- Email: [email protected]
[62] Quantitative assay for signal peptidase
784
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[62]
3. The effect should appear with brief exposure to the analog. If cells are exposed to analogs for many hours, the protein processing mechanism may be altered, and the levels of many enzymes may change because incorporation of analogs generally increases the turnover of proteins and increases their susceptibility to protea s e s 65 Acknowledgments We thank Drs. Robert Abeles, Herman Gershon, Robert Handschumacher, Kenneth Klein, Theodore Otani, Upendra Pandit, and Marco Rabinowitz for supplying amino acid analogs for our studies. 65 A. Goldberg and A. C. St. John, Annu. Rev. Biochem. 45, 747 (1976).
[62] Q u a n t i t a t i v e A s s a y for Signal P e p t i d a s e 1 By ROBERT C. JACKSON
Signal peptidase is the enzyme responsible for removing the signal peptide portion of nascent presecretory and presumably also prelysosomal and premembrane proteins during their cotranslational translocation across the rough-endoplasmic reticulum (RER) membrane. In many respects signal peptidase is a unique protease. It is an integral membrane protein whose substrate is not a full-length protein, but, rather, an incomplete polypeptide chain that is engaged in the translocation process. These unique properties of signal peptidase were the source of several obstacles to its assay. Signal peptidase activity was first detected by a cotranslational, or translocation-dependent assay. 2 In this assay the entire series of events that occurs at the RER membrane during translocation is reconstituted in vitro. The assay involves the translation of mRNA encoding a secretory protein in a cell-free in vitro translation system containing microsomal membranes. Polysomes bearing nascent presecretory proteins bind to elements on the cytoplasmic surface of the microsomes, and the nascent presecretory proteins are translocated across the microsomal membrane. During translocation across the membrane they become available to sigThis work was supported by United States Public Health Service Grant GM26763. 2 G. Blobel and B. Dobberstein, J. Cell Biol. 67, 852 (1975).
METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5
[62]
SIGNAL PEPTIDASE ASSAY
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nal peptidase and are cleaved. In this assay signal peptidase activity is coupled to both the translation and translocation of the presecretory protein; consequently, it is impossible to examine the signal peptidase reaction independently of these events. Furthermore, the translocation-dependent assay utilizes microsomal signal peptidase. Unless signal peptidase could be solubilized from the microsomal membrane and assayed in its soluble form, it could not be purified or characterized. Finally, the translocation-dependent assay is critically dependent on in vitro protein synthesis. The assay is, therefore, restricted to ionic conditions and detergent concentrations that are compatible with in vitro translation. These restrictions severely limit the flexibility of the translocation-dependent assay for enzyme purification purposes. These obstacles to the assay of signal peptidase were partially removed by the introduction of a posttranslational or translocation-independent assay. 3 In this assay the cleavage of full-length [35S]preprolactin to [35S]prolactin by detergent-solubilized signal peptidase proceeds in the absence of in vitro translation and polypeptide translocation. Products of the reaction are separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and detected by autoradiography or fluorography. Sequence analysis of the prolactin molecule produced in the translocation-independent assay substantiated that the amino-terminal sequence of the product is identical to that of authentic bovine prolactin, 3 thereby confirming the fidelity of posttranslational cleavage. Several other presecretory proteins, including pregrowth hormone, 3 prepromellitin, 4,5 and human preplacental lactogen, 6,7 have also been shown to serve as substrates in the translocation-independent assay. However, not all fulllength presecretory proteins are processed with equal efficiency; several are very poor substrates. This substrate-dependent variability was not altogether unexpected, since the full-length substrates utilized in the translocation-independent assay are not equivalent to the nascent substrates processed in vivo. Folding of the full-length presecretory molecule may obscure the cleavage site; hence, it is not surprising that the proportion of processable substrate molecules depends upon the folding pattern and, therefore, upon the primary sequence of a given presecretory protein. 3 R. Jackson and G. Blobel, Proc. Natl. Acad. Sci. U.S.A. 74, 5598 (1977). 4 G. Kreil, G. Mollay, R. Kaschnitz, L. Haiml, and U. Vilas, Ann. N. Y. Acad. Sci. 343, 338 (1980). 5 R. Kaschnitz and G. Kreil, Biochem. Biophys. Res. Commun. 83, 901 (1978). 6 A. W. Strauss, M. Zimmermann, I. Boime, B. Ashe, R. A. Mumford, and A. W. Alberts, Proc. Natl. Acad. Sci. U.S.A. 76, 4225 (1979). 7 A. W. Strauss, M. Zimmerman, R. A. Mumford, and A. W. Alberts, Ann. N. Y. Acad. Sci. 343, 168 (1980).
