[38] Biosynthesis of glycosylphosphatidylinositol anchors

[38]

B I O S Y N T H E S I S O F GPI A N C H O R S

[38] B i o s y n t h e s i s Glycosylphosphatidylinositol By

JOLANTA

VIDUGIRIENE

and

513

of Anchors

ANANT

K. MENON

Introduction Glycosylphosphatidylinositols (GPIs) are synthesized by all eukaryotic cells examined to date, and they are typically found covalently linked to cellsurface glycoproteins. GPIs serve as an important alternative mechanism for anchoring proteins to cell membranes, and a wide spectrum of functionally diverse proteins rely on a GPI anchor for membrane association. The GPI moiety is synthesized in the endoplasmic reticulum and then transferred to proteins containing a carboxyl-terminal GPI-attachment signal sequence. General information on the structure and biosynthesis of GPI anchors may be found in reviews 1-3 and in other chapters in this volume (see Fig. 1). The purpose of this chapter is to describe radiolabeling techniques for studying GPI biosynthesis through the use of cell lysates, subcellular fractions, and permeabilized cells. The methods described are used to generate well-characterized radiolabeled GPI biosynthetic intermediates, as well as to determine the topological arrangement of the lipids in the membrane bilayer. The description focuses on membrane preparations from African trypanosomes and mouse thymoma cells with comments and examples of applications involving other cell types. G l y c o s y l p h o s p h a t i d y l i n o s i t o l B i o s y n t h e s i s in M e m b r a n e P r e p a r a t i o n s a n d Permeabilized Ceils: G e n e r a l B a c k g r o u n d Glycosylphosphatidylinositols that have been synthesized in membrane preparations from mammalian ceils, trypanosomes, toxoplasma, and budding yeast correspond to the entire structure shown in Fig. 1A or to partially glycosylated lipid intermediates. The simplest model of the GPI biosynthetic pathway in bloodstream-form African trypanosomes 4,5 is shown in Fig. 1B; 1G. A. M. Cross, Annu. Rev. Cell Biol. 6, 1 (1990). 2M. C. Field and A. K. Menon, in "Lipid Modificationsof Proteins" (M. J. Schlesinger,ed.), p. 83. CRC Press, Boca Raton, Florida, 1993. 3 p. T. Englund,Annu. Rev. Biochem. 62,121 (1993);M. J. McConvilleand M. A. J. Ferguson, Bioehem. J. 294, 305 (1993). 4W. J. Masterson, T. L. Doering, G. W. Hart, and P. T. Englund, Cell (Cambridge, Mass.), 56, 793 (1989). 5 A. K. Menon, R. T. Schwarz,S. Mayor, and G. A. M. Cross,J. Biol. Chem. 265, 9033 (1990). METHODS IN ENZYMOLOGY,VOL. 250

Copyright © 1995 by AcademicPress, Inc. All rights of reproductionin any form reserved.

A

EtN-P-6Man(~l-2Manc~l-6Man(xl-4GlcN(~l-6myo-Inositol-P-LIPID

I

I

I

R1

R2

R3 SUBSTITUENTS

Trypanosoma brucei Toxoplasma gondii mammalian cells

B

R1

R2

-EtN-P

(zGal [~GalNAc EtN-P

R3 fatty acid fatty acid fatty acid

PI

UDP-GlcNAc GlcNAc-PI

GlcN-PI •

GDP-Man--~ dolichol-P-Man -~-

fatty acyl CoA ?

.,~---~GlcN-PI*

phospholipid ?

Man 3 GlcN-PI Man 3 GIcN-PI*

CDP-EtN - - " ~ PE EtN-P-Man 3GlcN-PI

INHIBITORS ~.o amphomycin -:~EDTA PMSF

EtN-P-Man 3 GlcN-PI*

EtN-P-Man 3GIcN-PI

7

gtN-P-Man 3 GlcN-PI*

I

Gal

UDP-Gal

FIG. 1. Glycosylphosphatidylinositol structure and biosynthesis. (A) GPIs that have been synthesized in vitro are shown. Mannose residues in the core glycan may be substituted with a-galactose residues [S. Mayor, A. K. Menon, and G. A. M. Cross, J. Biol. Chem. 267, 754 (1992)],/3-N-acetylgalactosamine [S. Tomavo, J.-F. Dubremetz, and R. T. Schwarz, J. Biol. Chem. 267, 21446 (1992)], or phosphoethanolamine [S. Hirose, L. Ravi, G. M. Prince, M. G. Rosenfeld, R. Silber, S. W. Andresen, S. V. Hazra, and M. E. Medof, Proc. Natl. Acad. Sci. U.S.A. 89, 6025 (1992)], depending on the membrane source. The inositol residue may be derivatized with an ester-linked fatty acid, rendering the structure resistant to hydrolysis by bacterial phosphatidylinositol-specific phospholipases IS. Mayor, A. K. Menon, and G. A. M. Cross, J. Biol. Chem. 265, 6164 (1990)]. (B) A model of GPI biosynthesis in bloodstreamform Trypanosoma brucei is shown. The mature phosphoethanolamine-containing GPIs undergo fatty acid remodeling reactions and are converted to dimyristoyl species [W. J. Masterson, J. Raper, T. L. Doering, G. W. Hart, and P. T. Englund, Cell (Cambridge, Mass.) 62, 73 (1990)]; remodeling may occur earlier if phosphoethanolamine addition is blocked [W. J. Masterson and M. A. J. Ferguson, EMBO J. 10, 2041 (1991)]. Inhibitors of particular reactions are shown and the sites of action indicated. CDP, Cytidine diphosphate; CoA, coenzyme A; EDTA, ethylenediaminetetraacetic acid; EtN, ethanolamine; Gal, galactose; GalNAc, Nacetylgalactosamine; GDP, guanosine diphosphate; GIcN, glucosamine; GlcNAc, N-acetylglucosamine; Man, mannose; P, phosphate; PI, phosphatidylinositol; PI*, phosphatidylinositol containing acylinositol; PE, phosphatidylethanolamine; PMSF, phenylmethylsulfonyl fluoride; UDP, uridine diphosphate.

[38]

BIOSYNTHESIS OF GPI ANCHORS

515

additional steps concerning side-chain modifications have to be introduced to adapt the scheme to mammalian cells and other cell systems, 6-8 and many details still remain to be worked out. GPI biosynthesis is initiated by transferring N-acetylglucosamine (GIcNAc) from U D P - G l c N A c to phosphatidylinositol (PI). 9 At least three gene products are involved in GlcNAc-PI synthesis as mutant cell lines belonging to three different complementation groups (A, C, H) are all defective in synthesis of the first GPI biosynthetic intermediate.l°,lla GlcNAc-PI is then deacetylated to give GlcN-PI, and acylated on the inositol ring to give GlcN-PI*. 5,12,I3 Elaboration of either GlcN-P! or GlcN-PI* proceeds by the sequential transfer of three mannose residues from dolichol-P-mannose 14,~4a and a capping phosphoethanolamine group from phosphatidylethanolamine. 15,I5a Thymoma cell mutants have been described that are defective in mannosylation and in the addition of the capping phosphoethanolamine: the class E mutant has a defective dolichol-P-mannose synthase, the class B mutant is defective in the third GPI mannosyltransferase, and the class F mutant is defective in the ethanolaminephosphotransferase. 6,11,~6Other GPI-defective cells are also available, 11b,~6amost notably human erythroleukemic K562 cells of the J and K complementation classes that are defective in GlcNAc-PI de-Nacetylation and possibly in GPI transfer to protein respectively. 16b Relatively little is known about the biosynthesis of the side-chain modifications found on the GPI glycan; only one report describes the direct radiolabeling 6 S. Hirose, G. M. Prince, D. Sevlever, L. Ravi, T. L. Rosenberry, E. Ueda, and M. E. Medof, J. Biol. Chem. 267, ]6968 (1992). 7 S. Tomavo, J.-F. Dubremetz, and R. T. Schwarz, J. Biol. Chem. 267, 21446 (1992). s M. C. Field, A. K. Menon, and G. A. M. Cross, J. Biol. Chem. 267, 5324 (1992). 9 T. L. Doering, W. J. Masterson, P. T. Englund, and G. W. Hart, J. Biol. Chem. 264, 11168 (1989). l0 V. L. Stevens and C. R. H. Raetz, J. Biol. Chem. 266, 10039 (1991). ~1E. Sugiyama, R. DeGasperi, M. Urakaze, H.-M. Chang, L. J. Thomas, R. Hyman, C. D. Warren, and E. T. H. Yeh, J. Biol. Chem. 266, 12119 (1991). 11a S. Hirose, R. P. Mohney, S. C. Mutka, L. Ravi, D. Singleton, G. Perry, A. M. Tartakoff, and M. E. Medof, J. Biol. Chem. 267, 5272 (1992). 11b T. Kinoshita and J. Takeda, Parasitology Today 107 139 (1994). 12 M. Urakaze, T. Kamitani, R. DeGasperi, E. Sugiyama, H.-M. Chang, C. D. Warren, and E. T. H. Yeh, J. Biol. Chem. 267, 6459 (1992). 13 L. C. Costello and P. Orlean, J. Biol. Chem. 2677 8599 (1992). 14 A. K. Menon, S. Mayor, and R. T. Schwarz, E M B O J. 9, 4249 (1990). 1hap. Orlean, Mol. Cell Biol. 10, 5796 (1990). 15 A. K. Menon, M. Eppinger, S. Mayor, and R. T. Schwarz, E M B O J. 12, 1907 (1993). 15a A. K. Menon and V. L. Stevens, J. Biol. Chem. 267, 15277 (1992). 16 A. Puoti and A. Conzelmann, J. Biol. Chem. 2687 7215 (1993). 16a S. D. Leidich, D. A. Drapp, and P. Orlean, J. Biol. Chem. 269, 10193 (1994). ~6bR. P. Mohney, J. J. Knez, L. Ravi, D. Sevlever, T. L. Rosenberry, S. Hirose, and M. E. Medof, J. Biol. Chem. 269, 6536 (1994).

