G-Protein-Coupled Receptor Regulation of Phospholipase D

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[20] G-Protein-Coupled Receptor Regulation of Phospholipase D By GUANGWEI DL', ANDREW J. MORRIS, VICKI A. SCIORRA, ANI) MICHAELA. F R O H M A N Introduction Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline (reviewed in Frohman el al.I). PA has been implicated in signal transduction, membrane vesicular trafficking. cytoskeleton reorganization, and cell proliferation (reviewed in Jones el al. 2 and Liscovitch el a/. 3). PLD activity is present in ninny manmmlian ceils and tissues and is upregulated in response to a wide variety of agonists that signal through heterotrimeric G-protein-coupled or tyrosine kinase receptors. Receptor stimula/ion initiates multiple signal transduction cascades, ultimately including activalion of protein kinase C (PKC), ADP-ribosylation factor (ARF), and Rho family members, which have been well characterized as activators of PLD in in v#ro and in ~'i~,oassay systems. Because ARK Rho. and PKC stimulate multiple downstream effector pathways that ultimately regulate cellular morphology, proliferation, and secretion, thcre has been intense interest in determining the relationship of PLD stimulation through each activator to these cell biological events. The regulation of mammalian PLD by G-protein-coupled receptors is complex and not fully understood at present. The two isolbrms, PLDI and PLD2, are seemingly activated differently, ahhough the details and degree of difference are not yet resolved. Despite the consensus that PKC, ARK and Rho family members directly and potently stimulate PLD (specifically PLD1) in l,ilro, 4 the relative importance of each effector class in stimulation of PLD in fifo, and whether synergy between them is required, remain topics of current research interest in many laboratories. Such studies can be approached through the manipulation of PKC, Rho, or ARF activity using overexpression of activated or inactive alleles, toxins, or pharmacological agents to activate or inhibit them (reviewed in Liscovitch el al.3). Although traditional and valuable, these approaches involve complicated interpretations, because manipulation of PKC, Rho, and ARF levels of activity affects

/ M. A. Frohman. T.-C. Sung. and A. J. Morris. Biochim. Bi~q,hys. Acta 1439, 175 (1990). 2 |9. Jones, C. Morgan, and S. CockcrofL

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3 M. kiscovitch, M. Czarny, G. Fiucci, and X. Tang, Biochem. J. 345, 401 (2000). 4 S. M. H a m m o n d , J. M. Jenco, S. Nakashima, K. Cadwalladcr, S. Cook, Y. Nozawa, M. A. g r o h m a n . and A. J, Morris, J. Biol. Chem. 272, 3860 (1997). Cop}right ~ 2002 b} AcademicPress All rights o["leproductionin any {'o1"1/Ilegel'~Cd ME'[HOI)S IN i{NZYMOI O(]h, \'OL ]45

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many things aside from PLD regulation (discussed in more detail in Zhang et al. 5 and D u e t al.(~). As an alternative approach, we have generated alleles of PLD that exhibit altered regulation including mutants that are inactive, 7 selectively nonresponsive to PKC 5 and/or Rho, ~ or insensitive to phosphatidylinositol 4,5bisphosphate [PI(4,5)P2]. 8 Using these molecular reagents, we have demonstrated that signal integration through both PKC and Rho via direct contact is required for significant physiological in vivo activation of PLD1 through G-protein-coupled receptors. The caveat to this approach is the one common to all overexpression studies, that the results obtained may not mirror with precision the regulation of the endogenous proteins. Our laboratory uses two main experimental systems, in vitro and in vivo cellbased assays, to study PLD activation through G-protein-coupled receptors. The in vitro system is used to assess the interaction and direct activation of PLD by its regulators, all of which have been well characterized as important components in the signaling pathways mediated by G-protein-coupled receptors. On the basis of the molecular reagents we create and characterize in the in vitro system, the in vivo system is then used to explore more complicated questions regarding signaling in intact cells. This article discusses some of these assays. Standard protocols to measure PLD activity in vitro and in vivo have been described previously 9 and are not described here. Protocols Expression and Purification of Glu-Glu-Tagged Phospholipase D Isoform 1, Using Baculoviral System In our previous experience, human PLDI protein purified using the baculoviral system [either untagged or with a hexahistidine (Hiss) tag] exhibited limited stability. Activity decreased noticeably each day and little was left after 3 - 4 days. To assist with purification, we explored expression of PLD 1 with a Glu-Glu tag (MEYMPMEG) fused to the amino terminus. The purified protein was unexpectedly found to be relatively stable; no detectable loss of enzyme activity is observed over a period of storage at 4 :~for 10 days. One possibility for the improved protein

