Proc. Natl. Acad. Sci. USA Vol. 77, No. 9, pp. 5192-5196, September 1980 Biochemistry Autoradiographic detection of animal cell membrane mutants altered in phosphatidylcholine synthesis (Chinese hamster ovary cells/immobilized colonies/filter paper replica plating/phospholipid metabolism/CDP-choline synthetase) JEFFREY D. ESKO AND CHRISTIAN R. H. RAETZ* Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706 Communicated by Eugene P. Kennedy, June 19,1980 ABSTRACT We have screened approximately 20,000 colo- tecting mutants of Chinese hamster ovary (CHO) cell colonies nies ofChinese hamster ovary cells immobilized on filter paper immobilized on discs of filter paper (8). This makes possible the [Esko, J. D. & Raetz, C. R. H. (1978) Proc NatL Acad. Sci. USA analysis of 104-105 colonies for specific biochemical or phe- 75, 1190-1193] for strains unable to incorporate [methl-"'CJ- choline into trichloroacetic acid-precipitable phospholipid at notypic defects by replica plating or in situ enzymatic assays. 40'C. Mutant 58, identified in this way, was specifically de- Previously, we isolated several myoinositol auxotrophs of CHO fective in choline incorporation, and other isolates were also cells without prior enrichment and demonstrated that myo- blocked in thymidine and leucine incorporation into DNA and inositol starvation of these strains resulted in the virtual elimi- protein, respectively. Further analysis of mutant 58 revealed that nation of phosphatidylinositol from subcellular membranes (8, the strain grew almost normally at 330C, the permissive tem- 9). Robbins (10) has described a histochemical assay for de- perature, but divided only once at 40'C, the restrictive tem- in CHO cells attached to filter perature. After a 20-hr incubation at 400C, the phosphatidyl- tecting a-mannosidase activity choline level dropped from 41% to 20% in the mutant whereas paper and has isolated several variants lacking this lysosomal other phospholipids, including sphingomyelin, continued to enzyme. accumulate. Wild-type cells contained approximately 50% We now report an autoradiographic screening technique for phosphatidyicholine at both temperatures. Anion-exchange identifying CHO colonies unable to generate choline-linked chromatography of the water-soluble choline metabolites ex- phospholipids in vivo. Strain 58, deficient in CDP-choline and tracted from mutant 58 revealed that phosphorylcholine accu- phosphatidylcholine synthesis at the nonpermissive tempera- mulation increased from 6 nmol/mg of protein at 330C to 42 nmol/mg of protein at 40'C whereas CDP-choline decreased ture, is strikingly defective in CDP-choline synthetase (cho- from 0.42 nmol to less than 0.07 nmol per mg of protein. Phos- linephosphate cytidylyltransferase; CTP:cholinephosphate phorylcholine also increased in wild-type cells shifed from 330C cytidylyltransferase, EC 2.7.7.15) assayed in vitro and accu- to 40°C (from 1.8 nmol to 16 nmol per mg of protein), but the mulates high levels of phosphorylcholine in vivo. Mutant 58 level of CDP-choline was not altered (from 0.52 nmol to 0.58 provides a new approach to studies of the regulation of mem- nmol per mg of protein). Enzymatic assays of extracts prepared brane phosphatidylcholine content and creates the possibility from mutant and wild-type cells revealed a reduction of CTP: phosphorylcholine cytidylyltransferase (EC 2.7.7.15) activity of isolating and mapping the genes involved in mammalian (CDP-choline synthetase) in the mutant to 1/40th that in the wild phosphatidylcholine metabolism. type, and mixing experiments excluded the production of an- tagonists to CDP-choline synthesis in the mutant. Thus, the in- EXPERIMENTAL PROCEDURES ability of the mutant to generate normal amounts of phospha- Materials. [32P]Orthophosphate (carrier-free), [methyl- tidylcholine in vivo was correlated with an enzymatic lesion 14C]choline, [methyl-14C]phosphorylcholine, [methyl-14C]- in the biosynthesis of CDP-choline in vitro. thymidine, and L-[1-14C]leucine were obtained from New Phosphatidylcholine is a major structural component of most England Nuclear. All other materials were reagent grade and cells possessing internal membranes (1, 2), representing about obtained from Sigma. Ham's F12 culture medium, trypsin, half of the membrane phospholipid. Its synthesis appears to be Mycostatin, Fungizone, and fetal bovine serum were obtained localized on the cytoplasmic face of the endoplasmic reticulum from GIBCO. Organic solvents were distilled prior to use. (3, 4), but little is known about the biochemical and genetic Methods. Strain CHO-K1 was cultured as described (8). For factors that control the relative amount and distribution of this mutant