516

GPI-ANCHOREDPROTEINS

[381

of a side-chain component in vitro. 17The completed GPI structure in bloodstream trypanosomes undergoes fatty acid remodeling reactions in which the glycerol-linked fatty acids are replaced by myristic acid. 18 GPI biosynthesis in vitro can be inhibited by various reagents as indicated in Fig. lB. With the exception of the GlcNAc-PI de-N-acetylase which has been partially purified, 18a none of the GPI biosynthetic enzymes have been isolated. P r e p a r a t i o n of Lysates, S u b c e l l u l a r F r a c t i o n s , a n d Permeabilized Cells for G l y c o s y l p h o s p h a t i d y l i n o s i t o l Labeling a n d D e t e r m i n a t i o n of T r a n s b i l a y e r D i s t r i b u t i o n Relatively crude membrane preparations are capable of synthesizing GPIs from radiolabeled precursors (e.g., sugar nucleotides) and endogenous substrates. We have made extensive use of total membrane preparations and microsomal fractions from African trypanosomes and mouse or human thymoma cells to study GPI biosynthesis. In general, the cells are disrupted by nitrogen cavitation or hypotonic lysis, and membranes are recovered in a single centrifugation step or by differential centrifugation. If enriched subcellular fractions are desired, the lysate is clarified by centrifugation to remove microbodies, and the postnuclear supernatant is fractionated by centrifugation through a discontinuous sucrose gradient. The various membrane preparations can be stored frozen at - 7 0 ° for many months with no significant loss of activity. For topology assays, it is important that the membranes are handled appropriately to give material consisting of a population of sealed vesicles, and that the integrity of the membrane vesicles is tested before and after experimental manipulation. GPIs may also he synthesized in permeabilized cells which are prepared by streptolysin O treatment. [Note: Microsomal fractions prepared from Chinese hamster ovary ( C H O ) ceils, canine pancreas, or rat liver show little (<20%~compared to thymoma cell microsomes) or no detectable GPI biosynthetic activity in the assays described in this chapter.]

Membrane Preparations from Thymoma Cells and Trypanosomes Cells, Reagents, and Equipment BW5147.3 mouse thymoma cells, maintained in suspension culture in Dulbecco's modified Eagle's medium ( D M E M ) supplemented with 17S. Mayor, A. K. Menon, and G. A. M. Cross, J. Biol. Chem. 267, 754 (1992). 18W. J. Masterson, J. Raper, T. L. Doering, G. W. Hart, and P. T. Englund, Cell (Cambridge, Mass.) 62, 73 (1990). 18aK. G. Milne, R. A. Field, W. J. Masterson, S. Conaz, J. S. Brimacombe, and M. A. J. Ferguson, J. Biol. Chem. 269, 16403 (1994).

[38]

BIOSYNTHESIS OF GPI ANCHORS

517

10% fetal bovine serum, 100 U/ml penicillin, and 100/~g/ml streptomycin in an atmosphere of 5% CO2 at 37° Bloodstream-form African trypanosomes (Trypanosoma brucei, e.g., strain 427, variant clone 117, isolated from the blood of an infected rat by centrifugation, followed by passage over a column of DEAE-cellulose) 19 Potter-Elvehjem tissue grinder with Teflon pestle, 10-ml capacity (Cat. No. 358039; Wheaton Scientific, Inc.; may be obtained through VWR Scientific) Minibomb cell disruption chamber (Cat. No. 881455; Kontes Glass Company, Vineland, N J; may be obtained through VWR Scientific) Micrococcal nuclease from Staphylococcus aureus (Cat. No. 107921; Boehringer-Mannheim, Indianapolis, IN) Tunicamycin (Cat. No. 654380; Calbiochem, San Diego, CA) Buffer A: 0.25 M sucrose, 10 mM H E P E S - N a O H , pH 7.5, 1 mM dithiothreitol (DTT) Buffer B: 25 mM H E P E S - N a O H , pH 7.5, 100 mM sucrose, 50 mM potassium acetate, 1/~g/ml leupeptin, and 0.1 mM N~-p-tosyl-L-lysine chloromethyl ketone (TLCK) Buffer L: 100 mM H E P E S - N a O H , pH 7.5, 50 mM potassium chloride, 10 mM MgCI2, 1 /zg/ml leupeptin, 0.1 mM TLCK, and 20% (w/v) glycerol PBS (phosphate-buffered saline) Procedure (Thymoma Cell Membranes). Harvest the thymoma cells (2-5 × 108) from the growth medium by centrifugation (1000 g, 5 min); wash twice with PBS and once with buffer A at a concentration of 2-5 x 107 cells/ml. Resuspend the cells (optimal final concentration 5 x 107 cells/ ml) in buffer A (supplemented with 1/xg/ml leupeptin and 0.1 mM TLCK) and expose the cells to 400 psi N2 pressure for 30 min in a 15-ml minibomb cell disruption chamber on ice (operate the bomb per manufacturer's instructions but use two 3/16-inch tungsten carbide balls when assembling the outlet port, instead of one 3/16-inch ball and one 5/32-inch ball). All subsequent manipulations are to be carried out with the sample on ice. Release the pressure slowly, and collect the cell lysate dropwise. Transfer the lysate to a Potter-Elvehjem tissue grinder and complete the cell disruption with three strokes of a tight pestle. Add micrococcal nuclease (1.5 units/ml) and incubate the lysate for 20 min on ice. Confirm the extent of lysis by visual inspection of an aliquot of the preparation with a light microscope. 19M. C. Field and A. K. Menon, in "Lipid Modifications of Proteins: A Practical Approach" (N. M. Hooper and A. J. Turner, eds.), p. 155. IRL Press, Oxford, 1992.