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Despite these improvements in the assay, inactivation of signal peptidase during chromatographic procedures remained problematic, until it was recognized that detergent-solubilized signal peptidase specifically requires phosphatidylcholine as a cofactor and can be inactivated by delipidation during chromatographic separations. 8 The additional discovery that activity can be restored to delipidated signal peptidase by readdition of detergent-solubilized phosphatidylcholine allows chromatographic separations to be performed in the absence of phospholipid. 8 Recognition of the phosphatidylcholine requirement has allowed us to define the phospholipid and detergent conditions required for signal peptidase activity and to develop a quantitative assay, which is described below.
Principle [35S]Prolactin produced by posttranslational cleavage of [35S]preprolactin, in an assay containing 1.0% Triton X-100 and 2.5 m M phospholipid, is detected by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) followed by autoradiography and is quantitated by counting the portion of the gel containing the prolactin band or by densitometry of the autoradiograph.
Reagents Ethanol-extracted soybean phospholipid, 37.5 mM (30 mg/ml) in 40 mM triethanolamine-HC1, pH 7.5 Triton X-100, 20% (v/v) [35S]Methionine-labeled bovine pituitary translation products, synthesized in a wheat germ cell-free translation system Canine pancreatic signal peptidase in 1.0% (v/v) Triton X-100, 40 mM triethanolamine-HC1, pH 8.1, 0.02% NaN3 (w/v)
Preparation of Reagents
Signal Peptidase. Although signal peptidase can be extracted from canine pancreatic rough microsomes with several detergents, including sodium deoxycholate, Triton X-100, Nikkol (octaethyleneglycol dodecyl ether), and octyl glucoside, the assay described here is designed for use with enzyme prepared with Triton X-100; therefore, only those procedures resulting in Triton X-100-solubilized enzyme are described. Delipidated signal peptidase is prepared essentially as described by Jackson and White, 8 with the exception that the Sepharose CL-6B column is equilis R. J a c k s o n and W. R. White, J. Biol. Chem.
256, 2545 (1981).
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SIGNAL PEPTIDASE ASSAY
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brated in 1.0% Triton X-100 instead of 0.2% deoxycholate. All procedures are conducted on ice or in a cold room at 4°, except as otherwise noted. Canine pancreatic rough microsomes are prepared as described by Shields and Blobel. 9 The rough microsomes are either used immediately or frozen in liquid nitrogen and stored at - 8 0 ° for future use. The rough microsomes are resuspended with a Teflon pestle homogenizer in 50 mM NaCI, 40 mM triethanolamine-HC1, pH 8.1, to a concentration of 70 A280 units/ml (measured in 1.0% SDS). Sodium deoxycholate (10%, w/v, decolorized with activated charcoal and recrystallized three times from aqueous acetone) is added to a final concentration of 0.7%, and the solubilized membranes are centrifuged at 100,000 g for 4 hr at 4°. The resultant deoxycholate extract can be frozen in liquid nitrogen and stored at - 8 0 ° or used immediately. The deoxycholate extract is delipidated by gel filtration chromatography on a column of Sepharose CL-6B. A 3.0-ml aliquot of delipidation buffer (2.0% deoxycholate, 0.02% NAN3, 40 mM triethanolamine-HCl, pH 8.1) is loaded onto a column (1.6 × 100 cm) containing 200 ml of Sepharose CL-6B equilibrated with 40 mM triethanolamineHC1, pH 8.1, 1.0% Triton X-100, 0.02% NAN3. The column is pumped at a flow rate of 10 ml/hr, until the delipidation buffer enters the column. Meanwhile the deoxycholate concentration of the signal peptidase extract is increased to 2.0% by the addition of an appropriate volume of 10% deoxycholate. A 9.0-ml aliquot of this sample is loaded on to the column, and the column is eluted with equilibration buffer at 10 ml/hr. Fractions (3.0 ml each) containing signal peptidase activity (assessed qualitatively 8) are combined, frozen in liquid nitrogen, and stored at - 8 0 ° . Samples of signal peptidase can be stored in this manner for at least 1 month without loss of activity. Signal peptidase preparations containing endogenous RER phospholipids can be prepared by directly solubilizing rough microsomes or ethylenediaminetetraacetic acid (EDTA)-stripped microsomes 3 in 1.0% Triton X-100. [35S]Methionine-labeled bovine pituitary translation products are prepared in a wheat germ cell-free translation system, ~°,~l as previously described, 3 with two exceptions: The cell-free system is supplemented with placental ribonuclease inhibitor ~2and total pituitary RNA, prepared from bovine pituitaries, by phenol-chloroform-isoamyl alcohol extraction ~3 and lithium chloride precipitation, in is used in place of poly(A) + pituitary 9 D. l0 R. 11 B. 12 G. 13 H. 14 R.