5 y. Zhang, Y. A. Altshuller, S. A. Hammond, E Hayes, A. J. Morris, and M. A. Frohman, EMBO J. 18, 6339 (1999). 6 G. Du, Y. M. Altshuller, Y. Kim, J. M. Han, S. H. Ryu, A. J. Morris, and M. A. Frohmam Mol. Biol. Cell 11, 4359 (2000). 7 T. C. Sung, R. Roper, Y. Zhang, S. A. Rudge, R. TemeL S. M. Hammond. A. J. Morris. B. Moss, J. Engebrecht, and M. A. Frohman, EMBO J. 16, 4519 (1997). 8 V. A. Sciorra, S. A. Rudge, G. D. Prestwich, M. A. Frohman, J. Engebrecht, and A. J. Morris, EMBO ,L 18, 5911 (1999). M. A. Frohman, Y. Kanaho, Y. Zhang, and A. J. Morris, Methods Enzymol. 325, 177 (2000).

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stability may lie in the elution method rather than the tag. The current protocol uses peptide competition to recover the purified protein rather than changes in pH. The purification and concentration of the PLD stimulators PKC, Rho, and ARF have been described previously)

Ptto,vt~lzolipase D lxq/brm 1 Evpression and Purification 1. The full-length PLD1 wild-type and mutant cDNAs are subcloned into a pFASTBAC vector (Life Technologies, Rockville, MD) modified to contain the sequence MEYMPMEG (Glu-Glu tag) at the amino terminus. I° Recombinant bacmids are prepared by transfection of DH10Bac cells with the pFASTBAC plasmids. Recombinant baculoviruses are amplified and propagated by standard procedures as described in the manual about the Bac-to-Bac baculovirus expression system (Life Technologies). High-titer stocks of recombinant baculoviruses for expression of Glu-Glu-tagged wild-type and mutant PLDI and PLD2 alleles are available on request. 2. To express PLD 1, monolayers of exponentially growing Spodopteraf)ug, iperda (Sfg) cells (3 × 107 cells/225-cm e flask, cultured at 27 in complete Grace's medium supplemented with glutamme, lactalbumm, Yeastolale (flom Life Technologies), and 10% (v/v) fetal bovine serum containing penicillin (100 U/ml) and streptomycin (I 00 mg/ml); generally, two flasks of cells are used for each purification] are infected with recombinant baculoviruses at a multiplicity of 10 lk~r I hr with gentle rocking. [If large amounts of protein are required (e.g., for concentration). 300-500 ml of the cells at a density of 1 x 10° cells/ml is seeded in a 500-ml spinner l]ask and is infected with recombinant baculoviruses at a multiplicity of I0. The infected cells are grown for 48 hr without removing the virus, and then the procedure described below is scaled up correspondingly.] 3. The virus-containing medium is then removed and replaced with fresh supplemenled complete Grace's medium. The infected cells are grown fl~r 48 hr, shaken off the flask, pelleted at 2000g for 5 min at 4', and washed once with ice-cold PBS. 4. The cells are then lysed on ice by the addition of 5 ml of ice-cold lysis buffer per 225-cm e flask. After 30 rain on ice, the cell lysate is centrifuged at 50,000£, for 30 rain at 4 . 5. While the cenlrifugafion step is in progress, 0.3 ml of anti-Glu-Glu tag immunoaffinity resin (kindly provided by N. Pryor, Onyx Corp., Richmond, CA; the immunoaflinity resin is commercially available from BAbCo, Berkeley, CA) is washed with 5-10 ml of lysis buffer in a 15-ml tube several times (spin down lhe resin by gentle centrifugafion to remove the lysis buffer, and then add flesh lysis buffer). ro

I~. R. Stcphens, A. Eguinoa. H. Erdjumcnt Bromage, M. Lui, F. Cooke, J. Coadwell, A. S. Smrcka. M. Thclcn, K. Cadwallader, I~ Tempst, and P. T. ltawkins, ('ell 89, 105 {19971.

