Department of Biological Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK1
Institut für Biochemie und Lebensmittelchemie, Technische Universität Graz, Petersgasse 12/II, A-8010 Graz, Austria2
Genetics and Microbiology Dept, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK3
Institut für Pflanzenphysiologie, Karl-Franzens Universität, Schubertstrasse 51, A-8010 Graz, Austria4
Author for correspondence: Michael Schweizer. Tel: +44 131 451 3186. Fax: +44 131 451 3009. e-mail: M.Schweizer{at}hw.ac.uk
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ABSTRACT |
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Keywords: phosphoribosyl-pyrophosphate synthetase (Prs), subcellular localization of Prs1p, cell integrity, Saccharomyces cerevisiae
Abbreviations: DIC, differential interference contrast; 5-FOA, 5-fluoroorotic acid; GFP, green fluorescent protein; NHR, non-homologous region; PRPP, 5-phosphoribosyl 1()-pyrophosphate; Prs, 5-phosphoribosyl-1(
)-pyrophosphate synthetase; RLU, relative light units
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INTRODUCTION |
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Cells are impermeable to PRPP. However, Eschericha coli prs mutants are fully rescued if supplemented with purine, pyrimidine, pyridine nucleotides, histidine and tryptophan (Hove-Jensen, 1989 ), indicating that in this organism Prs is not required for a function other than PRPP synthesis, although it has been suggested that the enzyme may be implicated in the initiation of DNA replication in E. coli (Sakakibara, 1992
). In humans, superactivity of Prs correlates with gouty arthritis and in some cases, disorders of the nervous system (Becker et al., 1988
, 1995
).
PRS genes have been cloned and sequenced from 19 organisms; both rat and human have two ubiquitously expressed PRS genes, with a third testis-specific gene found in man (Taira et al., 1990 ). Saccharomyces cerevisiae harbours five unlinked structural genes encoding Prs polypeptides Prs1pPrs5p. Within this PRS multigene family, PRS2, PRS3 and PRS4 exhibit more sequence similarity to each other than to either PRS1 or PRS5 (Carter et al., 1994
, 1997
; Hernando et al., 1998
, 1999
). Characterization of yeast strains deleted individually for each of the PRS genes indicates that, although none of them is essential for viability, the contribution of each of the gene products to the metabolic status of the cell appears to differ both qualitatively and quantitatively. Disruption of PRS1 or PRS3 results in a severely reduced growth rate and markedly decreased total Prs enzyme activity (Carter et al., 1997
; Hernando et al., 1999
), suggesting that these polypeptides have a key structural or regulatory role in PRPP synthesis.
The predicted Prs polypeptides Prs2p, Prs3p and Prs4p are 318355 amino acids long and have molecular masses of 34·7, 35·1 and 39·0kDa, respectively. Prs1p (427 amino acids, 47·0 kDa) contains a unique in-frame insertion of 105 amino acids, termed the non-homologous region (NHR) 1-1. Prs5p possesses two NHRs, one of 116 amino acids (NHR5-2) located between the divalent-cation-binding site and the PRPP-binding site, as is the case for NHR1-1 present in Prs1p. The other NHR, NHR5-1, consisting of 70 amino acids, is located directly in front of the divalent-cation-binding site and thus Prs5p with 496 amino acids is the largest polypeptide of the family (predicted molecular mass 53·5 kDa). The NHRs are not similar to each other or to any other sequences in the databases. The significance and function of the NHRs is at present unknown.
In the present work, by using antisera against two different epitopes of Prs1p, one outside (AK41) and the other within (AK39) the NHR1-1 of Prs1p, we determined the subcellular distribution of Prs1p by immunofluorescence analysis and by cellular fractionation. In fixed cells, both antisera associated with cytoplasmic structures. In living cells, a functional green fluorescent protein (GFP)Prs1p fusion also localized to granular cytoplasmic structures. The fact that certain PRS deletants are hypersensitive to caffeine and respond to the addition of caffeine to a liquid culture by releasing alkaline phosphatase more readily into the medium than the wild-type points to a possible relationship between Prs and the structure and/or function of the cell wall.