518

GPI-ANCHOREDPROTEINS

[381

Centrifuge the lysate at 10,000 g for 15 min at 4°. The pellet, which can be discarded, typically contains about 35% of the total lysate protein, most (70-90%) of the lysosomal and peroxisomal marker enzyme activities (/3hexosaminidase and catalase, respectively), and some markers for plasma membrane (-35% of total alkaline phosphodiesterase I activity), Golgi ( - 5 % of total a-mannosidase II activity), and endoplasmic reticulum (ER, -30% of total dolichol-P-mannose synthase activity); marker enzyme assays are performed as described elsewhere. 2°-22 Layer the postnuclear supernatant (PNS) over a 2-ml cushion of 0.5 M sucrose, centrifuge at 100,000 g for 60 min at 4°. Resuspend the pelletted microsomes at a concentration of 2-5 × 108 cell equivalents/ml in Buffer A; resuspension is most simply achieved by gentle pipetting. Store the microsomes in aliquots at - 7 0 °. If it is necessary to work with a microsome fraction enriched in ERderived vesicles, layer the supernatant (PNS) on top of a series of sucrose steps: 3.6 ml of 38% sucrose (bottom layer), 1.8 ml of 30% sucrose, and 1.8 ml of 20% sucrose [all prepared in 10 mM HEPES-NaOH, pH 7.5, 1 mM dithiothreitol (DTT)]. Centrifuge in a Beckman (Palo Alto, CA) 70Ti rotor at 43,000 rpm for 2 hr. Collect fractions from the top of the tube and resuspend the pellet in 3 ml of buffer A. The fractions may be assayed for various organelle-specific marker enzyme activities2°-22 if desired. The ER fractions are clearly separated from Golgi and plasma membrane: the pellet contains rough ER, and the region immediately above the pellet up to the 30-38% sucrose interface contains smooth ER. The two fractions together contain approximately 45% of the protein and about 90% of the ER marker enzyme (dolichol-P-mannose synthase)21 content of the PNS. Resuspend the fractions as above and store in aliquots at -70 °. Both fractions are active in GPI biosynthesis.22 Procedure (Trypanosorne L ysates and Microsomes). Resuspend the trypanosomes in RPMI 1640 culture medium at a density no greater than 108 cells/ml. Incubate the cells with 400 ng/ml tunicamycin (added from a 1 mg/ml stock solution in 95% ethanol) for 15-30 min at 37 °. Recover the cells by centrifugation (1000 g, 5 min, 4°), wash twice with ice-cold PBS, and proceed with cell disruption as follows. To prepare simple lysates4 use a hypotonic lysis procedure (A); for preparation of a microsomal fraction, cell disruption must be more thorough and is achieved by nitrogen cavitation and/or hypotonic lysis followed by passage through a needle and homogenization (B). 20 B. Storrie and E. A. Madden, this series, Vol. 182, p. 203. 21 W. A. Braell, A n a l Biochem. 170, 328 (1988). 22 j. Vidugiriene and A. K. Menon, J. Cell BioL 121, 987 (1993).

[38]

BIOSYNTHESIS OF GPI ANCHORS

519

For procedure A, lyse the cells by vortexing the pellet briefly with ice-cold water (containing 0.1 mM TLCK and 1 /xg/ml leupeptin) at a concentration of 109 cells/ml. After confirming the lysis by examining an aliquot of the preparation with a phase-contrast light microscope, add an equal volume of ice-cold buffer L. Snap-freeze 1-ml aliquots (5 × 108 cell equivalents) in plastic microcentrifuge tubes and store at - 7 0 °. When the lysates are thawed for GPI radiolabeling experiments (see below), they must be diluted in buffer L without glycerol and washed by centrifugation (DuPont SS-34 rotor, 12,000 g, 10 min, 4°) and resuspension: final resuspension should be at about 5 x 108 cells/ml in buffer L (minus glycerol) containing 5 mM MnC12.4 For procedure B involving cell disruption by nitrogen cavitation, go through two cycles of the procedure (pressurizing the minibomb and recovering the cell lysate) as described for thymoma cells. Alternatively, break the cells by hypotonic lysis. 22a Resuspend the cells at a density of 0.5-1 × 109 cells/ml in ice-cold hypotonic buffer (1 mM H E P E S - N a O H , pH 7.5, i mM EDTA) and, after a 10-min incubation on ice, pass the lysate through a 25-gauge needle three times. Then adjust the lysate to isotonicity (final concentrations of 25 mM H E P E S - N a O H , pH 7.5, 100 mM sucrose, 50 mM potassium acetate, 1 /xg/ml leupeptin, and 0.1 mM TLCK) and disrupt the cell ghosts further with 5 strokes in a Potter-Elvehjem homogenizer equipped with a tight-fitting Teflon pestle. Continue by centrifuging the homogenate (obtained by either disruption procedure) at 1000 g for 10 min at 4 °. Layer the supernatant on a cushion of 2 M sucrose (prepared in buffer B) and centrifuge at 100,000 g for 60 rain at 4 °. Carefully remove the bulk of the supernatant and resuspend the crude microsomes (recovered at the top of the sucrose cushion) in buffer B at a concentration of approximately 2 × 109 cell equivalents/ml. Divide the microsomes into aliquots in plastic microcentrifuge tubes, place the tubes in a dry ice-ethanol mixture or liquid nitrogen for rapid freezing, and store the aliquots at - 7 0 °. Starting from 109 trypanosomes (roughly 6 mg protein), recovery of protein in the microsome fraction is typically around 2.7 mg. [Note: The protease inhibitor PMSF should not be used in these lysis procedures because it has been shown to inhibit inositol acylation and phosphoethanolamine addition in trypanosomes (Fig. 1 B ) ; 23'23a PMSF is ineffective in blocking GPI assembly in mammalian cells. TM] 22a j. D. Bangs, L. Uyetake, M. J. Brickman, A. E. Balber, and J. C. Boothroyd, J. Cell Sci. 111, 1101 (1993). e3 W. J. Masterson and M. A. J. Ferguson, E M B O J. 10, 2041 (1991). 23a M. L. S. Giither, W. J. Masterson, and M. A. J. Ferguson, J. Biol. Chem. 269, 18694 (1994).

520

GPI-ANCHORED PROTEINS

[38]

Determination of Intactness of Microsomal Vesicles The intactness of the microsomal membrane barrier is critical for experiments concerning the transbilayer distribution of biosynthetic lipid intermediates and the sidedness of the GPI assembly process. Verification of the intactness of the microsomal vesicles is most easily achieved if the microsomes contain a conveniently assayed lumenal enzyme whose substrate cannot cross the membrane barrier; in other words, the latency of the enzyme activity is a measure of the intactness of the microsomal vesicle population. Comparison of enzyme activity in the presence and absence of detergent provides a quick method to assess microsome intactness and has been successfully used for rough E R vesicles derived from rat liver by determining the latency of mannose-6-phosphatase. 24'25 In the absence of convenient enzymatic assays as in the case of microsomes isolated from thymoma cell lines or trypanosomes, the integrity of membrane fractions can be determined by performing protease-protection experiments. We have carried out the procedure using BiP, an abundant E R lumenal protein, and tested to see if the microsomes are capable of protecting BiP from digestion by exogenously added protease. The amount of BiP in the microsomes before and after protease treatment is determined by Western blotting. The same approach can be used to examine the protection of any lumenal protein or the lumenal domain of an integral membrane protein, where specific antibody reagents are available.

Reagents Phenylmethylsulfonyl fluoride (PMSF; Cat. No. P-7626, Sigma Chemical Co., St. Louis, MO, or Cat. No. 236 608, Boehringer-Mannheim), prepared as a 0.2 M stock solution in ethanol or 2-propanol Proteinase K (Cat. No. 161 519, Boehringer-Mannheim) Trichloroacetic acid [TCA; prepare a 100% (w/v) stock solution by dissolving 500 g TCA in 227 ml water] Procedure. Dilute the membrane sample (roughly 2 × 106 cell equivalents, 5/zg protein) with ice-cold buffer B to a final volume of 100/xl. With the membranes on ice, add proteinase K (test eoncentrations in the range of 0-100 /zg/ml) and incubate for 30 min on ice. The following control samples should be included in the experiment: (i) add Triton X-100 (final concentration 0.25%) to disrupt the membranes before adding proteinase K, (ii) add PMSF (final concentration 3 mM) before adding proteinase K. Stop the proteolysis by adding PMSF to a final concentration of 3 mM. 24 W. J. Arion, this series, Vol. 174, p. 58. 25 j. S. Rush and C. J. Waechter, A n a l Biochern. 206, 328 (1992).

[381

B~OSVNTHESlSOF Gel ANCHORS

521

Then add ice-cold TCA (final concentration 7%) and leave the samples on ice for 15 min. Microcentrifuge the samples (10,000 g, 15 min, 4°), wash the precipitate with ice-cold acetone containing 1% TCA (leave the samples with acetone at -70 ° for 30 min), and recover the precipitate by microcentrifugation. Resuspend the precipitate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer 12 and resolve proteins by electrophoresis using 10% gels and standard SDS-PAGE protocols. 26 After electrophoretic separation proceed with standard Western blotting procedures 27 to detect and quantitate BiP. 27'27a We were able to procure antibodies to mammalian BiP from Drs. D. Bole (University of Michigan, Ann Arbor) and N. Brot (Roche Institute of Molecular Biology) and antibodies to trypanosome BiP from Dr. J. D. Bangs (University of Wisconsin--Madison Medical School); however, as mentioned above, antibody reagents to any ER lumenal protein will suffice for this purpose.

