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IDENTIFICATION OF TRANSPORT INTERMEDIATES
[33]
Acknowledgments L.H. was supported by a predoctoral fellowship from the National Science Foundation, and T.Y. by the Howard Hughes Medical Research Foundation. The work was supported by grants from the National Institutes of Health and the Howard Hughes Medical Research Foundation.
[33] P u r i f i c a t i o n o f S e c 4 P r o t e i n f r o m S a c c h a r o m y c e s Cerevisiae a n d Escherichia coli B y P E T E R NOVICK, M I C H E L L E D . G A R R E T T , PATRICK BRENNWALD, ALISA K . KABCENELL
and
Introduction Genetic analysis of protein export in the yeast Saccharomyces cerevisiae has identified a large number of genes whose protein products play essential roles in the many steps of transport of proteins from their site of synthesis on the endoplasmic reticulum to their ultimate release at the cell surface. In some cases, analysis of the sequence of the gene has revealed important clues to the possible mechanism of function of the gene product. One of the first such examples is the SEC4 gene, which is required at the final stage of transport to the cell surface) The sequence of SEC4 revealed that the encoded protein is a member of the ras superfamily, defined by the presence of conserved domains that together constitute a high-affinity guanine nucleotide-binding site. 2,a While all of the members of this superfamily share at least 30% sequence identify with each other, a subfamily, known commonly as the Rab proteins, share more extensive sequence identity with Sec4p2 Various lines of evidence suggest that members of the Rab protein family play roles analogous to Sec4p, regulating the many different vesicular transport events in eukaryotic cells: Sec4p undergoes a cycle of GTP binding and hydrolysis that may be coupled to a cycle of subcellular localization in which Sec4p first attaches to post-Golgi vesicles that go on to fuse with the plasma membrane. Sec4p then recycles through a soluble pool to reassociate with a new round of vesicles. Membrane attachment of Sec4p, Yptlp, and the many Rab ho-
P. Novick, C. Field, and R. Schekman, Cell 21, 205 (1980). 2 A. Salminen and P. Novick, Cell49, 527 (1987). 3 A. Valencia, P. Chardin, A. Wittinghofer, and C. Sander, Biochemistry 30, 4637 (1991). 4 W. E. Balch, Trends Biochem. Sci. 15, 473 (1990).
METHODSIN ENZYMOIX~Y, VOL. 219
Copyright© 1992by AcademicPress,Inc. Allfightsof r~productionin any formreserved.
[33]
PURIFICATIONOF See4 PROTEIN
353
mologs requires prior modification of carboxy-terminal cysteine moieties by the addition of isoprenyl lipids. One approach toward understanding the role of Sec4p and its many homologs is to pursue the accessory proteins that function in conjunction with them. These include proteins that stimulate the hydrolysis of bound GTP, 5,~ proteins that stimulate the release of bound GDP allowing exchange for GTP, proteins that modify the carboxy terminus to facilitate membrane association, 7 and proteins that allow dissociation from membranes for recycling, s To assay these accessory proteins it is useful to have available large amounts of pure protein. We present procedures here for producing and purifying large amounts of native Sec4p from either yeast or bacteria. The purification of Sec4p from yeast has been previously described 9 and the purification of Sec4 from bacteria has been adapted from the yeast protocol. Because bacteria lack prenylation enzymes, bacterially produced Sec4p is a convenient substrate for modification studies. Expression of Sec4p in S a c c h a r o m y c e s cerevisiae and Preparation of a Cell L y s a t e To produce large amounts of See4 protein in yeast, the gene is expressed under the control of the G A L l promoter. This promoter is strongly transcribed in the presence of galactose, but in the absence of the inducer expression is greatly reduced. ~° This is important because high levels of overexpression of Sec4p are detrimental to yeast and will ultimately arrest growth. The S E C 4 gene was placed under control of the G A L l promoter by first trimming the upstream noncoding region of S E C 4 to only 8 base pairs (bp) and then ligating it into the B a m H I site of the expression vector pNBI87. This construction has been described in detail. ~ Because this vector is maintained at only single copy number in yeast, the G A L 1 - S E C 4 construction was subcloned into YEp 13, a multicopy yeast vector containing the L E U 2 gene as a selectable marker ~2 to further increase the level of expression. The construction of this plasmid, designated pNB283 (see Fig. IA) has been described in detail. 9 pNB283 was introduced into the yeast strain NY603 ( M A T a leu2-3, 112 ura3-52 G A L + pep4::URA3) by alkali 5j. Becker,T. Tan, H. Trepte, and D. Gallwitz, Eur. Mol. Biol. J. 10, 785 (1991). 6E. S. Burnstein, K. Linko-Stentz, Z. Lu, and I. Macara, J. Biol. Chem. 266, 2689 (1991). 7G. Rossi,Y. Jiang, A. Newman, and S. Ferro-Novick,Nature (London) 351, 158 (1991). s S. Araki, A. Kikuchi, Y. Hata, M. Isomura, and Y. Takai, J. Biol. Chem. 265, 13007 (1990). 9 A. K. Kabeenell,B. Goud, J. Northup, and P. Novick,J. Biol. Chem. 265, 9366 (1990). ~oj. C. Schneiderand L. Guarente, this series, Vol. 194,p. 373. 11B. Goud, A. Salminen, N. C. Walworth, and P. Novick, Cell 53, 753 (1988). ~2j. R. Broach, J. N. Stathern, and J. B. Hicks, Gene $, 121 (1979).
354
IDENTIFICATION OF TRANSPORT INTERMEDIATES
,
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, 2,o
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pNB412 6.56 kb ~.b6 KD J
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~ S a l l 1.90
/ Pvu II 2.80 Sal I 3.10 FI~. 1. (A) Yeast See4 expression vector pNB238. The SEC4 gene has been inserted behind the GALl promoter on a vector maintained at high copy number in yeast. (B) Bacterial expression vector pNB412. The SEC4 gene has been ins~ed between the T7 promotor and terminator (Term).
