[28] Purification of N-ethylmaleimide-sensitive fusion protein

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I D E N T I F I C A T I O N OF T R A N S P O R T I N T E R M E D I A T E S

[28] Purification

of N-Ethylmaleimide-Sensitive Protein

[28]

Fusion

B y MARC R. BLOCK and JAMES E. ROTHMAN

Elucidation of the molecular mechanisms involved in vesicle budding, targeting, and fusion that occur during protein transport has been one of the major challenges of the last decade in the field of cellular biology. In vitro reconstitutions of most of the different steps of the endocytic and exocytic pathways (this volume, Section I) have enabled a biochemical study of the transport machinery. However, vesicular transport requires numerous protein components acting in concert. The removal of a single essential component along one purification step results in the complete loss of transport activity. The task of purifying transport components is greatly facilitated when one component can be eliminated at a time, transforming the overall assay into an assay specific for the single missing component. In a cell-free system that reconstitutes transport within the Golgi stack ~-4 Glick and Rothman 5 made the crucial discovery that Nethylmaleimide (NEM) selectively allowed the measurement of a single component even in the crudest fractions. This led to the first purification of a component of the transport machinery, named N-ethylmaleimidesensitive fusion protein (NSF), according to its function (Block et al.6). Herein, we describe the detailed procedure for purification of NSF from Chinese hamster ovary (CHO) cells, together with the main guidelines for the purification of this component from other sources. Assay for N S F Activity One of the peculiarities of NSF activity is that it can be found in soluble (cytoplasmic) and membrane-associated forms in CHO cells. However, little activity was present in the original preparation of the cytosol fractions, due to the high instability of soluble NSF. This allowed the original discovery that the mild treatment of vesicular stomatitis virus (VSV)infected 15B C H O and uninfected CHO Golgi membranes with NEM (15 E. Fries and J. E. Rothman, Proc. Natl. Acad. Sci. USA 77, 3870 (1980). 2W. E. Balch, W. G. Dunphy, W. A. Braell, and J. E. Rothman, Cell39, 405 (1984). 3 W. E. Balch, B. S. Glick, andJ. E. Rothman, Cell39, 525 (1984). 4 L. Orci, B. S. Glick, and J. E. Rothman, Cell46, 171 (1986). 5B. S. Glick and J. E. Rothman, Nature (London) 326, 309 (1987). 6M. R. Block,B. S. Glick, C. A. Wilcox,F. T. Wieland,and J. E. Rothman, Proc. Natl. Acad. Sci. USA 85, 7852 (1988). METHODS IN ENZYMOLOGY, VOL. 219

Copyright © 1992 by Academic Press, Inc. All l~hts of reproduction in any form reserved.

[28]

