J. Promzoo/, 39(6), 1992, pp. 7 19-723 0 1992 by the Society of Protozoologists

Immobilization Antigens from Tetrahymena thermophila Are Glycosyl-Phosphatidylinositol-LinkedProteins YOUNG-GYU KO and GUY A. THOMPSON, JR.' Department of Botany, University of Texas, dustin, Texas 78713 ABSTRACT. We have studied four strains of Tetrahymena thermophila, each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to ['H]ethanolamine or ['Hlmyristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamideelectrophoresis gels. Furthermore, antibodies raised to the 1-antigens of the T. fhrrmophilustrains selectively immunoprecipitatedradioactive proteins having molecular mass identical to that of the i-antigen characteristicfor that particular strain. The lipid moieties labeled by [3H]myristatewere not susceptible to hydrolysis by exogenous phosphatidylinositol-specificphospholipase C from bacteria. However, when protein extraction was camed out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [ZH]rnyristatewere rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-linked glycosyl-phospha-

tidylinositol anchors

T

HE expression of certain unique surface proteins in protozoans is strongly influenced by the cellular environment. The most widely studied example is the variant surface glycoprotein that coats the cell surface of trypanosomes such as Tr.vpanosoma hrucei, the parasitic organism that causes African sleeping sickness [6]. Under pressure from the host's immune defense system or other environmental factors, the predominating variant surface glycoprotein in a trypanosome population can be easily supplanted by a different variant, i.e. one not recognized by circulating antibodies. By 1985 it was recognized that variant surface glycoproteins are linked to the trypanosome surface membrane by a glycosyl-phosphatidylinositol(GPI) anchor consisting of phosphoryiethanolamine, mannose, and glucosamine units linked in sequence to a phosphatidylinositol molecule tightly embedded in the membrane. The variant GPI-anchored proteins on the trypanosome surface are thought by some to have evolved as a protection against the inhospitable environment within the host bloodstream (see discussion by Cross [6]). However, it is now becoming apparent that free-living protozoa also contain GPI-linked proteins that are capable of genetic variation. Considerable evidence has been reported suggesting that the variable surface antigen ofthe ciliate Paramecium primaurelia is attached to the cell surface by a GPI moiety [4]. Recently our laboratory described a 24-kDa GPIanchored protein from the related ciliate Tetruhymrna mimhres [ 13, 18, 221. During these initial studies, small growth temperature-dependent changes in the electrophoretic mobility of this protein [ 131 raised the possibility that it too may be a product of a multigene family capable of differentially expressing alternate proteins. A different species of Tetrahymena, T. thermophila, is ideally suited for examining this question. When grown over a broad temperature range, T. themophila expresses three variant types of proteins. These proteins have been termed immobilization antigens (i-antigens) because incubation with antibodies raised against the particular surface antigen being expressed blocks the cell's ability to swim [2, 81. Ser H, the i-antigen type specifically expressed between 20" and 35" C , has been studied in some detail and is represented in nature by four similar but nonidentical alleles, H I , H2, H3, and H4, in closely related T. thermophila strains. The experiments described here provide convincing evidence that each of these proteins contains a GPI anchor. MATERIALS AND METHODS Materials. Phosphatidylinositol-specificphospholipase C (PIPLC) from Bacillus thuringiensis was kindly provided by Dr.

' To whom correspondence should be addressed.