[33]
PURIFICATIONOF See4 PROTEIN
355
cation treatment 13 and transformants were selected at 25 ° on minimal medium containing 0.7% (w/v) yeast nitrogen base without amino acids (Difco, Detroit, MI) and supplemented with 2% (w/v) glucose. A recipient strain harboring a disruption of the PEP4 gene was utilized as PEP4 encodes the vacuolar hydrolase protease A, which has been implicated in the activation of several other vacuolar zymogens?4,t5 Elimination of this enzyme, therefore, reduces the endogenous protease activity in a yeast lysate. Each time the resulting strain, NY671, is grown from a frozen stock it should be screened for the maintenance of the Pep4- phenotype by assaying for the absence of carboxypeptidase Y activity? 6 A stationary culture is prepared by inoculating 25 ml of minimal medium containing 2.5% (w/v) raffinose [from a 10% (w/v) stock] with a single colony of NY671 and incubating for 4 days at 25 ° with aeration. Raffinose will not induce the GALl promoter, nor will it interfere with rapid induction following subsequent transfer to galactose medium. This stationary culture is then used to inoculate 2 liters of minimal medium supplemented with raffinose and growth is continued at 25 o with aeration for 21 hr. The Atop of this exponential culture should be between 0.6 and 2.0. To induce expression of the episomal SEC4 gene, 1600 A60o units of the cell suspension is pelleted in the GH-3.7 rotor of a Beckman (Palo Alto, CA) GPR centrifuge by spinning for 10 min at 3000 rpm at room temperature, and the cells are resuspended in 4 liters of minimal medium containing 2% (w/v) galactose [from a 20% (w/v) stock], and incubated at 25 ° with shaking. After 24 hr of incubation, the cells, at an Atop of 1.3- 1.5, are harvested as above and washed once with deionized water. All subsequent steps are performed at 4 ° . The cell pellet is resuspended in 20 ml of lysis buffer [0.3 M sorbitol, 20 m M Tris-HCl, pH 8.0, 10 m M NaCI, 5 m M MgC12, 1 m M dithiothreitol (DTT), 1 m M phenylmethylsulfonyl fluoride (PMSF), and 1 #g/ml each of antipain, leupeptin, aprotinin, chymostatin, and pepstatin A] at 0 - 4 ° . The cell suspension is added to the prechilled 85-ml chamber of a Bead-beater (BioSpec Products, Bartlesville, OK) half-filled with 0.5-mm glass beads. Additional lysis buffer is added to fill the chamber completely and thereby exclude all air. The chamber is sealed, surrounded by an ice water bath, and the cells are homogenized for a total of 3 min in six 30-sec ~3H. Ito, Y. Fukada, K. Murata, and A. Kimura, J. Bacteriol. 153, 163 (1983). 14G. Ammer, C. P. Hunter, J. H. Rothman, G. C. Saari, L. A. Vails, and T. H. Stevens, Mol. Cell. Biol. 6, 2409 (1986). '~ C. A. Woolford, L. B. Daniels, F. J. Park, E. W. Jones, J. N. Van ArsdeU, and M. A. Innis, Mol. Cell, Biol. 6, 2500 (1986). 16 E. W. Jones, this series, Vol. 194, p. 428.
356
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[33]
intervals. Between each 30-sec interval the chamber should be allowed to cool for 3 min. For a detailed discussion on the use of the BioSpec Bead-beater see Jazwinski. 17 The resulting lysate is recovered from the chamber, centrifuged at 3500 rpm for 10 min to remove unbroken cells and debris, and the supernatant spun in a Beckman Ti70 rotor at 34,000 rpm (90,000 g) for 60 min to pellet membranes. A layer of lipid forms at the top of the tubes and should be discarded. The high-speed supernatant should contain approximately 400 mg protein in a volume of 30-40 ml and can be used immediately or rapidly frozen in liquid nitrogen and stored at - 8 0 °. Expression of Sec4 Protein in Escherichia coli and Preparation of a Cell Lysate To express native Sec4 protein in Escherichia coli, we use the T7 expression system. 18 By this system the coding sequence of interest is inserted behind a T7 RNA polymerase promoter and then introduced into a bacterial strain containing a T7 RNA polymerase gene under control of the inducible lacUV5 promoter. Production ofT7 RNA polymerase in this strain is induced with isopropylthio-fl-D-galactoside (IPTG), which in turn yields high-level expression of the foreign gene. We have made use of the polymerase chain reaction (PCR) to engineer a restriction site such that we could ligate the SEC4 coding sequence in the optimal position behind the T7 promoter of the expression vector pET 1 ld. This vector contains the gene 10 ribosome-binding site with an NcoI site at the initiating AUG and a strong transcriptional terminator following the BamHI site. Two oligonucleotides have been employed for amplifying SEC4: one incorporates a BspHI site at the initiating AUG and the second incorporates a BamHI site downstream of the SEC4 termination codon. A BspHI site was chosen in primer I because digestion with this enzyme yields an overhang that is compatible with the NcoI site in pET1 ld and retains a serine as the second residue. The resulting construction is then used to transform BL2 I(DE3) cells that contain the IPTG-inducible T7 RNA polymerase gene. The transformants are plated on ZB (10 g Bacto-tryptone, 5 g NaC1, 15 g agar per liter) plates containing 100/lg/ml of ampicillin (Amp) and grown overnight at 37 °. To test the transformants for expression of Sec4p, multiple independent colonies are transferred from the ZB-Amp plate into 5 ml of ZB supplemented with 100/lg/ml ampicillin (Boehringer Mannheim, Indiat7 S. M. Jazwinski, this series, Vol. 182, p. 154. 18 F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, this series, Vol. 185, p. 60.