PURIFICATIONoF NSF

301

min at 0 °) was sufficient to eliminate most of the activity in the standard transport assay (Balch et aL2). Nevertheless, a residual transport activity was still present after NEM treatment. To decrease this background, the cytosol used in the NSF assay (8 mg/ml of protein) is now prepared as described in Balch and Rothman 7 with minor modifications: it is first desalted on a BioGel P-6DG column (Bio-Rad, Richmond, CA), and further incubated at 37 ° for 20 rain. In these conditions, NSF has a half-time of inactivation of 3 - 5 min and is readily destroyed in the preparation. A mixture of equal volumes of acceptor and donor Golgi membranes in 1 M sucrose, I0 m M Tris-HCl, pH 7.4, is incubated at 0 ° for 15 min with 1 m M NEM (final concentration) added from a fresh stock solution (50 mM). Then dithiothreitol (DTT) is added to 2 m M from a 0.1 M stock. This mixture of NEM-treated Golgi membranes may be refrozen in liquid nitrogen and stored at - 8 0 ° until use. Incubation mixtures of 50 #1 contain NEM-treated Golgi membranes (10/~1), 5 #1 of NSF-free cytosol, 10 p M palmityl-CoA, 50 p M ATP, 2 m M creatine phosphate, creatine kinase (7.3 international units/ml), 250 # M UTP, and 0.4 p M UDP[3H]GlcNAc (0.5 Ci) in an assay buffer containing 25 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-KOH (pH 7.0), 15 m M KCI, 2.5 m M magnesium acetate, and 0.2 M sucrose (derived from the Golgi fractions). The NSF fraction to be tested (up to 20 pl) is added last. After incubation at 37 ° for l hr, the VSV G protein is immunoprecipitated at 40 for at least 6 hr or alternatively at 30 ° for 2 hr as described in Balch et al. 3 Assays of all fractions must be performed in a predetermined linear range (0-0.2 mg/ml for crude NSF preparations). Biological Starting Material for NSF Purification Initially, Glick and Rothman 5 found that NSF could be removed from CHO Golgi membranes by adding small amounts of ATP. Actually, ATP not only induces NSF to stay in a soluble form, but it readily stabilizes it. Indeed, at 37 °, the half-time of thermal inactivation of NSF in solution is only 3 min without ATP, but it rises to 45 min when 300 p M ATP or ADP is added. This finding has been crucial for the success of the purification. Therefore in the purification procedure all our buffers are supplemented with 0.5 m M ATP when not stated otherwise. This addition allows a purification procedure lasting more than 24 hr at 4 ° with a minimal loss of activity. Even though NSF can be conveniently ATP extracted from CHO Golgi 7 W. E. Balch and J. E. Rothman, Arch, Biochem. Biophys. 240, 413 (1985),

302

IDENTIFICATION OF TRANSPORT INTERMEDIATES

[28]

membranes, this starting material results in an insufficient yield of NSF to be practical. Alternatively, we took advantage of the fact that NSF can be removed from CHO membranes with a moderate ionic force of 100 m M KCI without ATP to purify NSF from the crude supernatant (cytosol) fraction of CHO cells. The cytosol preparation has been modified to optimize the amount of NSF activity present in the extract. Routinely, a washed pellet of CHO cells (1 vol) is resuspended with 4 vol of a swelling buffer containing 20 m M piperazine-N,N'-bis(2-ethane sulfonic acid (PIPES)-KOH (pH 7.2), I0 m M MgC12, 5 m M ATP and homogenized in a solution containing 100 m M KCI and 5 m M ATP, 5 m M dithiothreitol, 1 m M phenylmethylsulfonyl fluoride (PMSF), 0.5 m M o-phenanthroline, leupeptin (10/tg/ml), and 1/IM pepstatin. The cells are allowed to swell 20 min on ice and then are disrupted in a Waring blender at high speed for 30 sec. Subsequently, KCI is added slowly to the homogenate while stirring to achieve a final concentration of 0.1 M from a 2.5 M stock solution. After low-speed centrifugation at 800 g for 15 minutes, the postnuclear supernatant may be frozen in 45-mi aliquots in liquid nitrogen and stored at - 8 0 °. Just before use, this postnuclear supernatant must be rapidly thawed at 37 ° and spun at 45,000 rpm in a 45 Ti (Beckman, Palo Alto, CA) rotor for 90 min. The clear supernatant is used as the starting material for NSF purification. Typically, it has an NSF activity of 2500 cpm of [3H]GlcNAc incorporated into VSV G protein per microgram of cytosolic protein. Initial Purification Steps: Elimination of Bulk Proteins

Guidelinesfor Successful NSF Purification Although NSF is stabilized with ADP or ATP, it remains a very unstable and fragile protein. Therefore, all the purification steps should be as short as possible and must be linked together whenever possible. All the biologically active fractions must be frozen in liquid nitrogen when not used and kept at - 8 0 °. The NSF activity can be further stabilized with 10% (v/v) glycerol or 1% (w/v) polyethylene glycol 4000 or 8000 (PEG), but it is destroyed by phosphate buffers and sulfate ions. Fast buffer changes cannot be achieved by gel filtration: NSF activity is completely lost when this chromatography step is used in the absence of PEG. With PEG, NSF becomes more resistant to gel filtration but the yields remain quite low. Alternative desalting procedures, such as fast dialysis using the ultrathin Spectrapor 2 dialysis tubing, must be preferred. Desalting is complete within 2 hr of dialysis at 4 ° with one buffer change, resulting in a high recovery of the activity.