screenings, cells were treated with 500 gtg of ethyl lipid. Most subcellular membranes contain different amounts methanesulfonate per ml at 370C for 18 hr (8) and shifted to of phosphatidylcholine (1) distributed unequally in their op- 330C, the permissive temperature, for several generations. After posing faces (5, 6), suggesting that mechanisms exist for its in- the cells were subcultured once at 330 C, they were stored in tracellular transport and insertion into preexisting membranes. liquid nitrogen and revived as needed. The isolation of the Because choline-linked phospholipids are also present in high mutant from this cell stock is described in the text. Filter paper abundance in serum lipoproteins (7), salvage of serum lipids and glass beads were prepared as described (8). Trypsin was and secretion may function along with de novo synthesis and used routinely to subculture cells (11), but for biochemical ex- degradation to set the cellular choline-lipid content. Enzymatic periments, cells were scraped from plastic culture dishes with and genetic studies of animal cell mutants unable to synthesize a rubber policeman and centrifuged at 600 X gav for 10 min. phosphatidylcholine would help to unravel the complex me- To make a crude lysate, we washed cell pellets once, resus- tabolism of this lipid, but such mutants have not been ob- pended them in 0.3 M sucrose containing 10 mM Tris-HCl (pH tained. 7.6), and froze them at -200C. Recently we described a rapid screening procedure for de- Radioactive labeling, extraction, and analysis of phospho- lipids were done as described (9) or were modified as indicated The publication costs of this article were defrayed in part by page in the table legends. Protein was measured according to Lowry charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviation: CHO cells, Chinese hamster ovary cells. this fact. * To whom reprint requests should be addressed. 5192 Downloaded by guest on September 29, 2021 Biochemistry: Esko and Raetz Proc. Natl. Acad. Sci. USA 77 (1980) 5193 RESULTS Autoradiographic Detection of Mutant Colonies Unable To Incorporate Choline into Phospholipid. When CHO cells were labeled for several hours with radioactive choline, chlo- roform extraction revealed that the label was incorporated ef- ficiently into choline-containing phospholipids, primarily phosphatidylcholine. If labeled cells were treated with tri- chloroacetic acid, over 98% of the acid-precipitable radioac- tivity was also chloroform soluble, indicating that [methyl- A 14C]choline was not a precursor for macromolecules other than membrane lipids (data not shown). Because of this specificity, the incorporation of labeled choline into acid-precipitable ~~~~~B material permitted the autoradiographic detection of phos- phatidylcholine synthesis in colonies attached to filter paper (Fig. 1). When CHO colonies derived from mutagen-treated cells were transferred to filter paper at 330C and then incubated for several hours with radioactive choline at 400C, autoradi- ography revealed that all of the colonies present on the disc were radioactive except for an occasional variant, indicated by the C 0 arrow (compare Fig. 1 A and B). Unlabeled colonies of this kind occurred with an approximate incidence of 1:5000. To repurify FIG. 1. [methyl- 14CJCholine autoradiography of CHO cell the putative mutants from adjacent wild-type cells (Fig. 1A), colonies immobilized on filter paper. Mutagen-treated cells were we retrieved the desired colonies from the original master plates dispersed with trypsin (11) and placed in 100-mm-diameter tissue- culture dishes to yield approximately 200 colonies per plate at-330C. by using glass cloning cylinders and trypsin (14). The cells were After 1 day, the cells were overlayed with a disc of Whatman no. 50 replated at different dilutions at 330C and again transferred filter paper and glass beads (8) and incubated at 330C for another 16 to filter paper as described in the legend of Fig. 1. Because the days. After the medium was aspirated and the beads were decanted, majority of colonies present after the first cloning were defec- the filter paper was removed from the dish with sterile tweezers and tive, as judged by choline autoradiography (Fig. 1 C and D), placed cell side up on a sterile metal or glass pan tilted at a 600 angle. further was not The surface tension of the residual medium held the disc firmly repurification generally necessary. Single against the pan. The paper was then rinsed vigorously with a 30-ml colonies were picked and used for the experiments described stream of medium lacking serum to remove loose cells. Next, it was below. The autoradiogram and stained filter paper corre- placed