[33]
PURIFICATION OF Sec4 PROTEIN
357
napolis, IN) and grown overnight at 37 ° until freshly saturated (less than 12 hr). Frozen stocks are prepared and stored at - 8 0 ° so that all subsequent cultures can be started directly from them. Fifty microliters of each overnight culture is then inoculated into 5 ml of ZB-Amp and grown at 37 ° for 3 - 4 hr until the A60ois between 0.4 and 0.6. IPTG is added to each tube to a final concentration of 0.4 m_M and cultures incubated at 37 ° for 2 hr with good aeration. One milliliter of each culture is harvested by centrifugation for 30 sec at top speed in a microfuge and the pellet is resuspended in 150 #1 of sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 rain. These samples are analyzed for expression of Sec4 protein by SDS-polyacrylamide gel electrophoresis (PAGE) on a 15% (w/v) SDSpolyacrylamide gel, which is then stained with Coomassie Brilliant blue. All four transformants that we analyzed showed some expression of a protein of the predicted size of Sec4p, but the levels varied quite dramatically. The identity of the overexpressed protein can be confirmed by immunoblots using anti-Sec4p sera. High-level expression of proteins in bacteria can lead to formation of an insoluble aggregate. Therefore the transformants showing the best expression of Sec4p are tested for solubility of the protein at varying temperatures and times of induction. For each transformant, two 50-ml cultures are prepared by inoculating 100 ml of ZB-Amp with 1 ml of an overnight culture and growing one 50-ml aliquot at 30 ° and the other at 37 °, until the A60oof the cells is between 0.4 and 0.6. Induction is then started by the addition of IPTG (0.4 mM, final concentration) and growth is continued at the same temperature with the removal of 5-ml aliquots from each culture 1, 2, and 3 hr after the start of induction. These samples are harvested by centrifugation in the GH-3.7 rotor of a Beckman GPR centrifuge at 3000 rpm at 4 °. The pellets are resuspended in ice-cold lysis buffer (20 m M Tris-HC1, pH 8.0, 100 m M NaC1, 5 m M MgC12, 1 m M DTT, 1 m M PMSF, and 1 gg/ml each of antipain, leupeptin, aprotinin, chymostatin, and pepstatin A) and transferred to 1.5-ml microfuge tubes. The samples are then sonicated on ice for 30 sec, in three 10-sec intervals with cooling on ice for I min between each 10-sec sonication (we used a Sonifier cell disruptor (Branson, Danbury, CT) model W185 on setting 5.5). The resulting sonicate is centrifuged at top speed in a microfuge and the pellet resuspended in a volume of lysis buffer equal to that of the sonic,ate supernatant. Equal volumes of sonic,ate supernatant and pellet fractions can then be analysed by SDS-PAGE and Coomassie Brilliant blue staining of the gel. We found that expression of Sec4p in the sonicate supernatant (i.e., soluble Sec4p) was different for the two transformants that we tested and also varied with the temperature and time of induction. Consequently, for the transformant expressing the greatest amount of soluble Sec4p (strain
358
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[33]
NRB412d), we found that the optimal conditions for expression of this protein were growth of the cells at 30 ° and induction with IPTG for 1 hr at 30*. To produce a larger scale induction of Sec4p, 5 ml of ZB medium containing 0.1 mg/ml ampicillin is inoculated from a stock of the strain NRB412d stored at - 8 0 ° and grown overnight at 37 ° with aeration. This is then used to inoculate 500 ml of ZB broth containing 0.1 mg/ml ampicillin and growth is continued at 30 ° with aeration until the Ac,oois between 0.4 and 0.6. To induce expression of the SEC4 gene, 0.4 rnM IPTG (final concentration) is added to the culture and growth is continued for 1 hr at 30 ° with aeration. Following this induction the cells are harvested by centrifugation for 10 min at 5000 rpm at 4 ° (we used a Beckman J2.21 centrifuge with a JA-10 rotor). The cell pellet can be used immediately or rapidly frozen and stored at -80*. The pelleted cells are resuspended in 7 ml of ice-cold lysis buffer and the cell suspension is sonicated on ice for 6 rain, in three 2-rain intervals with cooling on ice for 1 min between each sonication. The resulting sonicate is then centrifuged at 11,000 rpm for 15 rain at 4 ° (we used a Beckman J2.21 centrifuge with a JA-20 rotor) to remove unbroken cells and debris. The supernatant should contain approximately 60 mg of protein and should be used immediately. Guanine Nucleotide-Binding Assay GTP-binding activity is used to monitor the Sec4 protein throughout its purification. While in principle this assay does not uniquely detect Sec4 because there are many other GTP-binding proteins in both S. cerevisiae and E. coli lysates, they are, in fact, quite minor in abundance with respect to Sec4p due to the high level of expression achieved. This is reflected by the observation that the level of soluble GTP-binding activity increases over 40-fold on Sec4p induction in yeast and over 150-fold on Sec4p induction in E. coli. Guanine nucleotide binding is assayed by filtration. ~9 Briefly, 5/zl of column fractions is diluted into 20 gl of buffer A [50 m M sodium 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 5 m M MgC12, pH 8.0, 200 m M NaCl, 1 m M EDTA, 1 m M DTT, and 0.1% (w/v) Lubrol 12A9]. The binding reaction is initiated with the addition of 25/zl of buffer A containing 4 g M GTPTS (Boehringer Mannheim, Indianapolis, IN) and [3~S]GTPTS (New England Nuclear, Boston, MA) at a specific activity of approximately 5000 cpm/pmol. Following a 60-rain incubation at 30 °, the reaction mixtures are diluted with 4 ml of ice-cold ~9j. K. Northup, M. D. Smigel, and A. G. Gilman, J. Biol. Chem. 257, 11416 (1982).
[33]
PURIFICATIONOF See4 PgOTmN
359
20 m M Tris-HC1, pH 8.0, 100 m M NaC1, 25 m M MgC12, and rapidly filtered through 25-mm type HA filters (Millipore, Bedford, MA). A sampling manifold and vaccuum pressure pump (Millipore) are convenient in this regard. Wild-type Sec4p isolated from either yeast or bacteria is predominantly bound to GDP. Because the off-rate of GDP from Sec4p is only about 4 rain in the presence of 5 m M MgCI2 at 30 °, no preincubation under conditions of micromolar MgC12 is necessary to increase the rate of nucleotide exchange, as it is with other members of the r a s superfamily. The filters are washed five times with 2 ml of cold buffer, dried under a heat lamp, and counted in 10 ml of Ecoscint scintillation fluid (National Diagnostics, Manville, N J). The specific radioactivity of the nucleotide is determined by diluting the radioactive mixture 1 : 80 and spotting 20 #1 ( 1 pmol) onto each of three filters, which are then dried and counted as above. Purification of Sec4p from Yeast Gel filtration is an effective initial purification step because Sec4p is a small protein that, when overproduced, accumulates in a soluble, monomeric form. Approximately 350 mg of protein derived from a high-speed supernatant fraction of NY671 cells in a volume of 30 ml is applied to an 800-ml Sephacryl S-100 column (Pharmacia, Piscataway, NJ; 5 × 44 cm). The column is eluted with TMD buffer (20 m M Tris-HC1, pH 8.0, 5 m M MgC12, 1 m M DTT) supplemented with 100 m M NaC1, and 9-ml fractions are collected. It is convenient, but not essential, to follow the A280 with an in-line UV detector and chart recorder. The peak of [35S]GTPTSbinding activity elutes as a symmetrical peak after the bulk of other cell proteins (Fig. 2). Relative to the yeast high-speed supernatant, this purification step gives approximately 20-fold purification with 82% recovery of activity in a volume of 38 ml (see Table I). Sec4p binds only weakly to DEAE-Sephacel (under our buffer conditions). Thus the second stage of the purification involves a step in which Sec4p fails to bind to this ion-exchange resin while a number of contaminating proteins are retained. The pooled Sephacryl S-100 fractions are combined and then diluted twofold with an equal volume of TMD buffer to a final concentration of 50 m M NaCl. It is then loaded onto a 50-ml DEAE-Sephacel column (2.5 × I 1.5 cm; Pharmacia) equilibrated in TMD buffer with 50 m M NaC1. The column is washed with this buffer, and 4.4-ml fractions are collected. The flow-through and wash fractions containing protein as measured by absorbance at 280 nm are combined. It is not necessary to assay for GTPTS binding during this step. In the final stage of purification the NaCI concentration is reduced to
360
IDENTIFICATION OF TRANSPORT INTERMEDIATES
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FIG. 2. Top." Elution profile of See4 activity (nmol GTPyS bound/ral) in a yeast lysate from a Sephacryl S-100 column. Bottom." Elution profile of S¢c4 activity (nmol GTPyS bound/ml) from a DEAE-Sephacel column on elution with a gradient of NaC1.