[28]

PURIFICATIONOF NSF

303

Concentrations of the fractions required before each analytical purification steps cannot be carried out with ammonium sulfate precipitation. Ultrafiltration on XM 300 membranes (Spectrum Medical Industries, Los Angeles, CA) was found to be a fast and convenient alternative and no loss of activity is usually observed. Surprisingly, this procedure results in little if any loss of the small molecular components and does not constitute an actual purification step.

Polyethylene Glycol Precipitation This is the most crucial step of the whole purification procedure, and the major source of variability; therefore it must be performed with the maximum of care. Overprecipitation or incomplete precipitation will result in a poor final purification. Because a great deal of variability is observed with PEG precipitation, depending on the initial protein concentration, pH, and temperature,s all these parameters have been tested and standardized in our procedure. The clear high-speed supernatant from CHO extract (described above) containing NSF activity is diluted with a solution made of 100 m M KC1, 20 m M PIPES-KOH, pH 7.2, 10 m M MgC12, 5 m M ATP, 5 m M dithiothreitol, to adjust the protein concentration to 5 mg/ml. The temperature is set at 0 °, and the pH precisely adjusted to 7.0 with 1 M KOH (pH electrode equilibrated at 20°). The appropriate volume of a 50% (w/v) PEG 4000 (Sigma, St. Louis, MO) must be added dropwise with vigorous stirring until a final concentration of 8% (w/v) is reached. Nevertheless, the total time of PEG addition should not exceed l min. The mixture is further incubated for 30 min at 0 ° with stirring. The precipitate is then pelleted for 15 min at 10,000 rpm in a JA-20 rotor (Beckman, Palo Alto, CA). The pellet is suspended in onethird of the initial volume in a solution of 100 m M KC1, 20 m M PIPESKOH, pH 7.0, 2 mMMgC12, 2 mMdithiothreitol, and 0.5 mMATP. The suspension is homogenized with a Dounce (Wheaton, Millville, NJ) homogenizer and then sonicated with three 10-sec bursts in a 3000-W Branson (Danbury, CT) water bath sonicator at 0 °. Undissolved material is subsequently removed by a second 15-min centrifugation at I0,000 rpm in the JA-20 rotor. At this point, you may freeze the supernatant in liquid nitrogen or proceed.

Ion-Exchange Chromatography The NSF activity does not bind to anionic or cationic exchange chromatography at pH 7 under our conditions, suggesting that this protein is 8 K. C. Ingham, Arch. Biochem. Biophys. 186, 106 (1978).

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IDENTIFICATION OF TRANSPORT INTERMEDIATES

[28]

close to its isoelectric point. This fact was used to design a fast and efficient purification step. Most proteins do bind to either anionic or cationic exchanger at pH 7 and moderate ionic force while NSF activity runs in the flow through. Routinely, DE-52 cellulose (Whatman, Clifton, NJ) is swollen in a solution of I M PIPES-KOH, pH 7.0, 0.5 M KCI and then poured into a column. The column volume is chosen to equal the sample volume to be treated. One-third of this volume of Sepharose fast-flow S resin (Pharmacia, Piscataway, NJ) is equilibrated with a 2 M KC1 solution and poured into a column. Both columns are connected in series and equilibrated with a solution of 20 m M PIPES-KOH, pH 7.0, 100 m M KC1, 2 m M MgCI2, 2 m M dithiothreitol, and 0.5 m M ATP until the pH and the conductivity of the eluate become identical to those of the starting buffer. It is noteworthy that ATP does not bind to the DE-52 column at the concentration of 100 m_M KC1. The resolubilized material from PEG precipitation is applied to the top of the DE-52 column. The protein concentration of the fractions is followed using the Bradford assay. 9 The flow-through protein peak is then concentrated 20-fold by ultrafiltration on an XM 300 membrane (Spectrum) at 4 ° with a pressure of 20 psi. At this point, some precipitation may occu~ in the concentrate and the insoluble material must be eliminated with a new centrifugation for 15 min at 25,000 g at 4 °. The preparation may be frozen in liquid nitrogen at this point. Analytical Purification Steps Sedimentation on Glycerol Gradient

