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Cytosine Transport System in Saccharomyces Cerevisiae (8-Azidoadenine/Molecular Cloning/Membrane Protein/Glycosylation) RAINER SCHMIDT*T, MORRIS F

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Cytosine Transport System in Saccharomyces Cerevisiae (8-Azidoadenine/Molecular Cloning/Membrane Protein/Glycosylation) RAINER SCHMIDT*T, MORRIS F Proc. Nati. Acad. Sci. USA Vol. 81, pp. 6276-6280, October 1984 Biochemistry Photoaffinity labeling and characterization of the cloned purine- cytosine transport system in Saccharomyces cerevisiae (8-azidoadenine/molecular cloning/membrane protein/glycosylation) RAINER SCHMIDT*t, MORRIS F. MANOLSON*t, AND MARIE-REN1-E CHEVALLIER§ *Institut fuer Biophysik, Freie Universitaet Berlin, Thielallee 63-67, D-1000 Berlin 33, Federal Republic of Germany; and §Institut de Biologie Moldculaire et Cellulaire du Centre National de la Recherche Scientifique, 15, rue Rend Descartes, F-67084 Strasbourg, France Communicated by Boris Magasanik, June 11, 1984 ABSTRACT 8-Azido[2-3H]adenine was used as a photoaf- MATERIALS AND METHODS finity label for the purine-cytosine transport system. After Strains and Culture Conditions. S. cerevisiae RXII (a gift irradiation in the presence of the photoaffinity label, the cells of A. Kotyk, Prague) was used as the diploid wild-type were converted into protoplasts, their plasma membranes were strain. The mutant strain NC117spl (a leu2 adel fcy2-3) was purified, and the membrane proteins were extracted and sepa- obtained by a cross of NC99spl (a adel fcy2-3) (a derivative rated by NaDodSO4/PAGE. The radioactivity was specifically of FL100; ATCC 28383) x GRF18 (his3 leu2). The strains incorporated into a protein with a molecular weight of 120,000. were cultivated in GYNP medium (2% glucose/1% Difco Photoaffinity labeling of this protein could be blocked by yeast nitrogen base/0.5% peptone) containing hypoxanthine irradiation in the presence of natural substrates for the trans- at 10 ,g/ml at 30°C with agitation. port system. The molecular weight as determined by NaDod- When cells from the stationary growth phase were pre- S04/PAGE was found to be twice the value calculated from pared for maximum uptake, a 50-ml culture was preincu- mRNA analysis of the cloned gene. Incubation of exponentially bated in 250 ml of glucose/citrate buffer (50 mM sodium growing cells with tunicamycin, an antibiotic that inhibits citrate, pH 5.5/2% glucose) at 30°C under aeration for 60 glycosylation of proteins, resulted in a 40% decrease in the min. overall initial uptake rate, which correlates with the reduction To transform E. coli, strain BJ5183 (F- recBC sbcB endoI of the labeled Mr 120,000 protein. Treatment of the extracted gal met str hsdR thi bio) was used as receptor strain. labeled plasma membrane proteins with glycosidic enzymes Cloning Procedure. Cloning was accomplished by using resulted in disappearance of the Mr 120,000 peak and the two yeast gene libraries, the construction of which is de- appearance of new peaks at Mr 60,000 and Mr 73,000. These scribed by Losson and Lacroute (9). The plasmid used as a findings indicate that the purine-cytosine transport protein is a vector was pJDB207. In addition to two distinct origins of glycoprotein. DNA replication, one of which is functional in E. coli and the other, in yeast, it contains the yeast LEU2 gene and the The uptake of the purine bases adenine, guanine, and genes conferring hypoxanthine and the pyrimidine base cytosine in Sac- ampicillin and tetracycline resistance to E. charomyces cerevisiae is catalyzed by a single energy-de- coli (10). The yeast strain NC117spl (a leu2 adel fcy2-3) was pendent transport system (1, 2). In vivo properties of the used as recipient in yeast transformation. Because of their transport system such as genetic characterization, energy fcy2 mutation, ade -/leu+ -transformed cells (except those coupling, and a possible mechanism of substrate binding to having recovered a functional purine-cytosine transport pro- the purine-cytosine transport system are described in refs. tein) are unable to grow in the presence of low concentra- 3-6. tions (3 ,g/ml) of hypoxanthine. For the further characterization of the transport system, DNA Preparation and Analysis. Plasmid DNA from E. coli we found 8-azidoadenine to be a suitable photoaffinity label. was prepared according to Clewell (11). Yeast DNA prepa- It has a high affinity (Km = 9 x 10-6 M) without being ration was carried out as described (12). Restriction en- translocated and it is bound covalently to the transport donuclease digestion, filling of protruding ends, and ligation protein on irradiation with UV light. Its synthesis and prop- of DNA were carried out as suggested by the suppliers. erties are described in ref. 7. Transformation. Yeast was transformed by the method of The purine-cytosine transport system was cloned on a Hinnen et al. (13), except that Zymolase 60,000 at a final multicopy plasmid that can replicate autonomously in yeast concentration of 50 ,ug/ml