the level at which Sec4p is retained on a DEAE-Sephacel column and it is then eluted with an NaC1 gradient. To lower the NaCI concentration without diluting the protein to the extent that Sec4p is denatured the pool is first concentrated to 5 ml by pressure titration in a stirred cell containing an Amicon (Danvers, MA) PM10 membrane and then diluted fivefold with TMD buffer for a final NaC1 concentration of I 0 mM. The pool is applied to a 30-ml DEAE-Sephacel column (2.5 × 7 cm) equilibrated in T M D buffer with 10 m M NaC1, and the adsorbed protein is eluted with a 150-ml linear gradient of 1 0 - 5 0 m M NaC1 in TMD buffer. Fractions of
[33]
PURIFICATIONOF See4 PROTEIN
PURIFICATION OF
TABLE I Sec4 FROM YEASTAND
361
BACTERIA
GTP~Sbinding Source and fraction Yeast Solublelysate S-100 DEAE flow-through DEAE pool Bacteria Soluble lysate S-100 DEAE flow-through
Total protein (nag)
Total Total Specific volume activity activity (ml) ( n m o l ) (nmol/mg)
350.0 14.8 7.9 2.1
40.0 38.0 95.0 2.8
60 2.9 1.6
6.0 8.0 1.5
232 191 159 65 21.8 24.9 19.5
Yield (%)
Purification (-fold)
0.7 12.9 20.2 30.9
100 82 69 28
1 20 31 47
0.36 8.6 12.1
100 114 89
1 24 34
2.8 ml are collected and alternate fractions are assayed for GTPyS-binding activity. The peak of GTPyS-binding activity elutes at approximately 20 m M NaC1 and is pooled and concentrated approximately 10-fold by pressure filtration in a stirred cell. Prior to storage at - 8 0 °, the purified Sec4 protein should be diluted one-fourth with 6.4 m M dipalmitoylphosphatidylcholine (Sigma, St. Louis, MO), 32 m M 3-[3-cholamidopropyldimethylammonio]-l-propane sulfonate (CHAPS; Calbiochem, San Diego, CA), and 50 m M NaC1 in T M D buffer and then rapidly frozen in small aliquots in sialyzed glass tubes. The addition of detergent and phospholipid appears to stabilize Sec4p against repeated freeze-thaw cycles. The final preparation contains only a single species as visualized by SDS-PAGE and silver staining. The overall purification is typically 47-fold with a yield of approximately 2 mg. Purification of Sec4p from Escherichia coli This procedure is performed as described above for purification of Sec4p from yeast except that column sizes are scaled down because the lysate is prepared from only 500 ml of cells and the final ion-exchange column is omitted. The 6 ml of supernatant (from centrifugation of the sonicate at 13,800 g) containing approximately 60 mg of protein is applied to a 170-ml Sephacryl S-100 column (1.5 cm X 96 cm; Pharmacia). The column is eluted with T M D buffer supplemented with 100 m M NaCI, 2.2-ml fractions are collected and the peak of GTPyS-binding activity
362
IDENTIVnCATmN OF TRANSPORT INTERMEDIATES
[34]
pooled. This step gives approximately 24-fold purification with excellent recovery of GTPyS-binding activity in a volume of 8 ml (see Table I). The pooled fractions are then diluted with an equal volume of TMD buffer to give a final concentration of 50 m M NaC1. This is loaded onto a 12-ml DEAE-Sephacel column (2.5 × 2.5 cm) equilibrated with TMD buffer containing 50 m M NaC1. The column is washed with the same buffer until the absorbance at 280 nm returns to baseline. The peak of GTP~,S-binding activity is pooled and concentrated approximately 18-fold to 1.5 ml by pressure filtration in a stirred cell containing an Amicon PM 10 membrane. The protein is then rapidly frozen and stored at - 80 o. The overall purification is approximately 34-fold (see Table I), with a yield of about 500/tg of active protein and a purity of approximately 80-90% as analyzed by SDS-PAGE and Coomassie Brilliant blue staining of the gel. The GDP off-rate, GTP on-rate, and intrinsic GTP hydrolysis rate of the bacterially produced Sec4p are quite similar to those found for Sec4p isolated from yeast. Acknowledgments This work was supported by Grants GM35370 and CA46218 to P.N. from the National Institutes of Health. M.G. was supported by the Lucille P. Markey Charitable Trust, P.B. was supported by the Damon Runyon Walter Winchell Cancer Research Fund, and A.K.K. was supported by the Jane Coffin Childs Memorial Fund for Medical Research and by a Swebilius Cancer Research Award.
[34] P r e p a r a t i o n o f R e c o m b i n a n t ADP-Ribosylation Factor
By PAUL A. RANDAZZO, OFRA WEISS, and RICHARD A. KArIN Introduction ADP-ribosylation factor (ARP0 proteins were originally identified and purified based on an in vitro activity as the protein cofactor required for efficient ADP-ribosylation of Gs by cholera toxin ~ (for recent reviews see Kahn3,4). Subsequently, ARF was shown to be a 21-kDa GTP-binding t L. S. Schleifer, R. A. Kahn, E. Hansld, J. K. Northup, P. C. Sternweis, and A. G. Gilman, J. Biol. Chem. 257, 20 (1982). 2 R. A. Kahn and A. G. Gilman, J. Biol. Chem. 259, 6228 (1984). R. A. Kahn, in "G Proteins" (L. Birnbaumer and R. Iyengar, eds.), p. 201. Academic Press, Orlando, Florida, 1990. 4 R. A. Kahn, this series, Vol. 195, p. 233.