Shields and G. Blobel, J. Biol. Chem. 253, 3753 (1978). Roman, J. D. Brooker, S. N. Seal, and A. Marcus, Nature (London) 260, 359 (1976). Dobberstein and G. Blobel, Biochem. Biophys. Res. Commun. 74, 1675 (1977). Scheele and P. Blackburn, Proc. Natl. Acad. Sci. U.S.A. 76, 4898 (1979). Aviv and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 69, 1408 (1972). E. Rhoads, J. Biol. Chem. 250, 8088 (1975).
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RNA. Stocks of bovine pituitary translation products can be stored for at least 2 months at - 8 0 ° before use. Trichloroacetic acid precipitable radioactivity was determined by spotting 3 /xl of the translation products on Whatman 3 MM filter paper disks. The disks were processed as described by Mans and Novelli 15 and counted in toluene-Liquifluor (New England Nuclear, Boston, Massachusetts). Ethanol-Extracted Soybean Phospholipid. This phospholipid is prepared from soybean asolectin (Associated Concentrates, Woodside, New York) as described by Kagawa and Racker, ~6 except that the acetone wash procedure is repeated three times. Five grams of the acetone-insoluble phospholipid pellet is dissolved in 50 ml of anhydrous ether, and 125 ml of absolute ethanol are added with vigorous stirring. Stirring is continued for 2 hr under nitrogen. After centrifugation (700 g for 10 min) the supernatant is evaporated under reduced pressure and the residue of phospholipid is dissolved in 10 ml of chloroform-methanol (4: 1, v/v) and stored at - 2 0 °. Phospholipid prepared by this procedure is composed primarily of phosphatidylcholine and phosphatidylethanolamine. 17 Liposomes are prepared as follows. An aliquot of the phospholipid is dried with a stream of nitrogen and lyophilized for 2 hr to remove the last traces of organic solvent. The dried phospholipid is dispersed in 40 mM triethanolamine-HCl, pH 8.1, 0.02% NaN3 by vortex mixing. Liposomes are prepared from the dispersed phospholipid by sonication (under nitrogen) in a Branson 220 sonic bath until a pale opalescent solution is produced. Stock solutions of liposomes are routinely stored at - 8 0 ° and resonicated before use. Assay Procedure A stock substrate mixture sufficient for assaying the desired number of samples is premixed and stored on ice. For instance, for the experiment illustrated in Fig. 1 (12 assay tubes) 350/xl of substrate mix, containing 1.0% Triton X-100, 40 m M triethanolamine-HCl, pH 8.1, 5.0 mM soybean phospholipid, and 2. I0 × 10 7 cpm of pituitary translation products, was prepared by mixing 174 ~1 of [35S]Met-labeled pituitary translation products, containing 1.21 × 105 trichloroacetic acid-precipitable counts per minute, 17.5/xl of 20% Triton X-100, 97.8/zl of distilled water, 14/xl of 1.0 M triethanolamine-HC1, pH 8.1, and 46.7 /zl of 37.5 mM soybean phospholipid, which had been dispersed by brief sonication (approximately 10 sec) in a Branson B-220 sonic bath before use. Each assay tube 15 R. J. Mans and G. D. Novelli, Arch. Biochem. Biophys. 94, 48 (1971). 16 y . Kagawa and E. Racker, J. Biol. Chem. 246, 5477 (1971). 17 C. Miller and E. Racker, J. Membr. Biol. 26, 319 (1976).