M. G. Low (Columbia University, New York, NY). Unpurified antisera raised in rabbits against four strains of T. thermophila homozygous for the i-antigens Ser H I , Ser H2, Ser H3, and Ser H4 were gifts from Dr. N. E. Williams (University of Iowa, Iowa City, IA). [ 1-3H]Ethan-1-01-2-amine hydrochloride (29.5 Ci/ mmol) and [9, 10-3H]myristic acid (22.4 Ci/mmol) were purchased from Amersham Corp. (Arlington Heights, IL). In vivo labeling and cell harvesting. The four strains of T. thermophila were grown to a density of 2-4 x lo5 cells/ml at 27" C in enriched medium [21]. Before labeling cells with [3H]myristic acid, they were washed twice with room temperature inorganic medium (47 mM NaCI, I mM KH,PO,, 4 mM K,HPO,, and 1 mM MgSO,) and then resuspended in inorganic medium to give a density of 5 x lo5 cells/ml. Ten milliliter aliquots were preincubated for 15 mln in 50-ml Erlenmeyer flasks before adding 125 pCi of [9,1O-3H]myristic acid in 10 pl of ethanol. The cells were then incubated at 27" C for 24 h. For labeling with [ 1 -3H]ethan- 1-01-2-amine hydrochloride, the density of the twice-washed cells was brought to 1 x lo6 cells/ml in inorganic medium, and 2 ml of cells were preincubated in a scintillation vial at 27" C for 15 min. After adding 20 NCi of aqueous [3H]-ethanolamine, the cells were incubated at 27" C for 24 h. All experiments were performed at least twice. Protein extraction. Aliquots of a labeled cell culture were rapidly chilled in a mixture of acetone and dry ice and washed twice with cold 10 mM Tris-HC1, pH 7.0, containing 1 mM para-chloromercuriphenylsulfonicacid (pCMPSA), 1 m M phenylmethylsulfonyl fluoride (PMSF), and 2 mM ZnCI, by centrifugation for 5 min at 900 g. Cell pellets were resuspended in 200 p1 cold, freshly prepared aqueous 1 mM pCMPSA and 1 mM PMSF, immediately treated with an equal volume of hot sodium dodecylsulfate (SDS) buffer (2 mM SDS, 22% glycerol, I mM PMSF, 0.5 pg aprotinin/ml, I mM pCMPSA and 100 mM Tris-HCI, pH 6.8), and boiled at 100" C for 5 min. After chilling the sample, fresh PMSF, pCMPSA and aprotinin equivalent to final concentrations of 1 mM, 1 mM, and 0.5 ,ug/ml, respectively, were added, and the clear supernatant was retained after microcentrifugation (17,000 g ) for 7 min. Protein concentrations were determined by the bichichoninic acid method [201. The supernatant was stored at - 70" C. In some cases proteins were extracted at 0" C for 30 min with lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM EDTA, 50 mM Tris-HCI, pH 8.5, 1 mM PMSF, 0.5 pg aprotininlml) without pCMPSA. After lysis, the sample was microcentrifuged at 17,000 g for 7 min, and the clear supernatant was stored at -70" C. Phosphatidylinositol-specificphospholipase C treatment. One hundred micrograms of total protein was precipitated with cold acetone to remove all PI-PLC inhibitors and resuspended in 100 pl of HEPES, pH 7.2, containing 0.1% sodium deoxycho-