METHODSIN ENZYMOLOGY,VOL 219
Copyright© 1992by AcademicPress,Inc. Allrightsof reproduction in any formreserved.
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[33]
Acknowledgments L.H. was supported by a predoctoral fellowship from the National Science Foundation, and T.Y. by the Howard Hughes Medical Research Foundation. The work was supported by grants from the National Institutes of Health and the Howard Hughes Medical Research Foundation.
[33] P u r i f i c a t i o n o f S e c 4 P r o t e i n f r o m S a c c h a r o m y c e s Cerevisiae a n d Escherichia coli B y P E T E R NOVICK, M I C H E L L E D . G A R R E T T , PATRICK BRENNWALD, ALISA K . KABCENELL
and
Introduction Genetic analysis of protein export in the yeast Saccharomyces cerevisiae has identified a large number of genes whose protein products play essential roles in the many steps of transport of proteins from their site of synthesis on the endoplasmic reticulum to their ultimate release at the cell surface. In some cases, analysis of the sequence of the gene has revealed important clues to the possible mechanism of function of the gene product. One of the first such examples is the SEC4 gene, which is required at the final stage of transport to the cell surface) The sequence of SEC4 revealed that the encoded protein is a member of the ras superfamily, defined by the presence of conserved domains that together constitute a high-affinity guanine nucleotide-binding site. 2,a While all of the members of this superfamily share at least 30% sequence identify with each other, a subfamily, known commonly as the Rab proteins, share more extensive sequence identity with Sec4p2 Various lines of evidence suggest that members of the Rab protein family play roles analogous to Sec4p, regulating the many different vesicular transport events in eukaryotic cells: Sec4p undergoes a cycle of GTP binding and hydrolysis that may be coupled to a cycle of subcellular localization in which Sec4p first attaches to post-Golgi vesicles that go on to fuse with the plasma membrane. Sec4p then recycles through a soluble pool to reassociate with a new round of vesicles. Membrane attachment of Sec4p, Yptlp, and the many Rab ho-
P. Novick, C. Field, and R. Schekman, Cell 21, 205 (1980). 2 A. Salminen and P. Novick, Cell49, 527 (1987). 3 A. Valencia, P. Chardin, A. Wittinghofer, and C. Sander, Biochemistry 30, 4637 (1991). 4 W. E. Balch, Trends Biochem. Sci. 15, 473 (1990).
METHODSIN ENZYMOIX~Y, VOL. 219
Copyright© 1992by AcademicPress,Inc. Allfightsof r~productionin any formreserved.
[33]
PURIFICATIONOF See4 PROTEIN
353
mologs requires prior modification of carboxy-terminal cysteine moieties by the addition of isoprenyl lipids. One approach toward understanding the role of Sec4p and its many homologs is to pursue the accessory proteins that function in conjunction with them. These include proteins that stimulate the hydrolysis of bound GTP, 5,~ proteins that stimulate the release of bound GDP allowing exchange for GTP, proteins that modify the carboxy terminus to facilitate membrane association, 7 and proteins that allow dissociation from membranes for recycling, s To assay these accessory proteins it is useful to have available large amounts of pure protein. We present procedures here for producing and purifying large amounts of native Sec4p from either yeast or bacteria. The purification of Sec4p from yeast has been previously described 9 and the purification of Sec4 from bacteria has been adapted from the yeast protocol. Because bacteria lack prenylation enzymes, bacterially produced Sec4p is a convenient substrate for modification studies. Expression of Sec4p in S a c c h a r o m y c e s cerevisiae and Preparation of a Cell L y s a t e To produce large amounts of See4 protein in yeast, the gene is expressed under the control of the G A L l promoter. This promoter is strongly transcribed in the presence of galactose, but in the absence of the inducer expression is greatly reduced. ~° This is important because high levels of overexpression of Sec4p are detrimental to yeast and will ultimately arrest growth. The S E C 4 gene was placed under control of the G A L l promoter by first trimming the upstream noncoding region of S E C 4 to only 8 base pairs (bp) and then ligating it into the B a m H I site of the expression vector pNBI87. This construction has been described in detail. ~ Because this vector is maintained at only single copy number in yeast, the G A L 1 - S E C 4 construction was subcloned into YEp 13, a multicopy yeast vector containing the L E U 2 gene as a selectable marker ~2 to further increase the level of expression. The construction of this plasmid, designated pNB283 (see Fig. IA) has been described in detail. 9 pNB283 was introduced into the yeast strain NY603 ( M A T a leu2-3, 112 ura3-52 G A L + pep4::URA3) by alkali 5j. Becker,T. Tan, H. Trepte, and D. Gallwitz, Eur. Mol. Biol. J. 10, 785 (1991). 6E. S. Burnstein, K. Linko-Stentz, Z. Lu, and I. Macara, J. Biol. Chem. 266, 2689 (1991). 7G. Rossi,Y. Jiang, A. Newman, and S. Ferro-Novick,Nature (London) 351, 158 (1991). s S. Araki, A. Kikuchi, Y. Hata, M. Isomura, and Y. Takai, J. Biol. Chem. 265, 13007 (1990). 9 A. K. Kabeenell,B. Goud, J. Northup, and P. Novick,J. Biol. Chem. 265, 9366 (1990). ~oj. C. Schneiderand L. Guarente, this series, Vol. 194,p. 373. 11B. Goud, A. Salminen, N. C. Walworth, and P. Novick, Cell 53, 753 (1988). ~2j. R. Broach, J. N. Stathern, and J. B. Hicks, Gene $, 121 (1979).
354
IDENTIFICATION OF TRANSPORT INTERMEDIATES
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/ Pvu II 2.80 Sal I 3.10 FI~. 1. (A) Yeast See4 expression vector pNB238. The SEC4 gene has been inserted behind the GALl promoter on a vector maintained at high copy number in yeast. (B) Bacterial expression vector pNB412. The SEC4 gene has been ins~ed between the T7 promotor and terminator (Term).