[62]
SIGNAL PEPTIDASE ASSAY
FIG. 1. Quantitation of the translocation-independent assay for signal peptidase. The indicated amounts (0-25/~1) of a sample of delipidated signal peptidase were assayed in the presence of 2.5 mM ethanol-extracted soybean phospholipid. The [35S]prolactin produced by the cleavage reaction was separated from other labeled proteins by electrophoresis in 15% polyacrylamide gels containing 0.1% SDS and detected by autoradiography. The amount of [35S]prolactin produced was quantitated by excising and counting the portion of the gel containing the prolactin band. Radioactivity in the prolactin region of control samples (no enzyme) was subtracted from each experimental sample. Bars represent the range of duplicate samples.
50 4C
,"? O
3c
x E ~¢J
789
2(i
Signal Peptidase (ju[)
receives 25/~l of this substrate mix and 25/zl of a solution containing the indicated amount of delipidated signal peptidase in 1% Triton X-100, 40 mM triethanolamine-HCl, pH 8.1. After 90 min of incubation at 25°, the reaction is terminated by the addition of 25/zl of concentrated (3 ×) gel 50,
A
40
'?_o 30 E
~- 20
O
I0
5
[0
15
20
25
Signal Peplidase (#1)
FIG. 2. The concentration of [35S]preprolactin in the assay is rate limiting. The indicated amounts (0-25/~l) of a sample of delipidated signal peptidase were assayed in the presence of 2.5 mM ethanol-extracted soybean phospholipid. Each assay tube received either 1.5 x 10~ cpm of translation products (0 0) or 0.75 x l06 cpm of translation products ( × - - x ) . The [3~S]prolactin produced by the cleavage reaction was separated from other labeled proteins by electrophoresis in 15% polyacrylamide gels containing 0.1% SDS and detected by autoradiography (panel B, see next page). The amount of [35S]prolactin produced was quantitated by excising and counting the portion of the gel containing the prolactin band. The percentage of [35S]preprolactin converted to [35S]prolactin was determined by excising and counting the [3~S]preprolactin band as well as the [35S]prolactin band. By this method it was determined that 15.1% of the total available [~SS]preprolactin in the sample receiving 25/xl of signal peptidase and 1.5 × 106 cpm of translation products was processed to [35S]prolactin. PL, prolactin; pPL, preprolactin.
790
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0
5
10
15
25
_pPL .........PL
B FIG. 2B.
[62]
SIGNAL PEPTIDASE ASSAY
791
electrophoresis sample buffer containing 8% (w/v) SDS, 60 mM dithiothreitol, 12 mM EDTA, 48% sucrose, 0.24 M Tris-HCl, pH 8.8, and 0.04% Bromphenol Blue. The samples are heated in a boiling water bath for 3 min, cooled to room temperature, and alkylated by the addition of 10 /zl of 1.0 M iodoacetamide, followed by a 20-min incubation at 37°. If the Bromphenol blue indicator dye in any of the samples turns yellow owing to hydrolytic release of HI from iodoacetamide, the sample pH is restored to the correct range by adding 1.0 M Tris base (1-2/A) until the indicator dye turns blue. [35S]Met-prolactin is separated from [35S]Met-preprolactin and other labeled translation products by slab gel electrophoresis in SDS. 18Our gels contain 15% acrylamide and 0.4% bisacrylamide. They are 1.2 mm thick and are cast between 26.5 x 35.5 cm glass plates. Each gel contains 20 sample wells (1.5 cm deep × 1.0 cm wide). Each sample (85/zl) is loaded into a single well. Wells not receiving a sample are loaded with 85 ~1 of sample buffer (2.7% SDS, 4 m M EDTA, 0.08 M Tris-HC1, pH 8.8, 14% sucrose, 0.013% Bromphenol Blue). Gels are run at 23 mA for 15 hr. The gels are fixed, and free [35S]methionine is removed by washing each gel three times (1 hr per wash) in 500 ml of a solution containing 50% methanol, 10% acetic acid, and 5% glycerol. The washed gels are dried onto Whatman 3 MM filter paper. The border of filter paper around the dried gel is marked in several places with radioactive ink (approximately 25,000 cpm of 35S per microliter) and an autoradiograph of the dried gel is produced by exposing the gel to an 11 x 14 in sheet of Kodak X-RP-1 X-ray film for 2 days. The amount of [35S]prolactin produced in the assay is quantitated either by directly counting the portion of the gel containing the prolactin band or by densitometry of the autoradiograph. For direct quantitation, the autoradiograph is placed on a light box and the dried gel is positioned over it, so that the ink spots on the border of the dried gel correspond exactly with their replicas on the autoradiograph. The portion of the gel corresponding to the prolactin band (see Fig. 2B) on the underlying autoradiograph is traced in pencil and excised with scissors. The prolactin band from each lane is deposited into a 20-ml scintillation vial, hydrated with 100 ~1 of distilled water for approximately 5 rain, and heated with 1.0 ml of 90% NCS tissue solubilizer (Amersham Corp., Arlington Heights, Illinois) at 55° for 6 hr. Samples are cooled to room temperature, acidified by the addition of 50 ~1 of glacial acetic acid, and counted in 10 ml of Aquasol-2 (New England Nuclear, Boston, Massachusetts). Samples that receive no enzyme, but are otherwise identical to the experimental samples, are used to control for background radioactivity. 18 U. K. Laemmli, Nature (London) 227, 680 (1970).