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METHODS |
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A 909 bp fragment of PRS1 was amplified by PCR using YN94-1 DNA as template with the primers PRS1 start, +4(PRS1)[5'-CGTAAGTGTAAAATTTTTGT-3']+23(PRS1), and P2, +912(PRS1)[5'-AGAGAACTGGGATTTCC-3']+896(PRS1). The 5' end of the amplification product starts at the second codon of PRS1 and was therefore complementary to the 3' end of the GFP PCR product.
Both PCR products were joined in a PCR-ligation reaction (Pearson et al., 1998 ) driven with an excess of the two outer primers, P1 and P2. To optimize the efficiency of integration of the 1659 bp GFP-PRS1' construct the length of the 5'-flanking region was increased. A 190 bp fragment of PRS1 5'-flanking sequence was amplified using primers P11, -208(PRS1)[5'-GATGATTAGCGAAGTTC-3']-192(PRS1), and P10, -18(PRS1)[5'-TTAAGACTATTAAACGGT-3']-35(PRS 1). By virtue of the 18 bp overlap with the 5' end of the GFP-PRS1' construct, the two products were fused by PCR using the primers P11 and P2 to give rise to the final product of 1831 bp. This PCR product was used after agarose gel electrophoresis purification (Sambrook et al., 1989
) to transform YN95-21 (Gietz & Woods, 1994
) successfully to 5-FOA resistance, creating the strain YN97-96.
YN97-96 carrying prs1::GFP-PRS1 was used to create four additional strains, individually deleted for PRS2, PRS3, PRS4 and PRS5. YN98-8 and YN98-10, which are deleted for PRS2 and PRS3, respectively, were created by transforming YN97-96 with the PCR products obtained from YN97-141 and YN96-52 using primers flanking the disrupted PRS2 and PRS3 loci (Carter et al., 1997
). YN98-12 and YN98-14 were created by the short flanking homology method (Wach et al., 1994
) using appropriate primers for the flanking regions of PRS4 and PRS5 in conjunction with pUG6 DNA (Güldener et al., 1996
) as the template. Deletions were checked by PCR and Southern blot analysis.
Microscopic techniques and immunofluoresecence.
For immunofluorescence, cells in the early exponential phase of growth were fixed in formaldehyde/methanol exactly as described by Wente et al. (1992) . Antisera AK39 (peptide 1) and AK41 (peptide 2), described by Carter et al. (1997)
, were pre-adsorbed onto fixed cells and subsequently used at a 1:2700 (AK39) and a 1:900 (AK41) dilution. Binding of AK39 or AK41 was detected using FITC-conjugated secondary antibody against rabbit IgG (Oncogene Science Diagnostics; diluted 1:100). Invertase was detected using a 1:2500 dilution of a rabbit anti-invertase antiserum generously provided by R. Schekman, Berkely, CA. Nucleic acid was stained by DAPI (4',6-diamidino-2-phenylindole dihydrochloride). Epifluorescence analysis of wild-type and haploid strains deleted for the PRS genes indicated was performed using a Zeiss Axiovert 35 microscope (Carl Zeiss) and photographs were taken using a x100 objective with Kodak T-Max 100 PRO (Eastman Kodak).
Laser scanning microscopic analysis of tetraploid and haploid cells was performed using a Leica TCS 4d microscope and photographs were taken with a PL APO 63x1/1.40 objective on Kodak Ektachrome Elite 100 slide film (Eastman Kodak). Corresponding pictures were recorded with identical pinhole openings and amplification settings and were printed with the same exposure settings.
For electron microscopical investigations, cells were fixed in 4% paraformaldehyde/5% glutaraldehyde in 0·1 M cacodylate buffer pH 7·0 and 1 mM CaCl2 for 90 min at room temperature. Cells were then washed in buffer with 1 mM CaCl2 for 1 h and incubated for 1 h with a 2% aqueous solution of KMnO4. Fixed cells were washed in distilled H2O for 30 min and incubated in 1% sodium metaperiodate for 20 min. Samples were then rinsed in distilled H2O for 15 min and post-fixed for 2 h in 2% OsO4 buffered with 0·1 M cacodylate at pH 7·0. After another wash with buffer for 30 min, the samples were dehydrated in a graded series of ethanol washes (50100% with en bloc staining in 2% uranylactetate in 70% ethanol overnight) and embedded in Spurr resin. Ultrathin sections were stained with lead citrate and viewed with a Philips CM 10 electron microscope.