Even though NSF seems to be a large protein component, gel filtration does not give satisfactory results. Therefore, NSF is separated from the small proteins by velocity sedimentation through a glycerol gradient. Linear 10-35% (w/v) glycerol gradients (38 ml) are formed from the bottom of 40-ml Beckman Quick-Seal tubes with thin stainless steel tubing. Three gradients can be formed simultaneously using an automatic Beckman gradient maker. The buffer throughout the gradient is 20 m M HEPESKOH (pH 7), 100 m M KCI, 2 m M dithiothreitol, 2 m M MgC12, 0.5 m M ATP. The concentrated DE-52 fast-flow S Sepharose flow-through peak is layered on the top of each gradient in 2-ml portions by using a peristaltic pump. The gradients are centrifuged in sealed tubes in a VTi 50 rotor (Beckman) at 50,000 rpm for 2.5 hr with a slow acceleration and decelera9 M. M. Bradford, Anal. Biochem. 72, 248 (1976).

[28]

PURIFICATION

oF NSF

305

tion program. The use of a vertical rotor rather than a swinging bucket rotor reduces the length of the run from 16 to 2.5 hr and results in a considerable improvement of the yield at this step. Typically, three gradients are required to process NSF derived from 450 ml of cytosol. Fractions (2 ml) are collected from the bottom of the tube. The reproducibility of the gradients made with the Beckman apparatus allows us to collect three gradients at a time using a three-way peristaltic pump and, subsequently, to reduce the overall duration of the preparation. The protein concentration and NSF activity (in 0. l-/tl samples) of each fraction are measured. Only the most active fractions (Fig. I) are pooled, yielding about 1.5 mg/ml of protein per milliliter. At this stage a protein doublet of 76 and 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) dearly follows the NSF activity peak. Nevertheless, the 70-kDa protein is neither a component nor a proteolytic fragment of NSF but a main contaminant of the preparation. It will be eliminated by the next purification step. Whenever the volume of the pooled fractions ex-

'

I

X

I

I

2

Sedimentation

Z

o

o o

lO FRACTION

20 NUMBER

FIG. 1. Glycerol gradient step in the purification of NSF. A 10-35% (v/v) glycerol gradient was prepared and centrifugedas mentionedin the text. Then 0.1 and 5 pl of each 2-ml fractionwere assayedfor NSF activity(solidsymbols)and protein concentration(open symbols),respectively.The double-headedarrow representsthe fractionof the activitypeak that is pooledand used for the next purificationstep.

306

IDENTIFICATION OF TRANSPORT INTERMEDIATES

[9-8]

ceeds 10 ml, the preparation is then concentrated to 8 ml or less by ultrafiltration on an XM 300 membrane (Spectrum). This partially purified NSF preparation can be conveniently stored frozen in liquid nitrogen at this stage.