[33]
PURIFICATIONOF See4 PROTEIN
355
cation treatment 13 and transformants were selected at 25 ° on minimal medium containing 0.7% (w/v) yeast nitrogen base without amino acids (Difco, Detroit, MI) and supplemented with 2% (w/v) glucose. A recipient strain harboring a disruption of the PEP4 gene was utilized as PEP4 encodes the vacuolar hydrolase protease A, which has been implicated in the activation of several other vacuolar zymogens?4,t5 Elimination of this enzyme, therefore, reduces the endogenous protease activity in a yeast lysate. Each time the resulting strain, NY671, is grown from a frozen stock it should be screened for the maintenance of the Pep4- phenotype by assaying for the absence of carboxypeptidase Y activity? 6 A stationary culture is prepared by inoculating 25 ml of minimal medium containing 2.5% (w/v) raffinose [from a 10% (w/v) stock] with a single colony of NY671 and incubating for 4 days at 25 ° with aeration. Raffinose will not induce the GALl promoter, nor will it interfere with rapid induction following subsequent transfer to galactose medium. This stationary culture is then used to inoculate 2 liters of minimal medium supplemented with raffinose and growth is continued at 25 o with aeration for 21 hr. The Atop of this exponential culture should be between 0.6 and 2.0. To induce expression of the episomal SEC4 gene, 1600 A60o units of the cell suspension is pelleted in the GH-3.7 rotor of a Beckman (Palo Alto, CA) GPR centrifuge by spinning for 10 min at 3000 rpm at room temperature, and the cells are resuspended in 4 liters of minimal medium containing 2% (w/v) galactose [from a 20% (w/v) stock], and incubated at 25 ° with shaking. After 24 hr of incubation, the cells, at an Atop of 1.3- 1.5, are harvested as above and washed once with deionized water. All subsequent steps are performed at 4 ° . The cell pellet is resuspended in 20 ml of lysis buffer [0.3 M sorbitol, 20 m M Tris-HCl, pH 8.0, 10 m M NaCI, 5 m M MgC12, 1 m M dithiothreitol (DTT), 1 m M phenylmethylsulfonyl fluoride (PMSF), and 1 #g/ml each of antipain, leupeptin, aprotinin, chymostatin, and pepstatin A] at 0 - 4 ° . The cell suspension is added to the prechilled 85-ml chamber of a Bead-beater (BioSpec Products, Bartlesville, OK) half-filled with 0.5-mm glass beads. Additional lysis buffer is added to fill the chamber completely and thereby exclude all air. The chamber is sealed, surrounded by an ice water bath, and the cells are homogenized for a total of 3 min in six 30-sec ~3H. Ito, Y. Fukada, K. Murata, and A. Kimura, J. Bacteriol. 153, 163 (1983). 14G. Ammer, C. P. Hunter, J. H. Rothman, G. C. Saari, L. A. Vails, and T. H. Stevens, Mol. Cell. Biol. 6, 2409 (1986). '~ C. A. Woolford, L. B. Daniels, F. J. Park, E. W. Jones, J. N. Van ArsdeU, and M. A. Innis, Mol. Cell, Biol. 6, 2500 (1986). 16 E. W. Jones, this series, Vol. 194, p. 428.
356
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[33]
intervals. Between each 30-sec interval the chamber should be allowed to cool for 3 min. For a detailed discussion on the use of the BioSpec Bead-beater see Jazwinski. 17 The resulting lysate is recovered from the chamber, centrifuged at 3500 rpm for 10 min to remove unbroken cells and debris, and the supernatant spun in a Beckman Ti70 rotor at 34,000 rpm (90,000 g) for 60 min to pellet membranes. A layer of lipid forms at the top of the tubes and should be discarded. The high-speed supernatant should contain approximately 400 mg protein in a volume of 30-40 ml and can be used immediately or rapidly frozen in liquid nitrogen and stored at - 8 0 °. Expression of Sec4 Protein in Escherichia coli and Preparation of a Cell Lysate To express native Sec4 protein in Escherichia coli, we use the T7 expression system. 18 By this system the coding sequence of interest is inserted behind a T7 RNA polymerase promoter and then introduced into a bacterial strain containing a T7 RNA polymerase gene under control of the inducible lacUV5 promoter. Production ofT7 RNA polymerase in this strain is induced with isopropylthio-fl-D-galactoside (IPTG), which in turn yields high-level expression of the foreign gene. We have made use of the polymerase chain reaction (PCR) to engineer a restriction site such that we could ligate the SEC4 coding sequence in the optimal position behind the T7 promoter of the expression vector pET 1 ld. This vector contains the gene 10 ribosome-binding site with an NcoI site at the initiating AUG and a strong transcriptional terminator following the BamHI site. Two oligonucleotides have been employed for amplifying SEC4: one incorporates a BspHI site at the initiating AUG and the second incorporates a BamHI site downstream of the SEC4 termination codon. A BspHI site was chosen in primer I because digestion with this enzyme yields an overhang that is compatible with the NcoI site in pET1 ld and retains a serine as the second residue. The resulting construction is then used to transform BL2 I(DE3) cells that contain the IPTG-inducible T7 RNA polymerase gene. The transformants are plated on ZB (10 g Bacto-tryptone, 5 g NaC1, 15 g agar per liter) plates containing 100/lg/ml of ampicillin (Amp) and grown overnight at 37 °. To test the transformants for expression of Sec4p, multiple independent colonies are transferred from the ZB-Amp plate into 5 ml of ZB supplemented with 100/lg/ml ampicillin (Boehringer Mannheim, Indiat7 S. M. Jazwinski, this series, Vol. 182, p. 154. 18 F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, this series, Vol. 185, p. 60.