Permeabilization of Thymoma Cell Plasma Membranes with Streptolysin 0 Streptolysin O (SLO), a streptococcal pore-forming toxin, can be used to permeabilize the plasma membrane without damaging the membranes of intracellular organelles.28,29 To limit the effects of SLO to the plasma membrane without affecting intracellular structures, the toxin is added to cells at 4° to allow binding to the cell surface, excess toxin is removed, and pore formation is induced by shifting the incubation temperature to 30°37°. The SLO-derived pores can be as large as 30 nm in diameter, 3° and they are large enough to permit cytosolic proteins exceeding 240 kDa to escape into the extracellular medium.31 Conversely, radioactive sugar nucleotides, phospholipases, and other reagents can be introduced into the cell and gain access to intracellular organelles via SLO poresfl2

Reagents BW5147.3 mouse thymoma cells (maintained in suspension culture in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100/xg/ml streptomycin in an atmosphere of 5% CO2 at 37°) 26 D. E. Garfin, this series, Vol. 182, p. 425. 27 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 27a j. Vidugiriene and A. K. Menon, J. Cell Biol. 127, 333 (1994). 28 G. Anhert-Hilger, W. Maeh, K. J. Fohr, and M. Gratzl, Methods Cell Ciol. 31, 63 (1989). 29 S. G. Miller and H.-P. H. Moore, this series, Vol. 219, p. 234. 3o S. Bhakdi, J. Tranum-Jensen, and A. Sziegoleit, Infect. lmmun. 47, 52 (1985). 31 D. Gravotta, M. Adesnik, and D. D. Sabatini, J. Cell Biol. 111, 2893 (1990).

522

GPI-ANCHOREDPROTEINS

[38]

Streptolysin O (SLO; Cat. No. DF0482-60, VWR Scientific; contains 100-120 hemolytic units per vial) Procedure. Dilute the contents of one vial of SLO with 10 ml water, add DTT to a final concentration of 2 mM, and incubate the sample at 37° for 10 min. The DTT-activated SLO has to be used immediately and cannot be stored. Plasma membrane permeabilization is achieved as follows. In the first step, the thymoma cells are harvested, washed once with ice-cold PBS, and incubated for 20 min on ice with a prechilled solution of activated SLO at a concentration of 50-100 U per 107 cells, with the cells at a concentration below 107 cells/ml. During the procedure some portion of the SLO is expected to bind/insert into the plasma membrane. Remove the excess SLO by centrifuging the cells at 1000 g for 5 min at 4°. Wash the cell pellet once with ice-cold PBS. Resuspend the cells in an appropriate ice-cold buffer (the choice of buffer depends on further experimental conditions), then shift the cells to 37 °. Permeabilization, assessed by loss of cytoplasmic marker enzymes,31 propidium iodide staining,29 or GPI biosynthesis from UDp[3H]GlcNAc (see below), 22 is achieved in 5-10 min. [Note: The amount of SLO required for effective permeabilization can vary dramatically depending on the cell line used. 29 Also, SLO obtained from Sigma was inactive under our experimental conditions.]

General Procedures for Synthesizing Radiolabeled Glycosylphosphatidylinositols in Vitro a n d Analyzing Radiolabeled Lipids Radiolabeled GPIs may be generated in vitro by incubating membranes with any of the following radioactive precursors: UDp[3H]GIcNAc, GDP[3H]mannose, dolichol-p-[3H]mannose, CDp[3H]ethanolamine, CDP[/3-32p]ethanolamine, [3H]myristic acid, [3H]palmitic acid, and UDP[3H]galactose. GPI labeling via [3H]inositol in vitro does not work despite the efficient synthesis of [3H]inositol-labeled PI. 5'8 The two sugar nucleotides UDP[3H]GlcNAc and GDp[3H]mannose are the most useful for radiolabeling purposes, and detailed procedures for their use and comments on labeling are provided in more detail in a later section. Here we describe the general experimental approach involved in radiolabeling GPIs using trypanosome and thymoma membrane preparations and in extracting and analyzing the labeled lipids. Original articles may be consulted for details of labeling using CDp[3H]ethanolamine,is CDP[fl-32p]ethanolamine, 15 UDp[3H]galactose, 17 and 3H-labeled fatty acids, 13,18 and for additional examples of radiolabeling using membrane preparations from HeLa

[38]

BIOSYNTHESIS OF GPI ANCHORS

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TABLE I INCORPORATION OF RADIOACTIVITY INTO GLYCOSYLPHOSPHATIDYLINOSITOLS1N VITROa Membranes

Radiolabel

['hymoma microsomes ~ermeabilized thymoma cells [¥ypanosome microsomes

E'rypanosome lysate

Addition

P2

P3

IV

III

II

I

V

VI

UDp[3H]GlcNAc t'

EDTA

**

**

**

**

**

**

4.9

UDp[3H]GlcNAc C

EDTA

**

**

**

**

**

**

27

UDp[3H]GlcNAc J UDp[3H]GlcNAc e GDP[3H]Man f GDp[3H]Man f GDp[3H]Man g Dolichol-p-[3H]Man g CDp[3H]EtN h

EDTA --PMSF ----

** 19.8 27.7 ** 220 10.8 7.7

** 7.6 8.7 ** 100 3.5 4.3

** 6.9 3.4 0.2 9.5 2.6 **

** 9.3 7.3 1.2 62 2.8 **

** 11.2 12 5.2 16 4.5 **

** ND 5.4 24 2.3 8.3 **

11

7 84

13 29.6

** ** ** ** **

** ** ** ** **

" D a t a in cpm x 10 3. Lipid nomenclature is as follows: P2, EtN-P-Man3GlcN-PI; P3, EtN-PMan3GIcN-PI*; IV, Man3GlcN-PI; III, ManzGlcN-PI; II, ManlGIcN-PI + Man3GlcN-PI*; I, Man2GIcN-PI*; V, GlcN-PI; VI, GlcNAc-PI. PI* represents PI containing acylinositol. **, Not labeled; ND, not determined. r, U D p [ 3 H ] G l c N A c (1 /zCi) incubated with 0.2 mg t h y m o m a microsomes in a 100-/zl reaction for 30 rain at 37 °. c UDP[3H]GlcNAc (1 p~Ci) incubated with SLO-permeabilized t h y m o m a cells (3 x 10 6 cell equivalents, 21 units SLO) in a 300-/~1 reaction for 30 min at 37 °. d U D p [ 3 H ] G l c N A c (1 /xCi) incubated with trypanosome microsomes (5 x 10 7 cell equivalents) in a 150-pA reaction for 30 min at 37 °. e UDP[3H]GlcNAc (1 ~Ci) incubated with trypanosome microsomes in a 100-/xl reaction for 30 min at 37 ° (incorporation of radioactivity into lipids V and VI is presented as the total in both species). f G D p [ 3 H ] M a n (1/xCi) incubated with trypanosome microsomes (108 cell equivalents) in a 100-/xl reaction (containing 0.5 m M PMSF where indicated) for 30 min at 37 °. g GDP[3H]Man (1.6 × 106 cpm) or dolichol-p-[3H]Man (3.3 x 105 cpm) incubated with trypanosome lysates (8 × 10 7 cell equivalents) in a 125-/xl reaction for 40 min at 37°; the dolichol-P-[3H]Man was delivered in Triton X-100 (final concentration in the incubation of 0.006%, w/v). h CDP[BH]EtN (1.4 × 10 7 cpm) incubated with a trypanosome lysate (1.2 × 10s cell equivalents) in a 250-/zl reaction for 30 min at 37°; approximately 1.2 × 10 6 cpm was recovered as phosphatidylethanolamine. c e l l s a n d l e u k o c y t e s , 23a,32 T o x o p l a s m a

iae. 13 R e p r e s e n t a t i v e

GPI

labeling

gondii, 7 and Saccharomyces

data are presented

in Table

cerevis-

I.