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-0.5
I00
I
80
-0.4
60
-0.3
4O
•0,2 -~ o
20
.0.1
% x
g Q
E
2b
40
6'o
go
160
Time (mini
FIG. 3. Production of [35S]prolactinas a function of time of reaction. Duplicate aliquots (50 tzl each) of a reaction mixture (700/zl) containing delipidated signal peptidase (350 ~1), bovine pituitary translation products (2.08 x 107 cpm), 1.0% Triton X-100, 2.0 mg/ml of ethanol-extracted soybean phospholipid, and 40 mM triethanolamine-HC1,pH 8.1, were removed at the indicated times and assayed for the productionof [3~S]prolactin.The amount of [35S]prolactin produced was determined by densitometry of a 2-day autoradiograph (x x) or by excising and counting the portion of the gel containing the prolactin band (O 0). Bars represent the range of duplicate samples. Radioactivity in the prolactin region of control samples (generally about 20,000-30,000 cpm) is subtracted from the radioactivity of each experimental sample. Alternatively, the assay is quantitated by densitometrically scanning each lane of the autoradiograph at 540 nm on a Gelman DCD-16 densitometer. The relative amount of processed prolactin produced is proportional to the area under the prolactin peak (or to the maximum absorbance of the prolactin peak, provided that the peak widths at half height are identical, which is usually the case). Both methods of quantitating the assay yield comparable results (see Fig. 3). Characteristics of the Signal Peptidase Reaction Utilizing a qualitative assay for signal peptidase, we have previously shown that signal peptidase can be inactivated by delipidation and that activity can be restored to the delipidated enzyme by readdition of phospholipid, s However, although phospholipid was required for signal peptidase activity, excess phospholipid proved to be inhibitory: Furthermore, the optimal phospholipid concentration was shown to increase with increasing detergent concentration. (In these previous experiments a mixture of detergents, sodium deoxycholate, and Nikkol, was used. The deoxycholate concentration remained constant while the Nikkol concentration was varied.)
[62]
SIGNAL PEPT1DASE ASSAY
793
In designing a quantitative assay for signal peptidase we chose to use Triton X-100 as the detergent, because it is both readily available and compatible with most protein purification techniques. The optimal concentration of ethanol-extracted soybean phospholipid to be used in an assay containing 1.0% Triton X-100 was determined to be 2.5 m M (i.e., approximately 2.0 mg/ml; see Fig. 4); consequently, we maintain a final phospholipid concentration of 2.5 mM in all assays. Samples containing endogenous phospholipid, e.g., detergent-solubilized RER or EDTAstripped RER, represent an unresolved problem, because the relative efficacies of soybean phospholipid and endogenous RER phospholipid have not as yet been determined. However, since the phospholipid optimum is broad (Fig. 4), a small amount of additional endogenous RER phospholipid probably does not significantly affect signal peptidase activity. The concentration of [35S]preprolactin in the assay can be estimated from the specific activity of the [35S]methionine used, the number of methionines in preprolactin, and proportion of the total radioactivity in [35S]preprolactin (assumed to be -50%, see Fig. 2B), to be on the order of 1 nM. This low substrate concentration has several consequences for the signal peptidase assay. First, since the substrate concentration is below 20-
/
IO --
x
L0-
t
E
o~
/ 0-
-4 Phospholipid(raM) FIG. 4. Determination of the optimal concentration of phospholipid for expression of signal peptidase activity. Canine pancreatic signal peptidase was prepared by deoxycholate extraction of rough microsomes and delipidated by gel filtration chromatography in Sepharose CL-6B equilibrated with 1.0% Triton X-100. Signal peptidase was assayed, in duplicate, by the translocation-independent assay, using [35S]preprolactin as substrate. Assays contained 1.0% Triton X-100, 1.5 x 106 cpm [3%]Met-labeled bovine pituitary translation products, 40 mM triethanolamine-HC1, pH 8.1, and the indicated concentration of ethanol-extracted soybean phospholipid, in a total volume of 50 tzl. The [3%]prolactin produced by the cleavage reaction was quantitated by excising and counting the portion of the gel containing the prolactin band (0 . 0). Bars represent the range of duplicate samples.
