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Enzyme Phosphatidylserine Synthase (Saccharomyces Cerevisae/Chol Gene/Transformation) V

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Enzyme Phosphatidylserine Synthase (Saccharomyces Cerevisae/Chol Gene/Transformation) V Proc. Nati. Acad. Sci. USA Vol. 80, pp. 7279-7283, December 1983 Genetics Isolation of the yeast structural gene for the membrane-associated enzyme phosphatidylserine synthase (Saccharomyces cerevisae/CHOl gene/transformation) V. A. LETTS*, L. S. KLIG*, M. BAE-LEEt, G. M. CARMANt, AND S. A. HENRY* *Departments of Genetics and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461; and tDepartment of Food Science, Cook College, New Jersey Agricultural Experimental Station, Rutgers University, New Brunswick, NJ 08903 Communicated by Frank Lilly, August 11, 1983 ABSTRACT The structural gene (CHOI) for phosphatidyl- Mammals, for example, synthesize PtdSer by an exchange re- serine synthase (CDPdiacylglycerol:L-serine O-phosphatidyl- action with PtdEtn (9). However, PtdSer synthase is found in transferase, EC 2.7.8.8) was isolated by genetic complementation E. coli and indeed the structural gene for the E. coli enzyme has in Saccharomyces cerevmae from a bank of yeast genomic DNA been cloned (10). Thus, cloning of the structural gene for the on a chimeric plasmid. The cloned DNA (4.0 kilobases long) was yeast enzyme will permit a detailed comparison of the structure shown to represent a unique sequence in the yeast genome. The and function of prokaryotic and eukaryotic genes and gene DNA sequence on an integrative plasmid was shown to recombine products. The availability of a clone of the CHOI gene will per- into the CHOi locus, confwrming its genetic identity. The chol yeast mit analysis of its regulation at the transcriptional level. Fur- strain transformed with this gene on an autonomously replicating thermore, the cloning of the CHOI gene provides us with the plasmid had significantly increased activity of the regulated mem- the levels of PtdSer synthase in the cell, brane-associated enzyme phosphatidylserine synthase. Partial pu- opportunity to increase rification of phosphatidylserine synthase from microsomes of this facilitating the purification and characterization of this mem- transformed strain confirmed that the membrane-bound enzyme brane-associated enzyme. was overproduced 6-to 7-fold as comparedwith the wild-type strain. The strain also synthesized the product phospholipid, phospha- MATERIALS AND METHODS tidylserine, at an increased rate. The transformed strain had al- tered proportions of a variety of other phospholipids, suggesting Strains. The haploid S. cerevsiae strain VAL2C (Mata leu2- that their synthesis is affected by the rate of synthesis of phos- 3 leu2-112 ade6 chol) was derived from a cross of strain DC5 phatidylserine in yeast. (Mata leu2-3 leu2-112 his3 cani-11, provided by J. Hicks) to strain VAL12 (MATa ade6 ural chol). Strain VAL12 was iso- The characterization of mutants altered in the synthesis of klted as an ethanolamine auxotroph from wild-type strain SHID phospholipids has given considerable insight into the regulation SC (MATa ade6 ural). Strain 399 [MATa arg4-17 (UAA), lysl- of phospholipid metabolism in Escherichia coli and Saccha- 1 (UAA), met8-1 (UAG), trpl-l (UAG), leu2 (UGA)] was ob- romyces cerevisiae (1, 2). Using yeast phospholipid mutants and tained from G. Fink and wild-type strain S288C was used for the transformation techniques now available, it is possible to the enzyme preparations. The E. coli strain used was C600 (F- clone by complementation a number of the structural and reg- leuB pro- thr-). ulatory genes involved in yeast lipid metabolism. These cloned E. coli cells were cultured in LB medium (11). Additions in- genes can then be used as probes to study the synthesis, reg- cluded ampicillin (20 ug/ml) and tetracycline (100 Ag/ml). ulation, and assembly of the membrane-associated enzymes of Conditions and media for growth and genetic analysis of yeast phospholipid synthesis. wild-type and chow strains have been described (3, 4). The chol mutants of S. cerevisiae require either ethanol- Clone Bank of Yeast Genomic DNA and Plasmids for amine or choline for growth and are deficient in the synthesis Transformation. Plasmid YEp13 is capable of autonomous rep- of the phospholipid, phosphatidylserine (PtdSer) (3-5). PtdSer lication in both E. coli and yeast and can be selected for in yeast is the normal precursor for both phosphatidylethanolamine strains of genotype leu2-, because the plasmid bears the yeast (PtdEtn) and phosphatidylcholine (PtdCho). However, under wild-type LEU2 gene (12). The plasmid can be selected in E. conditions of ethanolamine or choline supplementation, chol coli by ampicillin resistance because it contains the sequence mutants are able to use an alternative pathway described by of the bacterial plasmid pBR322. A clone bank (12) of genomic Kennedy and Weiss (6) for the synthesis of PtdEtn and PtdCho DNA from yeast strain AB320 carried on plasmid YEp13 was (3-5). All chow mutants have