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Fig. 1. Appearance of radioactivity in the 52-kDa region of SDSpolyacrylamide gels after incubating T. thermophilu H3 cells with [ZH]ethanolamine. late. The suspension was incubated at 37°C for 90 min following the addition of 3.4 units of B. thuringiensis PI-PLC. Proteins were again recovered by cold acetone precipitation. For in vivo PI-PLC treatment, 2 mi Tetruhymenu with a density of 1.0 x lo6 cells/ml were labeled with 40 pCi 113H]ethan-1-01-2-amine hydrochloride for 24 h in 25 mM HEPES, pH 7.2, 23.5 mM NaCI, 0.5 mM KH2P0,, 2 mM K2HP0,, and 0.5 mM MgSO,. After 24 h of incubation, 10 U/ml of PI-PLC was added, and the suspension was gently shaken for 90 min at 28" C. After centrifuging the cells at 1500 g for 5 min, proteins from the supernatant were precipitated with cold acetone. The cell pellet from this treatment was solubilized in lysis buffer. Immunoprecipitation. Two hundred micrograms of protein labeled with [9, 10-3H]myristicacid or [ l-3H]ethan- I-01-2-amine hydrochloride were diluted with 50 pi of lysis buffer (1% Triton X-100, 150 m M NaCI, 20 mM EDTA, 50 mM Tris-HCI, pH 8.5, 1 mM PMSF, 0.5 pg aprotinin/ml, and 1 mM pCMPSA), and the preparation was reacted with 10 pi of antibody against the appropriate i-antigen at room temperature for 1 h with gentle mixing. After adding 20 pl of protein A-Sepharose beads (10% protein A-Sepharose in lysis buffer), the incubation was continued at room temperature for 1 h. After washing the antibodyantigen-protein A complex three times with lysis buffer, 70 p1 of SDS sample buffer (1% SDS, 60 mM Tris-HC1, pH 6.8, 11% glycerol, 10% 0-mercaptoethanol, and 0.00 I O/o Bromophenol Blue) was added, and the mixture was boiled for 5 min to denature and release the antigen. Electrophoresis, gel slicing and autoradiography. Samples containing 100 pg of protein sample were electrophoresed alongside commercially obtained molecular weight standards (BioRad, Richmond, CA) on a 15% polyacrylamide discontinuous slab gel (1 60 x 130 x 1.5 mm3) at a constant 40 mA according to Laemmli [lo]. Following electrophoresis, gels were stained with 0.1% Coomassie Brilliant Blue R-250 in methanol : acetic acid : water (50: 10:40). After destaining in methanol :acetic acid : water (50:10:40), each lane of the Coomassie Blue-stained gel was frozen with dry ice and cut transversely into 1 mm slices with a gel slicer (Joyce Loebl, Burlington, MA). Each slice was incubated in a scintillation vial with 0.3 ml of 30% hydrogen peroxide at 50" C for 24 h before adding 10 ml of scintillation cocktail (Bio Safe 11, Research Product International Corp., Mt. Prospect, IL) and analyzing in a Packard Tricarb Model 300

Fig. 2. Fluorogram of whole cell proteins from four strains of T. thermophila labeled for 24 h with [3H]ethanolarnine.Arrows indicate positions of i-antigens. Strains are identified above the lanes. Right lane represents the Coomassie Blue staining (CS) pattern of an equivalent quantity of strain H3 whole cell proteins. The staining patterns of the other strains were indistinguishablefrom that of H3. scintillation counter (Packard Inst. Co., Downers Grove, IL). Alternatively, the destained gel was treated with autoradiography enhancer (Resolution, EM Corp, Chestnut Hill, MA) according to the manufacturer's instructions. Fluorograms were prepared after placing the dried gels next to hypersensitized Kodak X-Omat AR X-ray film at -70" C for up to 20 days. RESULTS Incorporation of (3H]ethanolamine into T. thermophila proteins. Incubation of T. thermophilu strain H3 with [3H]ethanolamine resulted in relatively slow incorporation of radioactivity into material that migrated with an apparent molecular weight of approximately 52 kDa on SDS-polyacrylamide electrophoretic gels, as calculated based on the migration of molecular weight markers (Fig. 1). Peak incorporation of radioactivity into this band was not reached until 24 h or more after isotope addition. Using 24 h as the optimal time of labeling, three other strains of T. thermophila, namely, H1, H2, and H4, were also labeled with 13H]ethanolamine. Fluorography showed the presence of several labeled bands, especially in H 2 (Fig. 2), but a major band in each case was located at the position where the i-antigen of that particular strain has been reported to run [8]. Thus the apparent molecular masses of these major bands in H1, H2, H3, and H4 strains are 52, 44, 52, and 49 kDa, respectively. Quantitative data on the relative labeling of various proteins with [3H]ethanolaminewere obtained by slicing each lane of the electrophoretic gel into 1 mm segments and measuring their radioactivity in a scintillation counter. Figure 3 shows the distribution of 3H in a gel equivalent to that illustrated in Fig. 2.