794
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that required to saturate signal peptidase, any decrease in the number of substrate counts per minute in the assay results in a proportional decrease in the number of product counts per minute accumulated. In Fig. 2A, it is shown that a twofold decrease in the amount of [asS]Met-labeled translation products added to the assay results in a similar decrease in the number of counts per minute of [35S]prolactin produced. This situation, wherein the rate of reaction and the amount of product accumulated are directly proportional to the substrate concentration, occurs only when the substrate concentration is much less than the Michaelis constant (i.e., IS] ~ Km). A second consequence of the limiting amount of substrate in the assay is that the rate of reaction decreases as the substrate is utilized; therefore the accumulation of ['S]prolactin as a function of reaction time deviates from linearity (see Fig. 3). However, since only a small fraction (approximately 15%, see legend to Fig. 2) of the available [35S]preprolactin is converted to [35S]prolactin by 25/xl of signal peptidase during a 90-min incubation, the deviation from linearity is tolerably small. Consequently, we incubate samples for 90 min in order to increase the total [35S]prolactin accumulation and enhance the sensitivity of the assay. As shown in Figs. 1 and 2, this longer incubation time does not seriously affect the linearity of the assay over the range of enzyme concentrations employed. Finally, the substrate-limiting conditions of the assay make it imperative that each assay tube receive an identical number of substrate counts per minute. When utilizing a single preparation of bovine pituitary translation product this represents no problem, since when the substrate concentration is much less than Km, the Michaelis-Menten equation reduces to a form in which the fraction of substrate converted to product is independent of the initial substrate concentration. Practically, this means that the results obtained with a particular preparation of translation products should not change as the substrate decays. On the other hand, the results obtained with two different preparations of translation products cannot be compared since the proportion of radioactivity in [35S]preprolactin may change (e.g., owing to the use of different preparations of pituitary RNA). Any change in the proportion of radioactivity in [35S]preprolactin will affect the results obtained by altering the number of counts per minute of [35S]preprolactin added to the assay. Problems of this sort can be avoided by comparing only signal peptidase activities obtained in a particular experiment with a single preparation of bovine pituitary translation products.
TARGETING" SELECTED TECHNIQUES
[62]
3. The effect should appear with brief exposure to the analog. If cells are exposed to analogs for many hours, the protein processing mechanism may be altered, and the levels of many enzymes may change because incorporation of analogs generally increases the turnover of proteins and increases their susceptibility to protea s e s 65 Acknowledgments We thank Drs. Robert Abeles, Herman Gershon, Robert Handschumacher, Kenneth Klein, Theodore Otani, Upendra Pandit, and Marco Rabinowitz for supplying amino acid analogs for our studies. 65 A. Goldberg and A. C. St. John, Annu. Rev. Biochem. 45, 747 (1976).
[62] Q u a n t i t a t i v e A s s a y for Signal P e p t i d a s e 1 By ROBERT C. JACKSON
Signal peptidase is the enzyme responsible for removing the signal peptide portion of nascent presecretory and presumably also prelysosomal and premembrane proteins during their cotranslational translocation across the rough-endoplasmic reticulum (RER) membrane. In many respects signal peptidase is a unique protease. It is an integral membrane protein whose substrate is not a full-length protein, but, rather, an incomplete polypeptide chain that is engaged in the translocation process. These unique properties of signal peptidase were the source of several obstacles to its assay. Signal peptidase activity was first detected by a cotranslational, or translocation-dependent assay. 2 In this assay the entire series of events that occurs at the RER membrane during translocation is reconstituted in vitro. The assay involves the translation of mRNA encoding a secretory protein in a cell-free in vitro translation system containing microsomal membranes. Polysomes bearing nascent presecretory proteins bind to elements on the cytoplasmic surface of the microsomes, and the nascent presecretory proteins are translocated across the microsomal membrane. During translocation across the membrane they become available to sigThis work was supported by United States Public Health Service Grant GM26763. 2 G. Blobel and B. Dobberstein, J. Cell Biol. 67, 852 (1975).
METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5
[62]
SIGNAL PEPTIDASE ASSAY
785
nal peptidase and are cleaved. In this assay signal peptidase activity is coupled to both the translation and translocation of the presecretory protein; consequently, it is impossible to examine the signal peptidase reaction independently of these events. Furthermore, the translocation-dependent assay utilizes microsomal signal peptidase. Unless signal peptidase could be solubilized from the microsomal membrane and assayed in its soluble form, it could not be purified or characterized. Finally, the translocation-dependent assay is critically dependent on in vitro protein synthesis. The assay is, therefore, restricted to ionic conditions and detergent concentrations that are compatible with in vitro translation. These restrictions severely limit the flexibility of the translocation-dependent assay for enzyme purification purposes. These obstacles to the assay of signal peptidase were partially removed by the introduction of a posttranslational or translocation-independent assay. 3 In this assay the cleavage of full-length [35S]preprolactin to [35S]prolactin by detergent-solubilized signal peptidase proceeds in the absence of in vitro translation and polypeptide translocation. Products of the reaction are separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and detected by autoradiography or fluorography. Sequence analysis of the prolactin molecule produced in the translocation-independent assay substantiated that the amino-terminal sequence of the product is identical to that of authentic bovine prolactin, 3 thereby confirming the fidelity of posttranslational cleavage. Several other presecretory proteins, including pregrowth hormone, 3 prepromellitin, 4,5 and human preplacental lactogen, 6,7 have also been shown to serve as substrates in the translocation-independent assay. However, not all fulllength presecretory proteins are processed with equal efficiency; several are very poor substrates. This substrate-dependent variability was not altogether unexpected, since the full-length substrates utilized in the translocation-independent assay are not equivalent to the nascent substrates processed in vivo. Folding of the full-length presecretory molecule may obscure the cleavage site; hence, it is not surprising that the proportion of processable substrate molecules depends upon the folding pattern and, therefore, upon the primary sequence of a given presecretory protein. 3 R. Jackson and G. Blobel, Proc. Natl. Acad. Sci. U.S.A. 74, 5598 (1977). 4 G. Kreil, G. Mollay, R. Kaschnitz, L. Haiml, and U. Vilas, Ann. N. Y. Acad. Sci. 343, 338 (1980). 5 R. Kaschnitz and G. Kreil, Biochem. Biophys. Res. Commun. 83, 901 (1978). 6 A. W. Strauss, M. Zimmermann, I. Boime, B. Ashe, R. A. Mumford, and A. W. Alberts, Proc. Natl. Acad. Sci. U.S.A. 76, 4225 (1979). 7 A. W. Strauss, M. Zimmerman, R. A. Mumford, and A. W. Alberts, Ann. N. Y. Acad. Sci. 343, 168 (1980).
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Despite these improvements in the assay, inactivation of signal peptidase during chromatographic procedures remained problematic, until it was recognized that detergent-solubilized signal peptidase specifically requires phosphatidylcholine as a cofactor and can be inactivated by delipidation during chromatographic separations. 8 The additional discovery that activity can be restored to delipidated signal peptidase by readdition of detergent-solubilized phosphatidylcholine allows chromatographic separations to be performed in the absence of phospholipid. 8 Recognition of the phosphatidylcholine requirement has allowed us to define the phospholipid and detergent conditions required for signal peptidase activity and to develop a quantitative assay, which is described below.
Principle [35S]Prolactin produced by posttranslational cleavage of [35S]preprolactin, in an assay containing 1.0% Triton X-100 and 2.5 m M phospholipid, is detected by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) followed by autoradiography and is quantitated by counting the portion of the gel containing the prolactin band or by densitometry of the autoradiograph.