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6. The lysate supernatant obtained (10 ml) is mixed with the immunoaffinity resin and rotated at 4 ° for at least 1 hr. The resin is then placed in a 10-ml Bio-Rad (Hercules, CA) disposable chromatography column. Remove the bottom cap and let the extract flow through. Use the flowthrough to wash the 15-ml tube and the sides of the column until all of the resin is packed (all steps at 4':). 7. Wash the column with 10 ml of lysis buffer, and then 10 ml of stock buffer, and finally 10 ml of salt wash buffer (all steps at 4 ) . 8. Move the column to room temperature. Wash the column with 5 ml of salt wash buffer. During the wash, prepare 0.9 ml of elution buffer. 9. Cap the column bottom. Resuspended the resin with 0.3 ml of elution buffer and let it stand for 2 min. Remove the bottom cap and recover the eluent. Elute twice more with 0.3 ml each time. 10. Check the purity of the protein by 8% (w/v) sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). A 5- to 10-#1 aliquot from each fraction is added to 2x SDS-PAGE sample loading buffer containing 8 M urea, denatured at room temperature, and then electrophoresed. The protein should be detectable by standard Coomassie blue staining. Alternatively, use ARF-stimulated PLD activity in each fraction to determine the respective amounts of active protein. 11. Measure the concentration of the protein by the Bradford method, using the Coomassie Plus-200 protein assay reagent (Pierce, Rockford, IL). Briefly, 20/41 of bovine serum albumin (BSA) standards or PLD samples is mixed with 200 #1 of Bradford reagent in a 96-well plate and the absorbances at 595 nm are measured with a plate reader. Use the standard curve from BSA to determine the concentration of PLD. 12. Create a dose-response curve, using the in vitro PLD assay, to choose the suitable protein concentration for further experiments. Usually, 10-20 ng of purified protein per sample is appropriate for the in vitro PLD assay. In our hands, two 225-cm 2 flasks should yield approximately 20-70 l~tg of purified PLD1 protein. Solutions

Stock buffer: 20 mM Tris-HC1, (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol (DTT), 20 # M leupeptin, 0.1 mM phenylmetbylsulfonyl flouride (PMSF), 0.1 mM benzamidine Lysis buffer: Stock buffer plus 1% (v/v) Nonidet P-40 (NP-40) (final concentration) Salt wash buffer: Stock buffer plus 400 mM NaC1 (final concentration) Elution buffer: Stock buffer plus 400 mM NaCI plus Glu-Glu peptide (100 #g/ml, final concentration; make fresh) Glu-Glu peptide: EYMPTD (the free amine of the E should be acetylated; the C terminus should be left as a carboxyl) Notes. An example of the purification procedure is shown in Fig. 1A-C for a mutant PLD1 protein denoted "mini-PLDl," which lacks the amino terminus

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FIG. I. Purification and characterization o1"mini-PLD. (A) Sequence alignment of PLDI and miniPLDI. (B) SDS-PAGE of purilied mini-PLDI. Lane I, molecular weight markers; lane 2, 10 l~g of purified mini-PLD1. (C) Activation of mini PLDI by PI(4,5)P2. Mini PLDI activity was delermined with sonicated lipid vesicles containing 2.6 mM PC, the indicated concentrations o1" PI(4,5)P2, and varying amounts of PE to give a final lipid concentration of 100 raM. Assays contained 3 t~M GTP;~S aclivated ARF1. (D) Binding of mini-PLI)l to PI(4.5)P2 containing sucrose-loaded liposomes. MiniPLD1 was incubated with the indicated concentrations of PC : PS : PE liposomes containing 5 mol~7{ PI~4.5)P> The vesicles were sedimented by ultracenlrifugalion and vesicle-bound protein was detected by Western blotting.

and the central " l o o p " region. As r e p o r t e d previously, jr the 7 0 - k D a m i n i - P L D l is catalytically active in a P I ( 4 , 5 ) P x - d e p e n d e n t m a n n e r (see Fig. I C), i n d i c a t i n g that the a m i n o - t e r m i n a l P H d o m a i n does not m e d i a t e the a c t i v i t y - d e p e n d e n t PIP2 interaction. Instead, the PI(4,5)P2 interaction is m e d i a t e d by a b i n d i n g site located b e t w e e n the 2 H K D motifs (not s h o w n ; see also Sciorra el al.8). F i g u r e 1B s h o w s the r e c o v e r y o f G l u - G l u - t a g g e d m i n i - P L D , u s i n g the protocol d e s c r i b e d a b o v e as visualized by C o o m a s s i e blue staining: virtually pure P L D protein can be g e n e r a t e d

JJ T.-C Sung, Y. Zhang, A. J. Morris, and M. A. Frohmam J. Biol. Uhem. 274, 3659 (1999).