Mono S Fast Protein Liquid Chromatography The concentrated glycerol fractions are dialyzed in Spectrapor 2 tubing against 100 vol of a solution of 20 m M H E P E S - K O H (pH 7.0), 10% (v/v) glycerol, 2 m M MgC12, 2 m M dithiothreitol, and 0.5 m M ATP for a total of 2 hr at 4 ° (with one buffer change). The dialysate is centrifuged for 10 min at 10,000 rpm in a JA-20 rotor, and 8-ml portions of the clear supernatant are injected into a 1-ml Mono S column (preequilibrated with the dialysis buffer) at a flow rate of 0.5 ml/min. After a 15-ml wash, NSF activity is eluted with a swallow 10-ml linear gradient of 0 - 1 0 0 m M KC1. Activity is mainly found in two 0.5-ml fractions corresponding to approximately 60 m M KC1. It is noteworthy that an initial KCI concentration of 10 m M in the sample to be injected (due to insufficient dialysis) hampers NSF binding to the column. The column is stripped with 2 M KCI before reuse. Two runs are necessary to process the entire preparation derived from 450 ml of cytosol. The NSF activity corresponds to an absorbance peak at 280 nm and SDS-PAGE analysis of the fractions reveals a single protein band of 76 kDa (Fig. 2). The NSF (0.15 mg/ml) can be frozen in aliquots in liquid nitrogen and stored at - 80 o. It can be repeatedly thawed and frozen in liquid nitrogen with little loss of activity. Activity of the purified NSF fraction is completely eliminated by treatment with 1 m M NEM at 0 °, and under the same conditions the 76-kDa polypeptide is labeled with [14C]NEM. It is stimulated by long-chain acylCoA in a fashion similar to crude NSF. Monoclonal IgM directed at the 76-kDa polypeptide inhibited specifically the in vitro transport assay in Golgi 6 and many other in vitro assays related to other steps of protein transport, ~°,~1 suggesting a general use of this protein within the cell. One can conclude that the 76-kDa polypeptide contains the NSF activity required to promote transport. Table I summarizes the purification procedure. The NSF is purified 1000-fold relative to cytosol with an overall yield of 12% in activity. This degree of purification may be an underestimate due to the inactivation of NSF at the different steps. 1oC. J. M. Beckers, M. R. Block, B. S. Glick, J. E. Rothman, and W. E. Balch, Nature (London) 339, 397 (1989). it R. Diaz, L. S. Mayorga, P. G. Weidman, J. E. Rothman, and P. D. Stab_l, Nature (London) 339, 398 (1989).

[28]

PURIFICATION OF N S F

A

'1

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I

307

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I'] 0.5

C,

0.4

x

150

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50

60

0

FRACTION NUMBER

B

41

42 43

44 45

46 47 48 MW (10 `3 ) 97.4

76 66.2

42.7

31

21.5

FIG. 2. Chromatography of NSF on a Mono S FPLC (fast protein liquid chromatography) column. (A) Elufion profile. (Q) NSF activity in 0.01-~1 sarnplcs (the assay is saturated in fractions 43 and 44); - - , absorbance at 280 nm, reflecting protein concentration; - - - , concentration of KCI. (B) SDS-polyacrylamidc(10%, w/v) gel clcctrophorcsis of the proteins present in the fractions containing NSF activity (Coomassic bluc staining).

308

IDENTIFICATION OF TRANSPORTINTERMEDIATES

[28]

TABLE I PURIFICATIONOF NSF FROMCHO CELL CYTOSOL

Step

Yield of activity (% of step 1)

Relative specific activity normalized to step 1

Yield of protein (mR)

Cytosol PEG 4000 precipitation DE-52 ttowthrough Sepharose fast-flow S flow-through Glycerol gradient FPLC on Mono S