Reagents Guanosine

diphosphate

804, DuPont-NEN, beled

Chemicals,

[3H]mannose Boston,

MA,

(15-35

Ci/mmol;

or ART-440,

Cat. No. NET-

American

Radiola-

I n c . , St. L o u i s , M O )

32 s. Hirose, L. Ravi, G. M. Prince, M. G. Rosenfeld, R. Silber, S. W. A n d r e s e n , S. V. Hazra, and M. E. Medof, Proc. Natl. Acad. Sci. U.S.A. 89, 6025 (1992).

524

GPI-ANCHORED PROTEINS

[38]

Uridine diphosphate N-acetyl[3H]glucosamine (5-25 Ci/mmol; Cat. No. NET-434, DuPont-NEN, or ART-128, American Radiolabeled Chemicals, Inc.) Uridine diphosphate [3H]galactose (30-50 Ci/mmol; Cat. No. NET758, DuPont-NEN) Dolichol-p-[3H]mannose 14 Cytidine diphosphate [3H]ethnolamine 15 Cytidine [/3-32p]diphosphate ethanolamine 15 [3H]Myristic acid (10-60 Ci/mmol; Cat. No. NET-830, DuPont-NEN) [3H]Palmitic acid (30-60 Ci/mmol; Cat. No. NET-043, DuPont-NEN) 0.1 M ATP, coenzyme A, and DTT solutions, stored in the form of small aliquots at - 2 0 ° Tunicamycin (Cat. No. 654380; Calbiochem), prepared as a stock solution of 1 mg/ml in 95% ethanol and stored frozen at - 2 0 ° 2× Assay buffer: 25 mM H E P E S - N a O H , pH 7.5, 50 mM KC1, 10 mM MgC12, 10 mM MnC12 Two-phase mixture of n-butanol and water (prepared on a reasonable scale, i.e., take at least 200 ml of each, shake thoroughly, and allow to separate into two phases; the upper phase is water-saturated n-butanol and the lower phase is n-butanol-saturated water) Procedure. Thaw the frozen membrane preparation rapidly and add an equal volume of ice-cold 2× assay buffer containing 0.1 mM TLCK and 1/xg/ml leupeptin (trypanosome lysates must be washed to remove glycerol as described and suspended directly in 1 × assay buffer). Alternatively, use 0.5-1 × 108 cell equivalents in a total volume of 150-250/xl per reaction, with the sample in a glass tube (e.g., 12 × 75 mm borosilicate disposable culture tube). Supplement the reaction mixture with ATP, CoA, and DTT (final concentration 1 mM each). Add tunicamycin (final concentration 0.1 /xg/ml) and preincubate the reaction mixture briefly (<5 min) at room temperature. Add 0.25-10 /xCi of GDp[3H]mannose, UDp[3H]GlcNAc, CDp[3H]ethanolamine, or other radiolabeled precursor (previously evaporated to dryness and resuspended in a small volume of assay buffer; this is necessary as the material is usually supplied in ethanol or an ethanolwater mixture), mix carefully by gentle vortexing, and incubate the sample at 37 ° for 5-60 min, or as appropriate. Terminate the reaction by adding an ice-cold mixture of chloroform/ methanol (1 : 1, v/v) to achieve a final composition of chloroform/methanol/ water (10 : 10 : 3, v/v/v; use 1.7 ml chloroform/methanol per 250/xl of aqueous reaction mixture). Mix by stirring with a glass Pasteur pipette (do not vortex or shake). Centrifuge the sample (1500 g, 10 min, 4°) to pellet the insoluble cell debris (the pellet may be difficult to see if few cell equivalents of membranes were used in the assay). Transfer the supernatant to a fresh

[38]

BIOSYNTHESIS OF GPI ANCHORS

525

tube and evaporate to dryness in a centrifugal concentrator. Resuspend the dried residue in a mixture of n-butanol and water (750/~1 of each will suffice), mix vigorously using a vortex mixer, and separate the phases by centrifugation (1000 g, 10 min, room temperature). A sharp separation of the two phases is seen at room temperature; the interface may be diffuse if the sample is cold. Transfer the butanol-rich upper phase to a fresh tube and reextract the lower aqueous phase with water-saturated n-butanol. Pool the butanol phases and back-extract with n-butanol-saturated water. The phase separation procedures are important as they serve to desalt the lipid samples and, except in the case of 3H-labeled fatty acid, to remove the input water-soluble radioactivity. All GPI species are quantitatively recovered in the butanol phase. A representative thin-layer chromatogram of radiolabeled GPIs obtained from a GDp[3H]mannose labeling experiment is shown in Fig. 2. A differential lipid extraction protocol may be used if the starting aqueous reaction volume is small (typically <70/.d) or if the reaction is rapidly frozen in a dry ice-ethanol mixture and lyophilized. Extract the sample with chloroform/methanol (CM, 2:1, v/v) initially (--1.5 ml of solvent is needed per 70-~1 aqueous reaction volume); centrifuge and transfer the supernatant to a fresh tube. Repeat the extraction at least once (less solvent may be used in the second and subsequent extractions) or until very little more radioactivity is recovered in the extract (<0.1% of the initial extract),

o

P2

I

I

P3 1V III II I I

I

I

f

I

I

& I.........

L o

!

10

15

20

m i g r a t i o n distance [cm] FIG. 2. Thin-layer chromatogram of GPIs labeled in trypanosome microsomes via GDP[3H]mannose. The distribution of radioactivity was scanned using a Berthold LB2842 thin-layer scanner. P2, EtN-P-Man3GlcN-PI; P3, EtN-P-Man3GlcN-PI*; IV, Man3GIcN-PI; III, Man2GIcN-PI; II, ManlGlcN-PI + Man3GIcN-PI*; I, Man2GIcN-PI*; o, origin; f, front. Conditions for TLC: silica 60 thin-layer plate, solvent system of chloroform/methanol/water (10 : 10 : 3, v/v/v).

526

GPI-ANCHOREDPROTEINS

[381

then pool the extracts. The solvent mixture extracts neutral lipids, simple phospholipids, some dolichol-linked oligosaccharides, nonphosphoethanolamine-containing GPIs, and also the bulk of the input radioactivity if the sample was incubated with 3H-labeled fatty acids. Set the CM extract aside and proceed by adding chloroform/methanol/water (CMW, 10:10:3, v/v/v) to the partially delipidated membrane debris to extract more polar lipids; repeat the CMW extraction procedure at least once. Desalt the CM extract by a Folch washing procedure: add 4 m M MgC12 (CM extract to MgCI2 ratio of 5 : 1, v/v), vortex, separate the phases by centrifugation, and reextract the chloroform-rich lower phase with mock upper phase (mock upper phase is chloroform/methanol/water/1 M MgC12, 6 : 96 : 94 : 0.366, v/v/v/v) at least once. Desalt the CMW extract by drying and b u t a n o l water phase separation as described above. Store the extracts in tightly capped tubes at 4 °, or at - 2 0 ° for longer periods. The in v i t r o labeled lipid species should be analyzed in a variety of ways to check if they contain elements of the GPI core structure (Fig. 1A). Thin-layer chromatography is the most useful way to resolve the labeled lipids and to test the effect of various diagnostic enzymatic and chemical treatments such as hydrolysis by specific phospholipases and cleavage by nitrous acid. Relatively little radioactivity [ - 1 0 0 0 counts/min (cpm)] is required for such analyses, particularly if the chromatograms are scanned using a machine such as the Berthold LB2842 Automatic T L C Linear Analyzer (Wallac Inc., Gaithersburg, MD). Detailed protocols for GPI analysis may be found in other chapters in this series 33 and elsewhere in this volume and in the general literature. 5,19,34-37 Suitable T L C solvent systems are summarized in Table II.

G l y c o s y l p h o s p h a t i d y l i n o s i t o l Labeling via Radioactive S u g a r Nucleotides Labeling of GPI via the radioactive sugar nucleotides UDp[3H]GIcNAc and GDp[3H]mannose is especially attractive as all or almost all the GPI lipid biosynthetic intermediates are labeled. Specific inhibitors can be used to block the biosynthetic pathway and to accumulate radiolabeled interme33A. K. Menon, this series, Vol. 230, p. 418. 34S. Mayor, A. K. Menon, G. A. M. Cross, M. A. J. Ferguson, R. A. Dwek, and T. W. Rademacher, 3. Biol. Chem. 265, 6164 (1990). 35S. Mayor, A. K. Menon, and G. A. M. Cross, J. Biol. Chem. 265, 6164 (1990). 36T. Kamitani, A. K. Menon, Y. Hallaq, C. D. Warren, and E. T. H. Yeh, J. Biol. Chem. 267, 24611 (1992). 37M. A. J. Ferguson, in "Lipid Modifications of Proteins: A Practical Approach" (N. M. Hooper and A. J. Turner, eds.), p. 191, IRL Press, Oxford, 1992.