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in a one-step procedure by this approach. Figure 1C demonstrates that the protein is active in the presence of PLD stimulators [GTP?'S-activated ARF1 and PI(4,5)P2].

Concentration and Buffer Exchange of Phospholipase D Isqform l by Ultrafiltration. For concentration and buffer exchange of purified PLD 1, we initially tried ultrafiltration powered by centrifugal force. We found that most of the protein was lost and little concentration occurred. More successful was the stirred-cell ultrafiltration system powered by nitrogen gas pressure forces described below. We have been able to concentrated full-length PLD1 from 2 0 - 5 0 to ~200/~g/ml successfully in 10 mM piperazine-N,N'-bis (2-ethanesulfonic acid) (PIPES), 50 mM potassium glutamate (pH 7.4), which is a buffer appropriate for microinjection.12 The advantage of the stirred-cell ultrafiltration system over the centrifugal filter device is that it avoids concentration polarization, which may have caused protein precipitation or adherence onto the membrane surface. All of the procedures below are performed at 4 '~ or in a cold room. 1. For buffer exchange and further concentration of PLD, the protein eluted from the column is diluted more than 10-fold into the desired buffer. To concentrate PLD without exchanging the buffer, the protein can be loaded directly into the stirred cell. 2. Load an Amicon P M I 0 membrane (Millipore, Bedford, MA) into the stirred cell (Millipore, model 8003) with the glossy side up. Handle the membrane carefully with powder-free gloves and hold by the edge to avoid scratching the smooth, glossy surface. Check carefully to make sure that the stirred cell is properly assembled and that no leakage is visible around the membrane when pressure is applied. 3. Pour the protein solution into the stirred cell (up to the maximum operating volume) and place the unit on a magnetic stirrer. Connect the cell to the nitrogen gas regulator and pressurize to 40-50 lb/in 2 (pressure should not exceed 70 lb/in2). 4. Monitor the flow rate and ultrafiltrate volume until the desired rate of concentration has been achieved. The cell is refilled when the volume has decreased by one-third if the total sample volume initially exceeded the cell capacity. 5. Turn offthe stirrer and the nitrogen flow to the cell. Slowly release the internal cell pressure. Disassemble the stirred cell and transfer the concentrated sample to a 1.5-ml centrifuge tube, using a Pipetman (Rainin, Woburn, MA).

Notes. This protocol is still in evolution. The recovery of PLD I is variable, and the factor(s) influencing this are not certain. More difficulty seems to be encountered when the starting protein concentration is low or when low-salt buffers are 12 N. Vitale, A.-S. Caumont-Primus, S. Chasserot-Golaz, G. Du, S. Wu, V. A. Sciorra, A. J. Morris, M. A. Frohman, and M.-F. Bader, E M B O J. 20, 2424 (2001).

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used for the exchange. In more recent preliminary experiments, Amicon YM10 membranes have exhibited better recoveries than the PM membranes described above.

Prepa/zttion qf Tisstw Culture Cells f i , Use in in Vivo Pho,v~holipase D Assay for G-Protein-Cotq~led Receptor Sig~utli/t£, Molecular Reagents. A wide variety of expression plasmids have been used for functional studies of PLD in mammalian cells. Our laboratories use the cytomegalovirus (CMV) promoter-driven plasmid pCGN, which appends an N-terminal hemagglutinin (HA) epitope to the PLDI and PLD2 proteins. To dissect the signal pathways regulating PLDI activation, we have generated a useful series of mutants, which includes inactive allele Kg98R, 7 an allele selectively nonresponsive to PKC P I M 8 7 : an allele selectively nonresponsive to Rho 1870R) and an allele selectively nonresponsive to both PKC and Rho PIM87/1870R) [:sing cultured mammalian cells with either endogenous, transiently expressed, or stably expressed G-protein-coupled receptors, PLDI activation can be studied by using these alleles. The receptors thus far used in our studies are the m l, m2, and m3 muscarinic acetylcholine receptors (mAChRs), the Edg2 and Edg4 lysopbosphatidic acid (LPA) receptors, and the angiotensin I1 (ATI a)receptor. Notes. In our system, we have been able to examine PLD signaling in combination with G-protein-coupled receptors that signal through Gq/GII and Gle/G~3 (Edg4, ATla, and ml and in3 mAChRs), but not receptors that signal through Gi only (Edg2 and m2 mAChR), for which only small agonist responses were observed. This suggests that different types of G-protein-coupled receptors have different potentials to activate P L D I ) Cell Maitltenance. Our laboratory uses HEK-293 and COS-7 cells to study the regulation of PLD through G-protein-coupled receptors. The cells are maintained in complete Dulbecco's modified Eagle's meditnn (DMEM) supplemented with l(Y/c fetal calf serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). In stone cases, we use HEK-293 cells stably expressing the m2 and m3 mAChRs to study lhe signaling. Those cells are maintained in DMEM containing G418 (0.5 rng/ml ). T/'an,sj?ctiott aml Stimulation o[ CelLs I. On day 1 (morning), the cells are passed in 35-ram dishes (3-4 x 105 ceils/ dish) and are grown for 2 0 - 2 4 hr. In some cases, especially when it is planned to transfect the HEK-293 cells with constitutively activated Rho, Rbo kinase, or A R E which can cause the cells to round up and detach from the tissue culture dishes, more reproducible assay data are achieved by using poly-L-lysine hydrobromide (Sigma, St. Louis, MO)-coated dishes instead of regular tissue culture (TC) dishes. For preparation of poly-L-lysine, dissolve it in sterile tissue culture-grade water at