100 70 70 35 38 12

1 2.5 12.5 29 206 1000

600 163 54 11 1.2 0.14

Hints for Purification of N S F from Other Biological Sources One can find NSF activity in many other biological sources, such as bovine brain cytosol, rat Golgi extract, and yeast cytosol22 However, in crude tissue extracts, the biological activity is often masked by a strong inhibitory activity that does not allow the use of our NSF assay. With a more purified preparation, such as rat Golgi extract, NSF can be easily detected and exhibits properties similar to CHO NSF, i.e., ATP stabilization of soluble NSF, NEM sensitivity (with some differences according to the species), and improved stability in buffers containing glycerol or PEG. Nevertheless, the extraction of NSF from the membranes in a soluble form may require slightly different conditions. For example, ATP alone is not sufficient to induce the release of rat NSF from Golgi membranes. However, it can be readily extracted using 0.5 m M KC1 in a buffer containing 20 m M P I P E S - K O H , pH 7.2, 2 mMMgClz, 2 m M dithiothreitol, 5 m M ATP, and protease inhibitors. With rat Golgi extract we have been able to follow the biological activity after velocity sedimentation on a glycerol gradient in a manner indistinguishable from CHO NSF. The SDS-PAGE revealed the presence of the 76/70-kDa polypeptide doublet following the activity peak (not shown). However, rat Golgi extract does not yield a sufficient amount of protein and is not a suitable preparation on which to perform the whole purification procedure. Because the procedure described above does not require the measurement of NSF activity until the fifth step of purification, the procedure can be achieved blindly up to this ~2D. W. Wilson, C. A. Wilcox, G. C. Flynn, E. Chen, W.-J. Kuang, W. J. Henzel, M. R. Block, A. Ulldch, and J. E. Rothman, Nature (London) 339, 355 (1989).

[29]

RECOMBINANT NSF FROM E. coli

309

point using a crude whole-tissue extract as starting material (where no NSF activity can be detected). After the sedimentation on glycerol gradient, we found that most of the inhibitory activity is removed and that the NSF peak could be followed during the final purification steps. Therefore, it is likely that mammalian NSF in tissue extracts can be purified using our procedure with minor modifications.

[29] E x p r e s s i o n a n d P u r i f i c a t i o n o f R e c o m b i n a n t N-Ethylmaleimide-Sensitive Fusion Protein from Escherichia coli B y DUNCAN W . WILSON a n d JAMES E. ROTHMAN

The N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) is a homotetramer of 76 kDa, initially purified from Chinese hamster ovary (CHO) cells t and essential for the fusion of transport vesicles with their cognate acceptor membrane 2 at many stages in the secretory and endocytic pathways. Studies in vitro and in vivo have implicated mammalian NSF and Sec 18p, the Saccharomyces cerevisiae homolog,3 in endoplasmic reticulum (ER)-to-Golgi traffic, 4 in at least two stages of intra-Golgi transport, and in endocytosis.5 For an overview, see Wilson et al. 6 and references therein. Here we describe techniques for the expression and purification of large quantities of active, recombinant CHO cell NSF from the bacterium Escherichia coll. Availability of unlimited amounts of this protein, and the opportunity to prepare genetically modified derivatives, will greatly facilitate analysis of the biochemical and molecular properties of NSF.

M. R. Block, B. S. Glick, C. A. Wilcox, F. T. Wieland, and J. E. Rothman, Proc. Natl. Acad. Sci. USA 85, 7852 (1988). 2 V. Malhotra, L. Orci, B. S. Glick, M. R. Block, andJ. E. Rothman, Cell54, 221 (I988). 3 D. W. Wilson, C. A. Wilcox, G. C. Flynn, E. Chen, W.-J. Kuang~ W. J. Henzel, M. R. Block, A. Ullrich, and J. E. Rothman, Nature (London) 339, 355 (1989). 4 C. J. Beckers, M. R. Block, B. S. Glick, J. E. Rothman, and W. E. Balch, Nature (London) 339, 397 (1989). s R. Diaz, L. S. Mayorga, P. J. Weidman, J. E. Rothman, and P. D. Stahl, Nature (London) 339, 398 (1989). 6 D. W. Wilson, S. W. Whiteheart, L. Orci, and J. E. Rothman, Trends Biochem. Sci. 16, 334 (1991).

METHODS IN ENZYMOLOGY, VOL. 219

Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.