-1436.
Jelinsky, S. A. & Samson, L. D. (1999). Global response of Saccharomyces cerevisiae to an alkylating agent. Proc Natl Acad Sci USA 96, 1486-1491.
Johnston, S. A. & Carlson, M. (1992). Regulation of carbon and phosphate utilization. In The Molecular and Cellular Biology of the Yeast Saccharomyces, Gene Expression , pp. 193-281. Edited by E. W. Jones, J. R. Pringle & J. R. Broach. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Karin, M., Najarian, R., Haslinger, A., Valenzuela, P., Welch, J. & Fogel, S. (1984). Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast. Proc Natl Acad Sci USA 81, 337-341.[Abstract]
Klein, P., Kanehisa, M. & DeLisi, C. (1985). The detection and classification of membrane-spanning proteins. Biochim Biophys Acta 815, 468-476.[Medline]
Malpertuy, A., Tekaia, F., Casaregola, S. & 21 other authors (2000). Genomic exploration of the hemiascomycetous yeasts. 19. Ascomycetes-specific genes. FEBS Lett 487, 113121.[Medline]
Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M. D., Klionsky, D. J., Ohsumi, M. & Ohsumi, Y. (1998). A protein conjugation system essential for autophagy. Nature 395, 395-398.[Medline]
Muhlrad, D., Hunter, R. & Parker, R. (1992). A rapid method for localized mutagenesis of yeast genes. Yeast 8, 79-82.[Medline]
Nelissen, B., Mordant, P., Jonniaux, J. L., De Wachter, R. & Goffeau, A. (1995). Phylogenetic classification of the major superfamily of membrane transport facilitators, as deduced from yeast genome sequencing. FEBS Lett 377, 232-236.[Medline]
Oliver, S. G., Winson, M. K., Kell, D. B. & Baganz, F. (1998). Systematic functional analysis of the yeast genome. Trends Biotechnol 16, 373-378.[Medline]
Ozcan, S., Dover, J., Rosenwald, A. G., Wolfl, S. & Johnston, M. (1996). Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression. Proc Natl Acad Sci USA 93, 12428-12432.
Petitjean, A., Hilger, F. & Tatchell, K. (1990). Comparison of thermosensitive alleles of the CDC25 gene involved in the cAMP metabolism of Saccharomyces cerevisiae. Genetics 124, 797-806.
Rachidi, N., Martinez, M. J., Barre, P. & Blondin, B. (2000). Saccharomyces cerevisiae PAU genes are induced by anaerobiosis. Mol Microbiol 35, 1421-1430.[Medline]
Russnak, R., Pereira, S. & Platt, T. (1996). RNA binding analysis of yeast REF2 and its two-hybrid interaction with a new gene product, FIR1. Gene Expr 6, 241-258.[Medline]
Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A. & Arnheim, N. (1985). Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sander, C. & Schneider, R. (1991). Database of homology-derived protein structures and the structural meaning of sequence alignment. Proteins 9, 56-68.[Medline]
Schneller, J. M., Schneider, C. & Stahl, A. J. (1978). Distinct nuclear genes for yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases. Biochem Biophys Res Commun 85, 1392-1399.[Medline]
Sherman, F., Fink, G. R. & Hicks, J. B. (1986). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Souciet, J., Aigle, M., Artiguenave, F. & 21 other authors (2000). Genomic exploration of the hemiascomycetous yeasts. 1. A set of yeast species for molecular evolution studies. FEBS Lett 487, 312.[Medline]
Takahashi, Y., Mizoi, J., Toh, E. A. & Kikuchi, Y. (2000). Yeast Ulp1, an Smt3-specific protease, associates with nucleoporins. J Biochem 128, 723-725.[Abstract]
Uetz, P., Giot, L., Cagney, G. & 17 other authors (2000). A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623627.[Medline]
Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. (1994). New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793-1808.[Medline]
Wagner, R., Straub, M. L., Souciet, J. L., Potier, S. & de Montigny, J. (2001). New plasmid system to select for Saccharomyces cerevisiae purine-cytosine permease affinity mutants. J Bacteriol 183, 4386-4388.
Wendland, B. & Emr, S. D. (1998). Pan1p, yeast Eps15, functions as a multivalent adaptor that coordinates protein-protein interactions essential for endocytosis. J Cell Biol 141, 71-84.
Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg, C. P. & Boles, E. (1999). Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett 464, 123-128.[Medline]
Received 5 December 2001;
revised 4 March 2002;
accepted 20 March 2002.