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0.1 mg/ml and store it at -20~'; pipette I ml/dish and incubate overnight at room temperature; aspirate and rinse once with sterile tissue culture-grade water. Allow the plates to dry with the lid off (in a sterile laminar flow hood). The plates can be stored dry and sterile for months. 2. On day 2 (the cells should be ~ 7 0 - 8 0 % confluent), the cells are transfected in the morning with PLDI alleles and/or G-protein-coupled receptors. Prepare the tbllowing solutions in 12 x 75 mm sterile tubes. Solution A: For each transfection, dilute 1 # g of each DNA (plasmid) in 100 l~ 1 of Opti-MEM I containing 6-8/zl of PLUS reagent (Life Technologies) and incubate for 15 rain at room temperature Solution B: For each transfection, dilute 4-6/zl of Lipofectamine reagent in 100/~l of Opti-MEM I (the cocktails of Opti-MEM I-diluted PLUS and LipofectAMINE reagent can be made and added to the individual tubes in advance of the DNA) Combine the DNA and lipid solutions, mix gently, and incubate at room temperature for 15 min. Wash the cells once with 1 ml of Opti-MEM 1. For each transfection, add 0.8 ml of Opti-MEM I to each tube containing the lipid-DNA complexes. Mix gently and overlay the diluted complex solution onto the washed cells. Incubate the cells at 37' in a CO2 incubator. 3. Four to 5 hr posttransfection, the medium is replaced with complete DMEM and the cells are incubated for an additional 22-24 hr. For in vivo PLD assays, the transfection mixtures are replaced with complete DMEM containing 2/.tCi of [3H]palmitic acid (from American Radiolabeled Chemicals, St. Louis, MO): To prepare [3H]palmitic acid, 50 #1 of a 10-mCi/ml stock is dried in a Speed-Vac concentrator. The [3H]palmitic acid is then resuspended in 100 ~1 of sterile TE (5 mCi/ml) and sonicated for 30 sec at high power in a water sonicator before use. Use 0.4/~1 of resuspended [3H]palmitic acid per milliliter of medium. 4. On day 3, the cells are ready for PLD assays. Briefly, the cells are washed with 1 ml of warm, fresh Opti-MEM I once, and incubated in the same medium for 1 to 2 hr, after which the medium is replaced with fresh Opti-MEM I containing 0.3% (v/v) l-butanol and optionally various receptor agonists (the preparation and working concentration of the agonists are listed below). After 30 min, the stimulating medium is replaced with 300/zl of ice-cold methanol to halt further synthesis of the labeled lipid end product. The dishes are placed on ice and processed for the in vivo PLD assay, which has been described in a previous volume of this series. 9

Preparation of Agonists Carbachol (carbamylcholine chloride; Sigma): Stock solution, 400 raM; store at room temperature. Working concentration, 1 mM

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LPA (I-oleoyl-2-hydroxy-s/l-glycero-3-phosphate, 18:1 lyso-PA: Avanti. Alabaster, AL): Store in chloroform at 25 mg/ml at -20". May precipitate and need to be warmed for use. To prepare it for LPA receptors, dry down LPA under nitrogen gas, then resuspend in BSA (0.1 mg/ml in H20 or medium) (e.g,, Opti-MEM I), using sonication to make a 10 mM stock solution. Use a 100 #M final concentration to stimulate cells Angiotensin 1I (Sigma): Sleek solution, l0 tzM (dissolve in sterile H20), store at - 2 0 Working concentration, 100 nM