[38]

BIOSYNTHESIS OF GPI ANCHORS

527

T A B L E II SOLVENT SYSTEMS FOR THIN-LAYER CHROMATOGRAPHY System a

Re Values of characterized lipids b

A

P2 (0.16), P3 (0.36), Man3GIcN-PI (0.45), Man2GlcN-PI (0.52) ManlGIcN-PI + Man3GlcN-PI* (0.58), ManzGlcN-PI* (0.62), trypanosome dolichol-Pmannose (0.66) P2 (0.34), P3 (0.51), Man3GlcN-PI (0.56), ManzGlcN-PI (0.59) MankGlcN-PI + Man3GIcN-PI* (0.62), Man2GlcN-PI* (0.65), trypanosome dolichol-Pmannose (0.73) P2 (0), P3 ( - 0 ) , Man3GIcN-PI (0.08), ManzGIcN-PI (0.15) ManlGlcN-PI + Man3GlcN-PI* (0.23), GlcNAc-PI (0.47), GlcN-PI (0.53), trypanosome dolichol-P-mannose (0.66) GlcNAc-PI (0.7), GlcN-PI (0.65), PI (0.75)

B

C

D

" Solvent systems are as follows (volume ratios): A, chloroform/methanol/water (10: 10:3, v/v/v); B, chloroform/methanol/water (4 : 4 : 1, v/v/v); C, chloroform/methanol/acetic acid/ water (25 : 15 : 4 : 2, v/v/v/v); D, chloroform/methanol/1 M NH4OH (10 : 10 : 3, v/v/v). Silica 60 thin-layer plates [Kieselgel 60, 10 × 20 cm (Cat. No. 5626) or 20 × 20 cm (Cat. No. 5721-7), E M Science, Gibbstown, NJ] are used in each case and should be activated by heating in a 120 ° oven for at least 1 hr before use. P2, Et-P-Man3 GIcN-PI, P3, Et-P-Man3 GIcN-PI*, PI* represents PI containing acylinositol (see Fig. 1A).

diates. Because radiolabeled dolichol-linked oligosaccharide species (dolichol-PP-GlcNAcl_2Mano_9Glco_3) can also be synthesized from the precursors, incubations must be performed in the presence of tunicamycin (the GPI pathway is insensitive to tunicamycin). 4'34 This section describes protocols for the in vitro labeling of GPIs via UDp[3H]GlcNAc and GDp[3H]mannose and discusses the use of specific inhibitors.

Labeling via UDP[~H]GlcNAc: Synthesis of GlcNAc-PI, GlcN-PI, GlcN-PI*, and Later Biosynthetic Intermediates The synthesis of GlcNAc-PI, GIcN-PI, and GlcN-PI* (Figs. 1B and 3) can be studied in thymoma or trypanosome membrane preparations, or in SLO-permeabilized thymoma cells, using UDp[3H]GlcNAc. Endogenous PI acts as the GlcNAc acceptor, although it is reportedly possible to stimulate GlcNAc-PI synthesis by adding purified PI to the membrane preparation. 1° The subsequent mannosylation steps can be blocked by omitting GDPmannose and including EDTA in the incubation mixture to inhibit the synthesis of dolichol-P-mannose (Fig. 1B). GlcNAc-PI is not synthesized by microsomes derived from mutant cells belonging to the A, C, or H complementation groups] °'H'ala

528

GPI-ANCHOREDPROTEINS

[381

GlcNAc-PI ,~

GlcN-PI

GlcN-PI* f

0

0

_J I

0

I

LA___ I

5 10 15 migration distance [cm]

20

FIo. 3. Thin-layer chromatogram of GPIs labeled via UDp[3H]OlcNAc in SLO-perrneabilized thyrnorna cells. The labeling incubation was carried out in the presence of EDTA to limit the incorporation of radioactivity to the three nonmannosylated GPIs indicated at the top of the chromatograrn. The distribution of radioactivity in the chrornatogram was scanned using a Berthold LB2842 thin-layer scanner, o, Origin; f, solvent front. Conditions for TLC: silica 60 thin-layer plate, solvent system of chloroforrn/rnethanol/1 M NH4OH (10:10:3, v/v/v).

Rapidly thaw microsomes (0.2 mg protein in 50/zl) and dilute t h e m with an equal volume of buffer (25 m M H E P E S - N a O H , p H 7.5, 10 m M E D T A , 1/zg/ml leupeptin, 0.1 m M T L C K ) . E v a p o r a t e 1/zCi of UDp[3H] G l c N A c to dryness in a glass tube and resuspend in < 5 /zl of water or buffer. Transfer the microsome suspension to the tube containing the U D p [ 3 H ] G I c N A c and incubate the sample at 37 ° (incorporation of radioactivity into G l c N A c - P I and GlcN-PI is linear for up to 2 hr and with up to 0.8 mg of microsomal protein). Stop the reaction by placing the tube on ice, and obtain a single phase lipid extract by adding 300 tzl of water and 1.5 ml of ice-cold cloroform/methanol (1:2 v/v) to achieve a final composition of water/chloroform/methanol of 4 : 5 : 10, by volume. Induce the formation of two phases by adding 0.5 ml water and 0.5 ml chloroform, vortex to mix, and separate the phases by centrifugation at 1000 g for 10 min at 4 °. R e m o v e the u p p e r phase and wash the lipid-containing chloroform-rich lower phase with m o c k u p p e r phase (water/chloroform/methanol, 0.8 : 1 : 2, v/v/v) to r e m o v e salts and any contaminating water-soluble radioactivity (unreacted U D p [ 3 H ] G I c N A c and b r e a k d o w n products). The extraction and desalting procedures described in the previous section may also be used.

[38]

BIOSYNTHESIS OF GPI ANCHORS

529

Take 10% of the lipid-containing lower phase directly for liquid scintillation counting. It is important to evaporate the organic solvents before adding liquid scintillation cocktail, as the solvents (particularly chloroform) will sometimes interefere with scintillation couting. Dry the remainder of the sample and dissolve in water-saturated n-butanol (at roughly 2000 cpm per 10/xl). Analyze the samples by TLC using silica gel 60 thin-layer plates and chloroform/methanol/1 M ammonium hydroxide (10:10:3, v/v/v) as the solvent system. The chloroform/methanol/water (10: 10:3, v/v/v) system which is most often used to analyze GPIs may also be used but is not recommended here as it does not effectively separate GlcNAc-PI from GlcN-PI. More than 95% of the incorporated radioactivity is found in two radiolabeled lipid species; the faster moving lipid is GlcNAc-PI and the slower moving species is GIcN-PI. The lipids can be isolated from the thinlayer plate I9 and analyzed by enzymatic and chemical treatments. Both lipids should be hydrolyzed by PI-specific phospholipase C (PI-PLC), and the more polar lipid should be efficiently converted to the faster moving species by N-acetylation. 5'9'19 Using 1 /xCi of UDp[3H]GlcNAc, yields of radiolabeled GlcNAc-PI/GlcN-PI in a standard incubation (37 °, 30 min) are 6-8 × 104 cpm and 2.8 × 105 cpm per 1 mg protein of thymoma and trypanosome microsomes, respectively. Yields may be improved somewhat by including the nucleotide pyrophosphatase inhibitor dimercaptopropanol in the incubation mixture, 38'39but we have found this to be unnecessary as we detect very little or no degradation of UDp[3H]GIcNAc. 5'22 Radiolabeled GlcNAc-PI and GlcN-PI can also be generated by incubating SLO-permeabilized cells with UDP[3H]GlcNAc. Resuspend approximately 106 SLO-permeabilized thymoma cells, typically in 0.25-0.5 ml of ice-cold PBS containing 8 mM EDTA, 4 mM DTT, 0.1 mM TLCK, 1/xg/ml leupeptin. Add 1.5/zCi UDp[3H]GlcNAc and shift the temperature to 37° to induce SLO pore formation and GlcNAc/GIcN-PI synthesis. After incubation (typically 90 min), place the cells on ice and extract and analyze the labeled lipids as described above. As mentioned above, pore formation and hence labeling efficiency depends on SLO concentration. For thymoma cells the maximum synthesis that we observed (i.e., 105 cpm per 1 0 7 cells) was achieved at a concentration of 60 U SLO per 107 cells. A representative thin-layer chromatogram is shown in Fig. 3. If mannosylated and phosphoethanolamine-containing radiolabeled GPI intermediates are to be synthesized from UDp[3H]GlcNAc, EDTA must be left out of the incubation buffer, and tunicamycin (0.1 /zg/ml) and nonradioactive GDPmannose (1 mM) must be included (see general 38 C. R. Faltynek, J. E. Silbert, and L. Hof, J. BioL Chem. 256, 7139 (1981). 39 V. g. Stevens, J. Biol. Chem. 268, 9718 (1993).