[33]
PURIFICATION OF Sec4 PROTEIN
357
napolis, IN) and grown overnight at 37 ° until freshly saturated (less than 12 hr). Frozen stocks are prepared and stored at - 8 0 ° so that all subsequent cultures can be started directly from them. Fifty microliters of each overnight culture is then inoculated into 5 ml of ZB-Amp and grown at 37 ° for 3 - 4 hr until the A60ois between 0.4 and 0.6. IPTG is added to each tube to a final concentration of 0.4 m_M and cultures incubated at 37 ° for 2 hr with good aeration. One milliliter of each culture is harvested by centrifugation for 30 sec at top speed in a microfuge and the pellet is resuspended in 150 #1 of sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 rain. These samples are analyzed for expression of Sec4 protein by SDS-polyacrylamide gel electrophoresis (PAGE) on a 15% (w/v) SDSpolyacrylamide gel, which is then stained with Coomassie Brilliant blue. All four transformants that we analyzed showed some expression of a protein of the predicted size of Sec4p, but the levels varied quite dramatically. The identity of the overexpressed protein can be confirmed by immunoblots using anti-Sec4p sera. High-level expression of proteins in bacteria can lead to formation of an insoluble aggregate. Therefore the transformants showing the best expression of Sec4p are tested for solubility of the protein at varying temperatures and times of induction. For each transformant, two 50-ml cultures are prepared by inoculating 100 ml of ZB-Amp with 1 ml of an overnight culture and growing one 50-ml aliquot at 30 ° and the other at 37 °, until the A60oof the cells is between 0.4 and 0.6. Induction is then started by the addition of IPTG (0.4 mM, final concentration) and growth is continued at the same temperature with the removal of 5-ml aliquots from each culture 1, 2, and 3 hr after the start of induction. These samples are harvested by centrifugation in the GH-3.7 rotor of a Beckman GPR centrifuge at 3000 rpm at 4 °. The pellets are resuspended in ice-cold lysis buffer (20 m M Tris-HC1, pH 8.0, 100 m M NaC1, 5 m M MgC12, 1 m M DTT, 1 m M PMSF, and 1 gg/ml each of antipain, leupeptin, aprotinin, chymostatin, and pepstatin A) and transferred to 1.5-ml microfuge tubes. The samples are then sonicated on ice for 30 sec, in three 10-sec intervals with cooling on ice for I min between each 10-sec sonication (we used a Sonifier cell disruptor (Branson, Danbury, CT) model W185 on setting 5.5). The resulting sonicate is centrifuged at top speed in a microfuge and the pellet resuspended in a volume of lysis buffer equal to that of the sonic,ate supernatant. Equal volumes of sonic,ate supernatant and pellet fractions can then be analysed by SDS-PAGE and Coomassie Brilliant blue staining of the gel. We found that expression of Sec4p in the sonicate supernatant (i.e., soluble Sec4p) was different for the two transformants that we tested and also varied with the temperature and time of induction. Consequently, for the transformant expressing the greatest amount of soluble Sec4p (strain
358
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[33]
NRB412d), we found that the optimal conditions for expression of this protein were growth of the cells at 30 ° and induction with IPTG for 1 hr at 30*. To produce a larger scale induction of Sec4p, 5 ml of ZB medium containing 0.1 mg/ml ampicillin is inoculated from a stock of the strain NRB412d stored at - 8 0 ° and grown overnight at 37 ° with aeration. This is then used to inoculate 500 ml of ZB broth containing 0.1 mg/ml ampicillin and growth is continued at 30 ° with aeration until the Ac,oois between 0.4 and 0.6. To induce expression of the SEC4 gene, 0.4 rnM IPTG (final concentration) is added to the culture and growth is continued for 1 hr at 30 ° with aeration. Following this induction the cells are harvested by centrifugation for 10 min at 5000 rpm at 4 ° (we used a Beckman J2.21 centrifuge with a JA-10 rotor). The cell pellet can be used immediately or rapidly frozen and stored at -80*. The pelleted cells are resuspended in 7 ml of ice-cold lysis buffer and the cell suspension is sonicated on ice for 6 rain, in three 2-rain intervals with cooling on ice for 1 min between each sonication. The resulting sonicate is then centrifuged at 11,000 rpm for 15 rain at 4 ° (we used a Beckman J2.21 centrifuge with a JA-20 rotor) to remove unbroken cells and debris. The supernatant should contain approximately 60 mg of protein and should be used immediately. Guanine Nucleotide-Binding Assay GTP-binding activity is used to monitor the Sec4 protein throughout its purification. While in principle this assay does not uniquely detect Sec4 because there are many other GTP-binding proteins in both S. cerevisiae and E. coli lysates, they are, in fact, quite minor in abundance with respect to Sec4p due to the high level of expression achieved. This is reflected by the observation that the level of soluble GTP-binding activity increases over 40-fold on Sec4p induction in yeast and over 150-fold on Sec4p induction in E. coli. Guanine nucleotide binding is assayed by filtration. ~9 Briefly, 5/zl of column fractions is diluted into 20 gl of buffer A [50 m M sodium 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 5 m M MgC12, pH 8.0, 200 m M NaCl, 1 m M EDTA, 1 m M DTT, and 0.1% (w/v) Lubrol 12A9]. The binding reaction is initiated with the addition of 25/zl of buffer A containing 4 g M GTPTS (Boehringer Mannheim, Indianapolis, IN) and [3~S]GTPTS (New England Nuclear, Boston, MA) at a specific activity of approximately 5000 cpm/pmol. Following a 60-rain incubation at 30 °, the reaction mixtures are diluted with 4 ml of ice-cold ~9j. K. Northup, M. D. Smigel, and A. G. Gilman, J. Biol. Chem. 257, 11416 (1982).