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KO & THOMPSON - T E T R A H Y M E N A I-ANTIGENS ARE GPI-LINKED PROTEINS

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gel slice number Fig. 3. Distribution of radioactivity in I-mm gel slices from 24 h ['Hlethanolamine-labeled H1, H2, H3, and H4 cells. Left panels show radioactivity in whole cell proteins, with the i-antigen characteristic of each strain indicated by an arrowhead. Right panels show radioactivity in the immunoprecipitate(RIP) from each strain. Peak at extreme right side of each panel represents phospholipids and PI glycans migrating with the gel dye front. Treatment of each crude cell preparation with polyclonal antibodies raised against its own i-antigen resulted in the selective irnmunoprecipitation of a radioactive protein which exhibited the molecular mass expected for that i-antigen (Fig. 3 , right panels). Coomassie Blue staining revealed only bands corresponding to the excess antibody (data not shown), indicating a pronounced enrichment of the radioactive protein through immunoprecipitation. Incorporation of 13H]myristateinto T. thermophila proteins. To further test the hypothesis that Tetrahyrnena i-antigens are GPI-anchored proteins, cells were labeled with [3H]myristate in inorganic medium for 24 h. Radioactivity was recovered in the cellular lipids, but only sparingly in the i-antigen region or other protein-containing regions of protein electrophoretic gels. When the [3H]myristate labeling experiments were repeated (taking pains to add the recognized GPI-specific phospholipase C inhibitor pCMPSA [9] during the cell harvesting step, and boiling the preparation within a few seconds following addition of the SDS lysis buffer), protein bands, especially those corresponding in molecular mass to i-antigens, were strongly labeled (Fig. 4). Treatment of these crude cell preparations with antibodies for i-antigens produced selective immunoprecipitation of a radio-

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gel slice number Fig. 4. Distribution of radioactivity in 1 mm gel slices from 24 h [ZH]myristate-labeledH 1, H2, H3, and H4 cells. Left panels show radioactivity in whole cell proteins, with the i-antigen characteristic of each strain [8] indicated by an arrowhead. Right panels show radioactivity in immunoprecipitates(RIP)prepared using a portion ofthe whole cell protein extract from each strain. Slight differences in H4 peak positioning in left and right panels are due to uneven running of the gel. Peak at the extreme right of each panel represents lipids and PI glycans migrating with the dye front. In some experiments the peak cresting near slice 50 of H3 whole cell extracts was much reduced in size.

active protein from each T. thermophila strain; each of these proteins corresponded to the molecular mass of its i-antigen. Effects of exogenous phospholipase C on the GPI-anchored proteins. When H 3 cells labeled for 24 h with [3H]ethanolamine were treated with exogenous B. thuringiensis PI-PLC, no release of radioactive i-antigen into the incubation medium was detected (Fig. 5). On the other hand, several other proteins labeled with [3H]ethanolamine were released. In order to determine whether the resistance of the i-antigen to PI-PLC in vivo was caused by a steric effect of protein packing or perhaps by the inaccessibility of the i-antigen in the native membrane for other reasons, proteins extracted from cells labeled with [3H]myristate were incubated with PI-PLC. No significant loss of radioactivity from the i-antigen band was observed after incubations of up to 90 min (data not shown). In view of the potent lipolytic activity present in the T. thermophila strains used here, we realized that the products recovered from the initial [3H]ethanolaminelabeling studies (Fig. 1-

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gel slice number Fig. 5 . Action of exogenous B. thuringiensis PI-PLC on intact H3 cells prelabeled for 24 h with [3H]ethanolarnine.Panels I and 3 represent the distribution of radioactivity in gel slices from the cell pellet and supernatant, respectively, of control cells incubated without PI-PLC for 90 min. Panels 2 and 4 represent radioactivity recovered in the cell pellet and supernatant incubated with PI-PLC for 90 min. Arrowheads indicate the i-antigen positions.