530

GPI-ANCHORED PROTEINS

[38]

labeling procedures above). In thymoma cell microsomes, labeling of mature GPIs is considerably enhanced by including, additionally, GTP, Mg 2+, and coenzyme A which act to stimulate de-N-acetylation of GlcNAc-PI and increase synthesis of the critical GlcN-PI* intermediate. 39,39a

Labeling via GDp[3H]Mannose and Dolichol-p-[3H]Mannose The three mannose residues in the GPI core glycan are derived from dolichol-P-mannose14 (Fig. 1B). GPI labeling from GDp[3H]mannose therefore involves [3H]mannose transfer to endogenous dolichol phosphate to give dolichol-P-[3H]mannose. This reaction is inhibited by EDTA and the lipopeptide antibiotic amphomycin14'4° (Fig. 1B), and it does not occur in the class E thymoma mutant cell line. 41 Exogenous dolichol phosphate stimulates synthesis of dolichol-p-[3H]mannose from GDp[3H]mannose and thus increases the incorporation of radioactivity into GPIs. 14 DolicholP-[3H]mannose may be used directly for labeling. 14 For radiolabeling via GDp[3H]mannose, follow the general labeling protocol described above but include 1 mM nonradioactive UDPGlcNAc in the incubation mixture. When using dolichol-P-[3H]mannose for GPI labeling, dissolve the dolichol-p-[3H]mannose in a small volume of 0.2% (w/v) Triton X-100 (it is best to let the sample remain at room temperature for a while before use, with occasional mixing on a vortex mixer) before adding membranes. The final concentration of detergent should be no more than about 0.005%, as detergent in the incubation mixture depresses the synthesis of phosphoethanolamine-containing GPI species, without any apparent effect on the synthesis of mannosylated GPI intermediates. 14 GDp[3H]mannose can be used for GPI synthesis in trypanosome lysates and microsomes,4&s'14,2323a and somewhat less efficiently in thymoma membrane preparations 39a and HeLa cell lysates.23a'32 In both systems the extent and kinetics of labeling depend critically on the amount and quality of membranes used, GDPmannose concentration, and incubation time. We typically use 107-108 cell equivalents of trypanosome microsomes in a 100-/xl reaction mixture containing 0.2-1/xCi of GDp[3H]mannose. Some degradation of GDp[3H]mannose occurs during the incubationS; it may be possible to limit this with dimercaptopropano138 or by using larger amounts of the sugar nucleotide. Supplementing the reaction with 1-2/xM of nonradioactive GDPmannose decreases the specific activity but improves the yield of labeled GPIs. GPI synthesis reaches a maximum in 30-40 min in trypanosome microsome preparations; a similar level of incorporation is 39a V. L. Stevens and H. Zhang, J. Biol. Chem. in press. 4o D. K. Banerjee, M. G. Seher, and C. J. Waeehter, Biochemistry 20, 1561 (1981). 41 A. C h a p m a n , K. Fujimoto, and S Kornfeld, J. Biol. Chem. 255, 4441 (1980).

[38]

BIOSYNTHESIS OF GPI ANCHORS

531

reached in unfractionated trypanosome lysates but after a period of about 2 hr. Blocking phosphoethanolamine addition (in the trypanosome system) by including PMSF in the reaction mixture23'23a results in the accumulation of the radiolabeled Man3GPI species and provides a good way to isolate significant radiochemical amounts of the lipid intermediate. Radiolabeling of mammalian GPIs by incubating HeLa cell lysates with GDp[aH]mannose is considerably less efficient and requires larger amounts of radioactivity and membranes; 23a'32 this efficiency may be improved by including GTP and Coenzyme A or fatty acyl Coenzyme A in the reaction mixture. 39'39a Transfer of Glycosylphosphatidylinositol to Endogenous Protein Acceptors Mature phosphoethanolamine-containing GPIs are transferred to newly synthesized proteins carrying the appropriate GPI signal sequence. 42 The reaction proceeds by cleavage of the carboxyl-terminal GPI signal sequence and attachment of the GPI precursor (via ethanolamine) to the newly exposed o~-carboxyl group of the polypeptide. The carboxyl-terminal cleavage reaction can be analyzed by using standard protein translation-translocation assays which are described in detail elsewhere in this volume. Direct evidence for GPI attachment to in vitro translocated protein has not been obtained because translation-translocation assays involve femtomole amounts of translated protein and specific labeling of GPI precursors in the membranes is low and involves 3H-labeled precursors that are not as readily detectable as 35S- or 32p-labeled compounds. However, a large body of indirect evidence suggests that this is indeed the case (see this Volume, Chapter 39). GPI addition to protein can be studied by demonstrating the transfer of labeled GPI precursors to endogenous protein acceptors. 43 The acceptor molecules presumably represent proteins that are partially processed at the time of membrane preparation and are thus properly positioned to accept and be modified by GPIs radiolabeled in vitro. 43 Incubate trypanosome microsomes (108 cell equivalents) for 30-90 min at 37° with 1.5/zCi GDP[3H]mannose and 1 mM UDPGlcNAc in 100/xl of reaction buffer as described above in the general labeling protocol. At the end of incubation take 10% of the sample for lipid extraction and analysis. To the rest of the sample add 300 /xl of 4 mM PMSF in water (dilute the PMSF just before use from a 0.2 M stock solution in 95% ethanol). Incubate on ice for 10 min and add 400/xl of ice-cold 14% (w/v) TCA. Leave the sample on ice for 30 min, then centrifuge (12,000 g, 15 min, 4°) to pellet the precipitate. Resuspend the pellet in 500 ~1 acetone 42 S. Udenfriend, R. Micanovic, and K. Kodukula, Cell Biol. Int. Rep. 15, 739 (1991). 43 S. Mayor, A. K. Menon, and G. A. M. Cross, J. Cell Biol. 114, 61 (1991).

532

GPI-ANCHOREDPROTEINS

[381

containing 1% T C A and leave the sample at - 7 0 ° for 30 min. Centrifuge the sample again, resuspend the pellet in S D S - P A G E sample buffer, 26 and analyse by S D S - P A G E . 26 Process the gel for fluorography 44 by impregnating it with En3Hance ( D u P o n t - N E N ) and exposing the dried gel to X-ray film (e.g., Kodak, Rochester, NY, X-Omat A R ) at - 7 0 °. Exposure times of about 2 weeks are usually required. Sensitivity may be increased by using preflashed films. 45 Typically only one radiolabeled band of approximately 55 kDa is detected, corresponding to trypanosome variant surface glycoprotein. 43

Topological Analysis of G l y c o s y l p h o s p h a t i d y l i n o s i t o l B i o s y n t h e s i s in E n d o p l a s m i c R e t i c u l u m The distribution of the GPI pathway between the two leaflets of the E R m e m b r a n e bilayer can be studied directly by analyzing the distribution of radiolabeled biosynthetic lipid intermediates using membrane-impermeant probes. Similar approaches have been used to describe the compartmentation of the dolichol-linked oligosaccharide pathway of N-linked glycosylation. 46 The choice of appropriate membrane-impermeant probes is limited as it is important to select reagents that specifically modify or bind to the lipid targets of interest without destroying the integrity of the membrane bilayer. Ideally, the reagent should be effective in a short incubation with the membranes on ice, and it should be possible to quench or remove the reagent prior to membrane solubilization or lipid extracton. In this section we describe methods for using bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and the jackbean lectin concanavalin A (Con A) for probing the transbilayer distribution of radiolabeled GPIs in total or ERderived microsomes. 22'27a The PI-PLC enzyme can be used to probe GPI species that do not contain acylinositol (Fig. 1A); Con A will bind GPIs containing more than 2 mannose residues irrespective of inositol acylation. 27a The reader is referred to the recent literature for discussions of other potential topological probes, 22'27a including GPI-specific reagents introduced into the cytoplasm of living cells for the purpose of probing the orientation of GPI structures. 46a

44W. M. Bonner, this series, Vol. 96, p. 215. 45R. A. Laskey and A. D. Mills, Eur. J. Biochem. 56, 335 (1975). 46C. Abeijon and C. B. Hirschberg, Trends Biochem. Sci. 17, 32 (1992). 46aK. Mensa-Wilmot,J. H. Lebowitz,K.-P-. Chang, A. AI-Qahtani, B. S. McGwire, S. Tucker, and J. C. Morris, Z Cell Biol. 124, 935 (1994).