[33]
PURIFICATIONOF See4 PgOTmN
359
20 m M Tris-HC1, pH 8.0, 100 m M NaC1, 25 m M MgC12, and rapidly filtered through 25-mm type HA filters (Millipore, Bedford, MA). A sampling manifold and vaccuum pressure pump (Millipore) are convenient in this regard. Wild-type Sec4p isolated from either yeast or bacteria is predominantly bound to GDP. Because the off-rate of GDP from Sec4p is only about 4 rain in the presence of 5 m M MgCI2 at 30 °, no preincubation under conditions of micromolar MgC12 is necessary to increase the rate of nucleotide exchange, as it is with other members of the r a s superfamily. The filters are washed five times with 2 ml of cold buffer, dried under a heat lamp, and counted in 10 ml of Ecoscint scintillation fluid (National Diagnostics, Manville, N J). The specific radioactivity of the nucleotide is determined by diluting the radioactive mixture 1 : 80 and spotting 20 #1 ( 1 pmol) onto each of three filters, which are then dried and counted as above. Purification of Sec4p from Yeast Gel filtration is an effective initial purification step because Sec4p is a small protein that, when overproduced, accumulates in a soluble, monomeric form. Approximately 350 mg of protein derived from a high-speed supernatant fraction of NY671 cells in a volume of 30 ml is applied to an 800-ml Sephacryl S-100 column (Pharmacia, Piscataway, NJ; 5 × 44 cm). The column is eluted with TMD buffer (20 m M Tris-HC1, pH 8.0, 5 m M MgC12, 1 m M DTT) supplemented with 100 m M NaC1, and 9-ml fractions are collected. It is convenient, but not essential, to follow the A280 with an in-line UV detector and chart recorder. The peak of [35S]GTPTSbinding activity elutes as a symmetrical peak after the bulk of other cell proteins (Fig. 2). Relative to the yeast high-speed supernatant, this purification step gives approximately 20-fold purification with 82% recovery of activity in a volume of 38 ml (see Table I). Sec4p binds only weakly to DEAE-Sephacel (under our buffer conditions). Thus the second stage of the purification involves a step in which Sec4p fails to bind to this ion-exchange resin while a number of contaminating proteins are retained. The pooled Sephacryl S-100 fractions are combined and then diluted twofold with an equal volume of TMD buffer to a final concentration of 50 m M NaCl. It is then loaded onto a 50-ml DEAE-Sephacel column (2.5 × I 1.5 cm; Pharmacia) equilibrated in TMD buffer with 50 m M NaC1. The column is washed with this buffer, and 4.4-ml fractions are collected. The flow-through and wash fractions containing protein as measured by absorbance at 280 nm are combined. It is not necessary to assay for GTPTS binding during this step. In the final stage of purification the NaCI concentration is reduced to
360
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[33]
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the level at which Sec4p is retained on a DEAE-Sephacel column and it is then eluted with an NaC1 gradient. To lower the NaCI concentration without diluting the protein to the extent that Sec4p is denatured the pool is first concentrated to 5 ml by pressure titration in a stirred cell containing an Amicon (Danvers, MA) PM10 membrane and then diluted fivefold with TMD buffer for a final NaC1 concentration of I 0 mM. The pool is applied to a 30-ml DEAE-Sephacel column (2.5 × 7 cm) equilibrated in T M D buffer with 10 m M NaC1, and the adsorbed protein is eluted with a 150-ml linear gradient of 1 0 - 5 0 m M NaC1 in TMD buffer. Fractions of
[33]
PURIFICATIONOF See4 PROTEIN
PURIFICATION OF
TABLE I Sec4 FROM YEASTAND
361
BACTERIA
GTP~Sbinding Source and fraction Yeast Solublelysate S-100 DEAE flow-through DEAE pool Bacteria Soluble lysate S-100 DEAE flow-through
Total protein (nag)
Total Total Specific volume activity activity (ml) ( n m o l ) (nmol/mg)
350.0 14.8 7.9 2.1
40.0 38.0 95.0 2.8
60 2.9 1.6
6.0 8.0 1.5
232 191 159 65 21.8 24.9 19.5
Yield (%)
Purification (-fold)
0.7 12.9 20.2 30.9
100 82 69 28
1 20 31 47
0.36 8.6 12.1
100 114 89
1 24 34
2.8 ml are collected and alternate fractions are assayed for GTPyS-binding activity. The peak of GTPyS-binding activity elutes at approximately 20 m M NaC1 and is pooled and concentrated approximately 10-fold by pressure filtration in a stirred cell. Prior to storage at - 8 0 °, the purified Sec4 protein should be diluted one-fourth with 6.4 m M dipalmitoylphosphatidylcholine (Sigma, St. Louis, MO), 32 m M 3-[3-cholamidopropyldimethylammonio]-l-propane sulfonate (CHAPS; Calbiochem, San Diego, CA), and 50 m M NaC1 in T M D buffer and then rapidly frozen in small aliquots in sialyzed glass tubes. The addition of detergent and phospholipid appears to stabilize Sec4p against repeated freeze-thaw cycles. The final preparation contains only a single species as visualized by SDS-PAGE and silver staining. The overall purification is typically 47-fold with a yield of approximately 2 mg. Purification of Sec4p from Escherichia coli This procedure is performed as described above for purification of Sec4p from yeast except that column sizes are scaled down because the lysate is prepared from only 500 ml of cells and the final ion-exchange column is omitted. The 6 ml of supernatant (from centrifugation of the sonicate at 13,800 g) containing approximately 60 mg of protein is applied to a 170-ml Sephacryl S-100 column (1.5 cm X 96 cm; Pharmacia). The column is eluted with T M D buffer supplemented with 100 m M NaCI, 2.2-ml fractions are collected and the peak of GTPyS-binding activity
362
IDENTIVnCATmN OF TRANSPORT INTERMEDIATES
[34]
pooled. This step gives approximately 24-fold purification with excellent recovery of GTPyS-binding activity in a volume of 8 ml (see Table I). The pooled fractions are then diluted with an equal volume of TMD buffer to give a final concentration of 50 m M NaC1. This is loaded onto a 12-ml DEAE-Sephacel column (2.5 × 2.5 cm) equilibrated with TMD buffer containing 50 m M NaC1. The column is washed with the same buffer until the absorbance at 280 nm returns to baseline. The peak of GTP~,S-binding activity is pooled and concentrated approximately 18-fold to 1.5 ml by pressure filtration in a stirred cell containing an Amicon PM 10 membrane. The protein is then rapidly frozen and stored at - 80 o. The overall purification is approximately 34-fold (see Table I), with a yield of about 500/tg of active protein and a purity of approximately 80-90% as analyzed by SDS-PAGE and Coomassie Brilliant blue staining of the gel. The GDP off-rate, GTP on-rate, and intrinsic GTP hydrolysis rate of the bacterially produced Sec4p are quite similar to those found for Sec4p isolated from yeast. Acknowledgments This work was supported by Grants GM35370 and CA46218 to P.N. from the National Institutes of Health. M.G. was supported by the Lucille P. Markey Charitable Trust, P.B. was supported by the Damon Runyon Walter Winchell Cancer Research Fund, and A.K.K. was supported by the Jane Coffin Childs Memorial Fund for Medical Research and by a Swebilius Cancer Research Award.
[34] P r e p a r a t i o n o f R e c o m b i n a n t ADP-Ribosylation Factor
By PAUL A. RANDAZZO, OFRA WEISS, and RICHARD A. KArIN Introduction ADP-ribosylation factor (ARP0 proteins were originally identified and purified based on an in vitro activity as the protein cofactor required for efficient ADP-ribosylation of Gs by cholera toxin ~ (for recent reviews see Kahn3,4). Subsequently, ARF was shown to be a 21-kDa GTP-binding t L. S. Schleifer, R. A. Kahn, E. Hansld, J. K. Northup, P. C. Sternweis, and A. G. Gilman, J. Biol. Chem. 257, 20 (1982). 2 R. A. Kahn and A. G. Gilman, J. Biol. Chem. 259, 6228 (1984). R. A. Kahn, in "G Proteins" (L. Birnbaumer and R. Iyengar, eds.), p. 201. Academic Press, Orlando, Florida, 1990. 4 R. A. Kahn, this series, Vol. 195, p. 233.
METHODSIN ENZYMOLOGY,VOL 219
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