Mea,\urenwm of Pllm'pITolil~a.~e D Bimline to Sucrose-Loaded PhosT~holil)id ~Vsicle,s PLD 1 and PLD2 are extrinsic membrane proteins. Interaction of these enzymes with phospholipid bilayers is therelore important for their biological activity. The generation of P L D I alleles for dissecfion of G protein receptor-coupled regulation

raises the possibility that the activities could be altered if the mutations affect the interaction of PLD1 with the membrane itself. Binding of the PLD enzymes to artificial membranes can be measured with sucrose loaded phospholipid vesicles. The PLD proteins bind to vesicles coznposed of a equal molar ratio of PC • PS • PE with an aflinity of approximately 200 I*M. This affinity is not increased by inclusion of 5 mol% PI in the vesicles. However, PLD1 and PLD2 bind to vesicles containing 5 moV2~ PI(4,5)P2 with an affinity thai is at lcasl 10-fold higher. Structural integrity of a short region rich in basic amino acid residues is required for high-aflinity binding of the enzymes Io Pl(4,5)Pecontaining lipid vesicles. The basic procedure used is adapted from lhat described by Buscr and McLaughlin, n-~ and has been described previously for studies of PLD2. s In brief, large unilamellar vesicles are loaded with sucrose bufl'er and then incubaled with the PLD proteins in an isotonic buffer solution. The higher density of the vesicleencapsulated solution allow's the vesicles to be sedimented by ultracentrifugalion, The P I D binding is quantitated by either measurements of calalytic activity reroaming in the supernatant or by SDS-PAGE of vesicle-hound proteins. Ge'm'ralion qtSucro.~e-l,oad~'d Pho.v)ltolipid t,~'sicles

I. l,ipids are combined l:rom sleek solutions in CHCI3. In general, it is besl to prepare about twice tl~e a m o u l l l el" lipid required for lhe binding experiments because of losses during the procedure. TIle standard composition of lhe vesicles used in these experiments is I • I : 1 P C ' P S :PE. A trace qUalltily o1 I~HIPC is inchlded in the lipid mixture to lnolfilor lecl)\,'cly.

I: (.. A. Buscr and S. Mcl xmghlin, :~hvl~od~Mol. fliol. 84, 267 (I 9!)8I.

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2. The lipid mixture is dried completely in a small glass flask, using a rotary evaporator. Complete drying of the lipids is essential for success of the procedure. 3. The lipids are resuspended by vortex mixing in 176 mM sucrose, I mM morpholinepropanesulfonic acid (MOPS, pH 7.6) (sucrose buffer). In general, the total lipid concentration at this stage will be about 1 mM. The lipid suspension is subjected to five freeze-thaw cycles in a liquid N2 bath with thawing at 3 0 . 4. Vesicles are prepared by extruding the suspension through two 0. l-/,tm pore size polycarbonate filters, using either a hand-held extruder (Avanti Polar Lipids) or an N2 driven extruder (Lipex, Vancouver, Canada) according to the manufacturer directions. 5. The extruded vesicles are diluted 5-fold into 100 mM KCI, 1 mM MOPS (pH 7.0) (salt buffer), sedimented by centrifugation (100,000g for 30 min at 4 ) , and resuspended in the same buffer. A sample is taken for scintillation counting to determine the concentration of vesicles.

Measuring Binding o[ Phospholipase D Proteins to Sucrose-Loaded Vesicles 1, PLD proteins are incubated with the vesicles on ice in siliconized 1.5-ml microcentrifuge tubes. The vesicles are diluted in salt buffer to give a range of final concentrations. 2. The vesicles are sedimented by centrifugation at 100,000 g for 30 rain at 4 . PLD protein remaining in the supernatant is determined by measurement of enzymatic activity. Proteins associated with the sedimented vesicles are solublized in SDS-PAGE sample buffer and analyzed by SDS-PAGE and Western blotting.

Notes. An example of the PLD protein-vesicle interaction is visualized by Western blotting as a function of liposome concentration in Fig. 1D, using miniPLDI. Despite loss of the PH domain, the mini-PLD1 still interacts with the vesicles in a concentration- and (not shown) Pl(4,5)Pe-dependent manner. Acknowledgments This work was supported by grants from the NIH to M.A.F. (GM54813) and A.J.M. (GM50388). G.D. is a fellow of the American Heart Association.