3) had probably been stripped of their lipid moiety during isolation. Selected experiments were repeated using the more stringent isolation conditions to block hydrolysis of the GPI-anchors from their parent proteins. The gel mobilities ofthe intact products were essentially the same as those observed in the first trials. DISCUSSION To test for the possible attachment of GPI-anchors to T. thermophila i-antigens, cells were grown in the presence of [3H]ethanolamineor [3H]fatty acid to label these two distinctive components of GPI glycans. In both cases a major radioactive band on electrophoretic gels (Fig. 3, 4) corresponded closely to the known molecular weight of the i-antigen known to be characteristic of each of four distinct T. thermophila strains (Fig. 2 and [S]). The somewhat delayed appearance of label in the i-antigen position (Fig. I ) followed a similar pattern of [3H]ethanolamine incorporation into PI-anchored proteins of T. mimhres [l3], where the radioactivity was shown to accumulate first in the cellular pool of free GPI glycans and then be transferred slowly into a protein-bound GPI anchor. Williams et al. [23] and Bolivar & Guiard-Maffia [3], using somewhat different labeling conditions, observed a more rapid incorporation of [35S]methionineinto the protein moiety of T. thermophila i-antigens, suggesting that this entity is utilized for i-antigen formation without delay. Using both radioactive precursors, the labeled putative i-antigens were recovered in a much higher state of purity following immunoprecipitation with authenticated i-antigen antibodies. The lack of Coomassie Blue staining in the region of the purified i-antigen indicated that T. thermophila, unlike Paramecium and trypanosomes, does not contain i-antigens in high abundance. During the course of these experiments, parallel studies of a similar nature were being carried out by Ron et al. [ 171. These workers measured the incorporation of [3H]mannose and [3H]glucosamine into immunoprecipitable i-antigens of the same four strains of T. thermophila that we have used. Such findings could, on their own, be interpreted in several ways since man-

nose and glucosamine might well be components of non-GPIanchored Tetrahymena glycoproteins. Now, however, the combined results of both laboratories show the incorporation into the i-antigens of ethanolamine, fatty acid, mannose, and glucosamine, which together account for four of the five major GPI components (inositol has not been sought). These findings, reinforced by our documentation of a GPI-anchored protein in T. mimbres [13, 18, 221, afford compelling evidence that the i-antigens contain a covalently-linked GPI chain. Our studies with Tetrahymena are in agreement with recent work by Capdeville and associates [4, 51 using a closely related ciliate, Paramecium primaurelia. Paramecium is covered by a variable surface antigen which at 250-300 kDa is much larger than the Tetrahymena i-antigens, but has many other features in common with them [5]. The Paramecium surface antigen incorporated [3H]myristate,and the covalently linked fatty acid was susceptible to hydrolysis by a very active endogenous lipase activity and by exogenous PI-PLC [4]. While these observations alone may not be sufficient to confirm that the Paramecium surface antigen is GPI-anchored, the similarities of the system to that in T. thermophila are notable. The procedures that we initially utilized for the preparation of T. thrrmophila GPI-anchored protein extracts activated a potent endogenous lipolytic activity reminiscent ofthat reported for Paramecium [4]. This lipolytic activity in the H strains, as evidenced by a minimal recovery of radioactivity in the i-antigen region of gels, was much greater than activity of the same kind previously noted in T. mirnbres [13, 181. By adding two known PI-PLC inhibitors, ZnCI, and pCMPSA [9], to the cell suspension during the final centriguation and lysis of the cells, degradation of the GPI-anchored proteins could be prevented. It appears that the GPI-anchored proteins are most susceptible to hydrolysis during the short period between cell disruption by detergents and inactivation of lipolytic activity by boiling, as is apparently true in Paramecium [4]. Addition of the inhibitors during the last centrifugation step is a precautionary step, taken in case some cell disruption occurred during pellet resuspension. Few details are known as yet regarding the endogenous hydrolytic activity degrading the T. thermophila GPI anchor moiety. The gel mobilities of the i-antigens were unchanged by the loss of the lipid moiety, as has been shown with GPI-anchored proteins of Dictyostelium [ 191. We are also uncertain as to why the i-antigens were not susceptible to attack by exogenous PIPLC either in vivo or in vitro. There are examples in the literature of GPI-anchored proteins resistant to PI-PLC (reviewed by Low [ 1 I]). The failure ofthe B. thuringiensis PI-PLC to attack the GPI anchor of human erythrocyte acetylcholinesterase [ 161 and certain of the Trypanosoma brucei surface glycoproteins [ 121 was caused by palmitoylation of the inositol ring. Our previous analysis of T. mimbres GPI-anchored proteins indicated that exogenous PI-PLC could hydrolyze approximately 30% of the glycan anchors in vivo [13]. Structural studies of the T. mimhres GPI glycans yielded data compatible with fatty acylation of the inositol ring of some molecules [ 181, but this observation has not yet been confirmed. On the basis of the precursor labeling patterns we have observed, especially with T. thermophila strains H2 and H3, other proteins are also labeled with GPI-anchor precursors (Fig. 2). The fact that some ofthese were solubilized by in vivo treatment with PI-PLC (Fig. 5), argues that they are located on the cell surface. Slightly different labeling patterns with [3H]myristate and [3H]ethanolamine(compare Fig. 3 & 4) may reflect a delayed retailoring of the GPI fatty acid components [ 121, a substitution of 2-aminoethylphosphonate for ethanolamine phosphate in certain anchors [7, 151, or other metabolic alterations. Continued time course studies will be necessary to understand the