was used to prepare the sphero- or in Escherichia coli. Cloning was necessary to (i) increase plasts. E. coli was transformed by the method of Cohen et al. the amount of transport protein in the yeast plasma mem- (14). brane, (ii) allow measurement of the length of the mRNA Measurement of the Initial Rate of Uptake for Purine Bases. transcript of the cloned gene, and (iii) enable sequencing of Initial uptake rates were measured as in ref. 6. Exponentially the gene. growing cells or stationary cells after 60 min ofpreincubation In this work, we used 8-azido[2-3H]adenine as a photoaf- were incubated for 1 min with ('4C]hypoxanthine. The cells finity label. The transport protein was labeled in vivo, the were separated from the reaction mixture by filtration over plasma membranes ofthe labeled cells were purified by using glass fiber filters. The filters were then assayed for radio- cationic silica microbeads (8), and the plasma membrane activity. proteins were extracted for further characterization. tTo whom requests for reprints should be addressed at: Centre International de Recherches Dermatologiques (CIRD), Sophia An- The publication costs of this article were defrayed in part by page charge tipolis, F-06565 Valbonne, France. payment. This article must therefore be hereby marked "advertisement" tPresent address: Biology Department, McGill University, Mon- in accordance with 18 U.S.C. §1734 solely to indicate this fact. treal, Canada. 6276 Downloaded by guest on September 29, 2021 Biochemistry: Schmidt et al. Proc. Natl. Acad. Sci. USA 81 (1984) 6277 BclI Synthesis of 8-Azido[2-3H]Adenine. Commercially avail- Sall Kpn I able 8-azido[2-3H]adenosine was used as starting material T' Pvuff Aval = 37 GBq). BamHlI IIECORI Clal S hi ' h ACIOa (250 ACi; 15 Ci/mmol in 2.5 ml of methanol; 1 Ci H AccI~IIoIT __ The methanol was evaporated under a gentle stream of SPtS~?tXbal T KpnlT Tt TET11r nitrogen and the residue was then suspended in 0.5 ml of 1 M T HCl and incubated for 15 min at 100'C. Thereafter, the H~i H~lll-~ sample was freeze-dried and dissolved in 2.2 ml of 0.01 M A 4kb B 2.7kb HCl. An almost 100% degradation into 8-azido[2-3H]adenine was confirmed by TLC. a< C~ 2.4kb Photoaffinity Labeling. Cells were harvested in the early D 3.2kb exponential phase of growth, washed once with distilled water, and then suspended in ice-cold distilled water (1 x 108 FIG. 1. Restriction map of yeast DNA insert into pNCII4-9. The cells per ml). One milliliter of this cell suspension was horizontal bars and their corresponding letters indicate that these pipetted into a glass dish (diameter, 5.5 cm), which was fragments were recloned into plasmid pJDB207 to test their com- stirrer. 8-Azido[2- plementation ability in yeast. Several HindIII sites found in frag- placed in an ice bath on a magnetic ment A are not shown on the map because their position was not 3H]adenine was added to a final concentration of 0.5 ,tM. For determined. testing the inhibitory effect of natural substrates of the pu- rine-cytosine transport system on specific binding of the photoaffinity label, these substrates were added in thousand- bated for 2.5 hr to reach a final cell titer of 3 x 107 cells per fold molar excess to the reaction mixture prior to irradiation. ml. After 5 min of incubation, the cell suspension was frozen by Chemicals. 8-Azido[2-3H]adenosine, the 14C-labeled mo- pouring liquid nitrogen over it. The glass dish was then lecular weight standards, Protosol, and Econofluor were transferred to an ethanol/dry ice bath. The sample was from New England Nuclear. 14C-labeled purine bases and irradiated for 5 min (1.9 mW/cm2) with a UVS-54 Miner- [14C]cytosine were purchased from Amersham Buchler. alight lamp (emission maximum, 254 nm). Thereafter the Tunicamycin was from Sigma. The glycosidic enzymes were sample was quickly thawed and the cells were separated H, a The whole pro- from Boehringer Mannheim except for endoglycosidase from the incubation mixture by filtration. product of Miles Laboratories. Restriction endonucleases cedure was carried out in the dark. and Plasma membranes were iso- were from Biolabs, Bethesda Research Laboratories, Plasma Membrane Isolation. Boehringer Mannheim. All other chemicals were of analyti- lated and purified as described in ref. 8. The cells were UVS- converted into protoplasts and their plasma membranes were cal grade or of the highest purity available. The lamp coated with cationic silica microbeads. After lysis, the plasma 54 Mineralight was from Ultra Violet Products (San Gabriel, membranes were washed free of other cell organelles. CA). NaDodSO4/PAGE. Electrophoresis was carried out as de- scribed by Laemmli (15). After photoaffinity labeling, the RESULTS plasma membrane obtained from 4 x 108 cells was incubated for 5 min at 100'C in denaturation buffer [5% (wt/vol) Na- Cloning of the Purine-Cytosine Transport Protein Gene. DodSO4/5% (vol/vol) 2-mercaptoethanol/10 mM Tris HCl, Transformation of strain NC117spl with two independently pH 8].
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