Subcellular fractionation.
Wild-type strain X2180-1A was used for fractionation experiments. One litre cultures in 2 litre flasks were incubated at 30 °C on a rotary shaker with vigorous aeration. For isolation of the plasma membrane, Golgi, nuclei and cytosol, cells were grown in YEPD. Mitochondria and microsomes were isolated from cells grown in 2% lactate medium. Cells were harvested by centrifugation and converted to spheroplasts as described by Daum et al. (1982) .
Subcellular fractionation was performed essentially as reviewed by Zinser & Daum (1995) . Briefly, plasma membranes were isolated according to Serrano (1988)
and approximately 100-fold enriched for plasma membrane H+-ATPase. Mitochondria were isolated according to Daum et al. (1982)
and were approximately 4-fold enriched for the major protein of the outer mitochondrial membrane, porin. Microsomes were obtained after clearing the post-mitochondrial supernatant as described by Gaigg et al. (1995)
. Isolation of the Kex2p-enriched (200-fold) Golgi fraction was as described by Leber et al. (1995)
. Nuclei were isolated by sucrose density-gradient centrifugation as described by Hurt et al. (1988)
and approximately 12-fold enriched for the ER marker BiP/Kar2p. Cytosol was obtained by 40% (NH4)2SO4 precipitation of the 10000 g supernatant obtained after removal of the crude nuclear pellet.
Protein and Western blot analysis.
Prior to quantification by the Lowry method, proteins were precipitated with 10% trichloroacetic acid and solubilized in 0·1% SDS, 0·1 M NaOH. Proteins were separated on a 10% SDS-PAGE gel and transferred to nitrocellulose filters (Hybond-C; Amersham Pharmacia Biotech). After blocking, blots were incubated at 4 °C overnight in a 1:2000 dilution of AK39 or AK41. The intensity of the immunoreaction was quantified after scanning the autoradiogram by using the wand tool present in the image analysis software NIH Image 1.60. Antisera against yeast plasma membrane H+-ATPase, Kex2 protease and BiP/Kar2p were obtained from G. Daum (Technical University of Graz, Austria) and were originally gifts from R. Serrano (Universidad Politecnica, Valencia, Spain), R. Fuller (University of Michigan Medical Center, Ann Arbor, MI, USA) and R. Schekman (University of California, Berkeley, CA, USA), respectively. Antisera against porin and the 40 kDa microsomal protein were obtained from G. Daum.
Caffeine sensitivity.
A single colony from a freshly grown YEPD culture was used to inoculate 10 ml YEPD broth. Cultures were grown overnight in a vertical rotary incubator. A constant inoculum of 2x104 cells was applied by means of a multiple inoculator on YEPD plates containing caffeine at the concentrations indicated. Plates were scored after incubation at 30 °C for 3 d.
Chemiluminescent determination of alkaline phosphatase release.
Overnight 10 ml cultures obtained as above were used to inoculate 200 ml YEPD broth in 500 ml Erlenmeyer flasks. Cultures were then shaken in an orbital incubator until an OD600 of 1·0 was reached, at which time either 10 ml of a 100 mM caffeine stock solution or 10 ml sterile distilled H2O was added. One millilitre samples were collected immediately and then at hourly intervals and the cells removed by centrifugation (13000 r.p.m., 3 min). Supernatants were stored at -20 °C until the assay was carried out. Alkaline phosphatase was assayed using a chemiluminescent detection reagent (Lumiphos-530 AP detection reagent; Amersham Pharmacia Biotech). One hundred microlitres of supernatant was mixed with an equal volume of Lumiphos-530 in a 5 ml plastic luminometer tube (Sarstedt) and incubated at room temperature. Relative light units (RLU) were measured using a Lumat LB 9501 luminometer (Berthold). Maximum RLU readings were obtained after 4560 min, therefore reaction rate (RLU min-1) was calculated from the linear part of a plot of several readings recorded between 10 and 90 min after the addition of the reagent.