very reduced levels of the enzyme donated by K. Nasmyth. The plasmid containing the CHOI DNA phosphatidylserine synthase (CDPdiacylglycerol:L-serine 0- will be referred to as YEpCHOI. A second vector, YIp28, ob- phosphatidyltransferase, EC 2.7.8.8). Thus, the CHOJ locus tained from D. Botstein was used to construct an integrating has the characteristics expected of the structural gene for this plasmid containing the yeast CHOI and LEU2 genes as well as enzyme (4). Yeast PtdSer synthase is a regulated integral mem- pBR322 sequences and is referred to as YIpCHOl. brane protein (7, 8). In this report, we describe the isolation of Spheroplasts of strain VAL2C were transformed with 100 jtg the yeast CHOI gene and present further evidence to sub- of plasmid DNA by the procedure of Hinnen et al. (13) and Leu+ stantiate the identification of CHOI as the structural gene for Cho' transformants were selected in agar plates lacking both PtdSer synthase. leucine and ethanolamine. E. coli strain C600 was transformed PtdSer synthase is not found in all eukaryotic organisms. with yeast DNA prepared from Leu+ Cho' transformants by The publication costs ofthis article were defrayed in partbypage charge Abbreviations: PtdSer, phosphatidylserine; PtdEtn, phosphatidyl- payment. This article must therefore be hereby marked "advertise- ethanolamine; PtdCho, phosphatidylcholine; kb, kilobase(s); PtdIns, ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. phosphatidylinositol. 7279 Downloaded by guest on September 27, 2021 7280 Genetics: Letts et al. Proc. Natl. Acad. Sci. USA 80 (1983) the method of Mandel and Higa (14) and ampicillin-resistant transformants were selected. Plasmid DNA was prepared from E. coli by cesium chloride/ ethidium bromide density gradient centrifugation (15). Restric- tion endonucleases were purchased from Bethesda Research Laboratories and were used as recommended by the supplier; after digestion, DNA fragments were analyzed by gel electro- phoresis on 0.7% agarose (16). Restriction fragment sizes were determined with reference to A and 4X174 DNA fragments of known sizes. DNA fragments were recovered from the agarose gel (17) and were ligated into modified plasmid YIp28 (cleaved EcoRI with appropriate restriction enzymes) using T4 ligase. PvuI Assay of PtdSer Synthase. PtdSer synthase activity was mea- sured by following the incorporation of 0.5 mM L-[3-3H]serine (10,000 cpm/nmol) into chloroform-soluble material in the presence of 50 mM Tris HCI, pH 8.0/0.6 mM MnCl2/0.2 mM CDP-diacylglycerol/4 mM Triton X-100 and enzyme protein in a total volume of 0.1 ml as described (7). A unit of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of 1 nmol of product per min under the assay con- ditions described above. Specific activity is defined as units/ Cla I EcoRI mg of protein. Protein was determined by the method of Brad- ford (18) with bovine serum albumin as the standard. FIG. 1. Restriction endonuclease map of the hybrid integrating plasmid (YIpCHO1) containing the CHO gene locus on a 4-kb DNA RESULTS fragment. The approximate positions of the ampicillin (ampR)-resis- tance gene and the origin (ori) of replication of pBR322-containing Isolation and Identification of the CHOI Gene. The yeast plasmids are based on the results of Boliver et al. (20). The following CHOI gene was isolated by screening for Leu+ Cho' yeast col- enzymes were also tested and found not to cut within the CHOl clone: onies after transformation of strain VAL2C with the yeast ge- BamHI, Pst I, Bgl II, Sst I, Xba II, Xho I, and Xor II. nomic DNA clone bank of Nasmyth and Reed (12). Putative Leu+ Cho' transformants were picked and retested for both present in strain 399 allowed us to detect the plasmid in all four leucine and ethanolamine prototrophy. One of these trans- spores of a given tetrad. In tetrads in which all four spores had formants was grown on nonselective (YEPD) medium for sev- a Leuu Cho' phenotype, all of the suppressible markers seg- eral days and then restreaked to isolate single colonies. The regated in a 2+:2- fashion. Thus, the plasmid that confers the phenotype of the individual colonies was then tested by replica Leu+ Cho' phenotype does not contain a nonsense suppressor. plating onto medium lacking leucine and ethanolamine. It was To show that the isolated yeast DNA was derived from the observed that 30-40% of the colonies were phenotypically leu- CHOI locus itself, the cloned DNA sequence was transferred cho- after the period of growth on nonselective medium. This to the integrative plasmid YIp28. This plasmid lacks 2-pum DNA instability of phenotype is characteristic of yeast cells that have and can stably transform yeast cells only by homologous re- been transformed with autonomously replicating plasmids (19). combination into the genome (13). The plasmid containing the The simultaneous loss of both the leu+ and cho' markers also putative CHOI DNA (YEpCHO1) was digested with endonu- confirms that the Leu+ transformants had not reverted at the cleases HindIII and Sal I (see Fig.
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