KO & THOMPSON- TETRAHYMENA I-ANTIGENS ARE GPI-LINKED PROTEINS

dynamics of the system and possible precursor-product relationships. A wealth o f published information is available on the distribution, genetic variation, and response t o environmental factors of the Tetrahymena i-antigens [I]. For example, the H3 antigen is not produced by cells growing a t 40" C, but is rapidly induced after shifting cells t o the permissive temperature of 28" C [23]. T h e appearance within 30 min of the newly induced H3 antigen o n the surface of these cells, first in the non-ciliated regions a n d then gradually along the surface of the cilia, has been closely followed by immunocytochemical labeling. N o clear physiological function of the Tetrahymena i-antigen has been shown. However, recent evidence reported by Bolivar & Guiard-Maffia [3] indicates that i-antigen may be transported t o the cell surface a s secretions from the cell's mucocysts a n d that some of t h e material may subsequently be internalized during the formation o f food vacuoles a t the cytopharynx. This likelihood of continuous rapid secretion, shedding, and partial reutilization of a major GPI-anchored protein may explain the relatively low abundance o f i-antigens associated with Tetrahymena at any given time, a n d suggests further that part of the i-antigen complement is sequestered away from the cell surface a t all times. T h e susceptibility of the i-antigen G P I to hydrolysis by endogenous phospholipase(s) raises the interesting possibility that such a cleavage is a normal step i n i-antigen release as it has recently been reported t o operate in releasing pancreatic glycoproteins [ 141. The realization that i-antigens have GPI anchors allows a convergence o f two well developed research areas that were previously thought t o be unrelated. Correlating the respective pools of information should expedite research o n these enigmatic proteins. ACKNOWLEDGMENTS We thank Norman Williams for the i-antigen-specific antibodies and for sharing unpublished observations. We also thank Martin Low for t h e PI-PLC. T h i s study was supported in part by grants from t h e Welch Foundation (F-350) and the National Science Foundation (DCB-8802838).

LITERATURE CITED 1. Allen, S. &Gibson, I. 1973. Genetics of Tetrahymena. In: Elliott,

A. M. (ed.), Biology of Tetrahymenu. Dowden, Hutchinson, and Ross, Stroudsburg, PA, pp. 307-373. 2. Bannon, G. A., Perkins-Dameron, R. & Allen-Nash, A. 1986. Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila. Mol. Cell Biol.. 6: 3240-3245. 3. Bolivar, I. & Guiard-Maffia, J. 1989. Cellular localization of the SerH surface antigen in Tetrahymena thermophila. J . Cell SCI.,94:343354. 4. Capdeville, Y., Cardoso de Almeida, M. L. & Deregnaucourt, C. 1987. The membrane-anchor ofParamecrum temperature-specific surface antigens is a glycosylinositolphospholipid. Biochem. Biophys. Rex Commun., 147:1219-1225.