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RESULTS |
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Preliminary analysis of the subcellular distribution of Prs1p in cells which overexpress it suggested that it is located in the cytoplasm. Overexpression of a protein can, however, potentially affect its subcellular distribution. We therefore analysed the distribution of Prs1p in wild-type cells. Since high-resolution laser scanning immunofluorescence analysis is limited by the small size of haploid yeast cells, large tetraploid cells were employed to allow detection of Prs1p at its normal level of expression (Fig. 1). This analysis revealed that both AK39 and AK41 antisera stain the cytoplasm in a granular pattern. This distribution is not specific for the tetraploid strain used since the same granular staining was also observed if diploid wild-type cells (YPH501; Table 1
) were stained with the antisera.
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DISCUSSION |
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Immunofluorescence analysis of the subcellular distribution of Prs1p in tetraploid wild-type cells suggests that Prs1p is a cytosolic enzyme predominantly found in granular structures that localize to the cell perimeter, below the plasma membrane. This arrangement was confirmed by analysing the subcellular localization of a functional GFPPrs1p fusion protein. The fact that Prs1p localization is not affected in secretory mutants indicates that these granular structures do not represent a post-Golgi compartment. To our knowledge, the anti-Prs1p staining pattern most closely resembles that observed with an antiserum against the cell cycle protein kinase Cdc28p, which has been localized to the yeast cytoplasmic matrix (Wittenberg et al., 1987 ).
Western blot analysis of protein extracts of subcellular fractions isolated from yeast homogenates revealed the presence of approximately equal amounts of Prs1p in all but the mitochondrial fraction. These findings might be interpreted to suggest that Prs1p exists in an aggregated form or is otherwise associated with insoluble cytoplasmic structures. Furthermore, ultrastructural analysis of triple PRS deletion strains did not reveal any obvious alterations of the nucleo- or cytoplasm that could be associated with loss of Prs1p. However, an alteration in the cells complement of Prs1pPrs5p appears to have consequences for the plasma membrane as shown by the increased frequency of plasma membrane invaginations. The postulated association of Prs with the plasma membrane may also be responsible for the caffeine sensitivity observed in various deletant strains. The purine analogue caffeine affects many cellular processes; caffeine sensitivity has been shown to be associated with defects in components of the MAP (mitogen-activated protein) kinase pathways (Cid et al., 1995 ; Stark, 1999
).
prs1
prs3 strains have the most caffeine-sensitive phenotype, failing to grow in the presence of 2 mM caffeine (Fig. 6
). Caffeine sensitivity can be reversed by incubation in 1 M sorbitol as an osmotic stabilizer (Hampsey, 1997
). Interestingly, PRS3 was isolated in a colony-sectoring assay to identify genes interacting with WHI2 whose deletion also causes caffeine sensitivity. However, Binley et al. (1999)
showed that mutants of PRS3 and WHI2 are not co-lethal. The same authors have documented that strains lacking
prs3 are affected not only in their ability to grow in the presence of 5 mM caffeine but also do not respond normally to nutrient deprivation, and are altered in ion homeostasis and in the organization of the actin cytoskeleton in stationary phase. The varying degrees of caffeine sensitivity observed in the PRS deletant strains may reflect inability to maintain cell integrity as suggested by the microscopic analysis of the plasma membrane (Fig. 5
). Further evidence of impairment of cell integrity is afforded by the demonstration that the loss of either PRS1 or PRS3 leads to the release of alkaline phosphatase in the presence of caffeine (Fig. 7
). This caffeine-associated release of alkaline phosphatase is prevented by the addition of 1 M sorbitol (data not shown). Deletants
prs2,
prs4 and
prs5, which are only slightly more sensitive to caffeine than the wild-type, do not show a caffeine-associated release of alkaline phosphatase. The osmotic remediability of the caffeine-sensitive phenotypes of certain PRS deletant strains and the release of alkaline phosphatase suggest that in some way an alteration of the Prs complement of the cell can affect cell integrity.
The results presented here and elsewhere (Hernando et al., 1999 ) suggest that the products of the five PRS genes could aggregate to form subcellular structures which may play a role in maintaining cell integrity. If one assumes that single and multiply deleted strains contain Prs complexes different from the wild-type, this could explain the alteration in caffeine sensitivity as reflecting a suboptimal maintenance of cell integrity.
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ACKNOWLEDGEMENTS |
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Received 10 April 2000;
revised 8 August 2000;
accepted 21 August 2000.
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