[38]

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Phosphatidylinositol-Specific Phospholipase C Treatment as Probe of Transbilayer Distribution of Enzyme-Sensitive Glycosylphosphatidylinositols in Microsomes Reagents Bacillus thuringiensis PI-PLC (Cat. No. GPI-02, Oxford Glycosystems, Rosedale, NY) or Bacillus cereus PI-PLC (Cat. No. 11403-069, Boehringer-Mannheim); unless otherwise specified, enzyme units are determined by assaying hydrolysis of [3H]phosphatidylinositol at pH 7.0 in 0.1% sodium deoxycholate,47 where 1 unit corresponds to 1/xmol [3H]PI hydrolyzed per minute Sodium deoxycholate (Cat. No. D 5670, Sigma) Buffer P: 0.25 M HEPES-NaOH, pH 7.5,100 mM sucrose, 25 mM KC1 Procedure. Label GPI lipids with UDp[3H]GlcNAc, GDp[3H]mannose, or other radioactive precursor using trypanosome or thymoma microsomes (roughly 108 cell equivalents) as described above. Although incubation times can vary between 5 and 60 min, shorter times (15 min) are recommended to preserve the sealed quality of the microsomes. To stop the reaction place the tube on ice and add a 1000-fold excess of nonradioactive substrate. Mix the sample by gentle pipetting and divide it into five aliquots. Each aliquot may be diluted with 100/xl buffer P if necessary for liquid handling. Add different concentrations of PI-PLC (in the range 0-2 units/ml) in the presence or absence of 0.1% sodium deoxycholate (final concentration) and incubate the sample for 20 min on ice; maximal hydrolysis of cytoplasmically disposed GPI lipids in trypanosome membranes is achieved with 0.2-0.5 units/ml of enzyme. Monitor the intactness of the microsomal vesicles prior to and after PI-PLC treatment by determining the extent to which a lumenal protein marker is protected from the action of exogenously added proteases (see above). Terminate the reaction by adding ice-cold buffer P to give a final volume of 250 ~1, then 1.7 ml of chloroform/methanol (1:1, v/v). Proceed with lipid extraction as described above. To determine the extent to which the PI-PLC susceptible GPIs (P2, Man3GIcN-PI, and Man2GIcN-PI) are hydrolyzed, take 10% of the lipid extract for liquid scintillation counting and analyze the rest by thin-layer chromatography (see Table II for appropriate solvent systems). Determine the amount of radioactivity in each GPI species before and after PI-PLC treatment: this may be done by scraping individual areas of the thin-layer 47M. G. Low,in "LipidModificationof Proteins:A PracticalApproach" (N. M. Hooperand A. J. Turner, eds.), p. 117. IRL Press, Oxford, 1992.

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chromatogram and assessing radioactivity by scintillation counting, or by scanning the chromatogram and using peak integration software. In intact membranes, hydrolysis of a particular lipid species implies that the lipid is located in the cytoplasmic (external) leaflet of the microsomal membrane bilayer. [Note: No systematic information is available on the effect of different detergents on the activity of the various bacterial PI-PLCs. Triton X-100 at concentrations greater than 0.1% (w/v) inhibits the B. thuringiensis enzyme. 47 If detergents other than sodium deoxycholate are to be used to solubilize the microsomes, the effect on PI-PLC activity must be checked.]

Concanavalin A Binding as Probe of Mannose-Containing Glycosylphosphatidylinositols The jackbean lectin Con A is a membrane-impermeant probe 48 which has been successfully used to probe the orientation of dolichol-linked oligosaccharides in E R vesicles. 49 Con A can bind specifically to GPI species containing more than two mannose residues, 27a consistent with its known specificity for other mannose-containing oligosaccharides. 5° The Con A binding assay is based on the difference in solubility of free GPIs (soluble in chloroform/methanol/water, 10:10:3, v/v/v) versus Con A-bound GPI complexes (not soluble in organic extracts). The binding occurs on ice and can be blocked specifically by methyl ot-o-mannopyranoside (ot-MeMan). Because sucrose interferes with the binding, membranes must be diluted into buffer without sucrose, or they must be pelleted and resuspended in buffer without sucrose prior to Con A treatment (the dilution procedure gives more reliable results).

Reagents Con A from Canavalia ensiformis (Type IV; Cat. No. C-2010, Sigma) Methyl a-o-mannopyranoside (o~-MeMan; Cat. No. M-6882, Sigma) Buffer C: 25 mM H E P E S - N a O H , pH 7.5, 25 m M KC1, 5 m M MgCI2, 5 m M MnCI2, 5 m M CaCI2 Procedure. Synthesize radiolabeled GPIs in intact microsomes as described in the section on PI-PLC treatment (above), except do not add excess nonradioactive substrate at the end of the labeling incubation. Transfer aliquots of the reaction mixture (typically 2 × 107 cell equivalents in 20/xl) to separate glass tubes and add buffer C to give a final volume of 100 ~1. For determining the extent of GPI binding to Con A in detergentdisrupted membranes, include Triton X-100 (final concentration 0.1%) in 48E. Rodriguez Boulan, G. Kreibich, and D. D. Sabatini, J. Cell BioL 78, 874 (1978). 49M. D. Snider and P. W. Robbins, J. BioL Chem. 257, 6796 (1982). 50S. Ogata, T. Muramatsu, and A. Kobata, J. Biochem. (Tokyo) 78, 687 (1975).

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some samples. Place the samples on ice and add Con A (up to 5 mg/ml final) from a 10 mg/ml stock solution prepared in buffer C to give a reaction volume of 200/xl (the sucrose content of the original microsomal suspension is thus reduced -10-fold). Incubate for 20 min on ice, then terminate the binding by adding 5/xl of 0.5 M oL-MeMan. Increase the sample volume to 250/xl by adding 45/xl calf serum (to ensure 100% precipitation of organic insoluble material during lipid extraction) and an appropriate amount of buffer (25 mM HEPES-NaOH, pH 7.5, 25 mM KC1). Proceed with lipid extraction by adding 1.7 ml chloroform/methanol (1 : 1, v/v). Control samples in which Con A is added after a-MeMan are important in checking that no binding occurs during the lipid extraction process. Pellet the precipitates by centrifugation (1500 g, 20 rain, 4°), carefully remove the supernatant, and reextract the pellet with chloroform/methanol/ water (10 : 10 : 3, v/v/v). Pool the extracts and process the sample by drying and butanol-water phase partitioning as described in the general radiolabeling protocol above. Take 10% of the final butanol phase for liquid scintillation counting and analyze the remainder by thin-layer chromatography (see Table I! for appropriate solvent systems). Depletion of a particular lipid species from the butanol phase implies that the lipid is located in the cytoplasmic (external) leaflet of the microsomal membrane bilayer and is available for binding to Con A. The result may be quantitated by comparing the extent of depletion in intact microsomes to that in detergent-solubilized preparations where the phosphoethanolamine-containing trypanosome GPIs and the two intermediates Man3 GlcN-PI and Man3 GlcN-PI* (Fig. 1) bind Con A with about 70% efficiency. Acknowledgments This work was supported by the University of Wisconsin--Madison, an award from the Irma T. Hirschl Trust (New York), and National Institutes of Health Grant AI28858. The procedures described here owe much to the contributions of numerous individuals; we particularly acknowledge Satyajit (Jitu) Mayor, Ralph Schwarz, Mark Field, and George Cross, all former colleagues at The Rockefeller University (New York). We are also grateful to Peter Gerrold, Ralph Schwarz, and Vicky Stevens for comments on the manuscript. A. K. M. is grateful to K. M. M. for help with manuscript editing.