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5. Capdeville, Y . ,Deregnaucourt, C. & Keller, A.-M. 1985. Surface antigens of Paramecium primaurelia. Exp. CelL Res., 161:495-508. 6. Cross, G. A. M. 1990. Cellular and genetic aspects of antigenic variation in trypanosomes. Ann. Rev. Immunol., 8:83-110. 7. Dearborn, D. G., Smith, S. & Korn, E. D. 1976. Lipophosphonoglycan of the plasma membrane of Acanthamoeba castellanii. J . Biol. Chem., 251:2976-2982. 8. Doerder, F. P. & Berkowitz, M. S. 1986. Purification and partial characterization ofthe H immobilization antigens of Tetrahymena rhermophila. J. Protozool., 33:204-208. 9. Jager, K., Stieger, S. & Brodbeck, U. 1991. Cholinesterase solubilizing factor from Cytophuga sp. is a phosphatidylinositol-specific phospholipase C. Biochim. Biophys. Acta, 1074:45-5 I . 10. Laemmli, U. K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227:680-685. 11. Low, M. G. 1989. Glycosyl-phosphatidylinositol:a versatile anchor for cell surface proteins. FASEB J., 3:1600-1608. 12. Mayor, S., Menon, A. K. & Cross, G. A. M. 1990. Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. J. Biol. Chem., 265:6 1 74-6 18 1. 13. Pak, Y., Ryals, P. E. & Thompson, G. A., Jr. 1991. Phosphatidylinositol glycan formation and utilization by the ciliate Tetrahymena mimbres. 1. Biol Chem., 266: 15054-1 5059. 14. Paul, E., Leblond, F. A. & LeBel, D. 199 1. In resting conditions, the pancreatic granule membrane protein GP-2 is secreted by cleavage of its glycosylphosphatidylinositol anchor. Biochem. J., 277379-88 1. 15. Previato, J. O., Gorin, P. A. J., Mazurek, M., Xavier, M. T., Fournet, B., Wieruszesk, J. M. & MendonSa-Previato, L. 1990. Pnmary structure of the ohgosaccharide chain of lipopeptidophosphoglycan of epimastigote forms of Trypanosoma cruzi. J. Biol. Chem.. 265: 25 18-2526. 16. Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G. & Rosenberry, T. L. 1988. Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. J. Biol. Chem., 263:18766-18775. 17. Ron, A,, Williams, N. E. & Doerder, F. P. 1992. The immobilization antigens of Tetrahymena thermophila are glycoproteins. J. Protozool., 39:508-5 10. 18. Ryals, P. E., Pak, Y . & Thompson, G. A,, Jr. 1991. Phosphatidylinositol-linked glycans and phosphatidylinositol-linked proteins of Tetrahymena mimbres. J. Biol. Chem., 266: 15048-1 5053. 19. Sadegi, H., da Silva, A. M. & Klein, C. 1988. Evidence that a glycolipid tail anchors antigen 117 to the plasma membrane of Dictyostelium discoideum cells. Proc. Natl. Acad. Sci. USA, 8555 12-55 15. 20. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. & Klenk, D. C. 1985. Measurements of protein using bichichoninic acid. Anal. Biochem., 150:76-85. 21. Thompson, G. A., Jr. 1967. Studies of membrane formation in Tetrahymena pyriformis. I. Rates of phospholipid biosynthesis. Biochemistry, 6:20 15-2022. 22. Weinhart, U., Thomas, J. R., Pak, Y., Thompson, G. A., Jr. & Ferguson, M. A. J. 199 1. Structural characterization of a novel glycosyl-phosphatidylinositolfrom the protozoan Tetrahymena mimbres. Biochem. J., 279:605-608. 23. Williams, N. E., Doerder, F. P. & Ron, A. 1985. Expression of a cell surface immobilization antigen during serotype transformation in Tetrahymena thermophila. Mol. Cell. Biol., 5: 1925-1932.

Received 4-6-92, 6-23-92;accepted 7-17-92