Reagents Ethanol-extracted soybean phospholipid, 37.5 mM (30 mg/ml) in 40 mM triethanolamine-HC1, pH 7.5 Triton X-100, 20% (v/v) [35S]Methionine-labeled bovine pituitary translation products, synthesized in a wheat germ cell-free translation system Canine pancreatic signal peptidase in 1.0% (v/v) Triton X-100, 40 mM triethanolamine-HC1, pH 8.1, 0.02% NaN3 (w/v)
Preparation of Reagents
Signal Peptidase. Although signal peptidase can be extracted from canine pancreatic rough microsomes with several detergents, including sodium deoxycholate, Triton X-100, Nikkol (octaethyleneglycol dodecyl ether), and octyl glucoside, the assay described here is designed for use with enzyme prepared with Triton X-100; therefore, only those procedures resulting in Triton X-100-solubilized enzyme are described. Delipidated signal peptidase is prepared essentially as described by Jackson and White, 8 with the exception that the Sepharose CL-6B column is equilis R. J a c k s o n and W. R. White, J. Biol. Chem.
256, 2545 (1981).
[62]
SIGNAL PEPTIDASE ASSAY
787
brated in 1.0% Triton X-100 instead of 0.2% deoxycholate. All procedures are conducted on ice or in a cold room at 4°, except as otherwise noted. Canine pancreatic rough microsomes are prepared as described by Shields and Blobel. 9 The rough microsomes are either used immediately or frozen in liquid nitrogen and stored at - 8 0 ° for future use. The rough microsomes are resuspended with a Teflon pestle homogenizer in 50 mM NaCI, 40 mM triethanolamine-HC1, pH 8.1, to a concentration of 70 A280 units/ml (measured in 1.0% SDS). Sodium deoxycholate (10%, w/v, decolorized with activated charcoal and recrystallized three times from aqueous acetone) is added to a final concentration of 0.7%, and the solubilized membranes are centrifuged at 100,000 g for 4 hr at 4°. The resultant deoxycholate extract can be frozen in liquid nitrogen and stored at - 8 0 ° or used immediately. The deoxycholate extract is delipidated by gel filtration chromatography on a column of Sepharose CL-6B. A 3.0-ml aliquot of delipidation buffer (2.0% deoxycholate, 0.02% NAN3, 40 mM triethanolamine-HCl, pH 8.1) is loaded onto a column (1.6 × 100 cm) containing 200 ml of Sepharose CL-6B equilibrated with 40 mM triethanolamineHC1, pH 8.1, 1.0% Triton X-100, 0.02% NAN3. The column is pumped at a flow rate of 10 ml/hr, until the delipidation buffer enters the column. Meanwhile the deoxycholate concentration of the signal peptidase extract is increased to 2.0% by the addition of an appropriate volume of 10% deoxycholate. A 9.0-ml aliquot of this sample is loaded on to the column, and the column is eluted with equilibration buffer at 10 ml/hr. Fractions (3.0 ml each) containing signal peptidase activity (assessed qualitatively 8) are combined, frozen in liquid nitrogen, and stored at - 8 0 ° . Samples of signal peptidase can be stored in this manner for at least 1 month without loss of activity. Signal peptidase preparations containing endogenous RER phospholipids can be prepared by directly solubilizing rough microsomes or ethylenediaminetetraacetic acid (EDTA)-stripped microsomes 3 in 1.0% Triton X-100. [35S]Methionine-labeled bovine pituitary translation products are prepared in a wheat germ cell-free translation system, ~°,~l as previously described, 3 with two exceptions: The cell-free system is supplemented with placental ribonuclease inhibitor ~2and total pituitary RNA, prepared from bovine pituitaries, by phenol-chloroform-isoamyl alcohol extraction ~3 and lithium chloride precipitation, in is used in place of poly(A) + pituitary 9 D. l0 R. 11 B. 12 G. 13 H. 14 R.
Shields and G. Blobel, J. Biol. Chem. 253, 3753 (1978). Roman, J. D. Brooker, S. N. Seal, and A. Marcus, Nature (London) 260, 359 (1976). Dobberstein and G. Blobel, Biochem. Biophys. Res. Commun. 74, 1675 (1977). Scheele and P. Blackburn, Proc. Natl. Acad. Sci. U.S.A. 76, 4898 (1979). Aviv and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 69, 1408 (1972). E. Rhoads, J. Biol. Chem. 250, 8088 (1975).
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TARGETING: SELECTED TECHNIQUES
[62]
RNA. Stocks of bovine pituitary translation products can be stored for at least 2 months at - 8 0 ° before use. Trichloroacetic acid precipitable radioactivity was determined by spotting 3 /xl of the translation products on Whatman 3 MM filter paper disks. The disks were processed as described by Mans and Novelli 15 and counted in toluene-Liquifluor (New England Nuclear, Boston, Massachusetts). Ethanol-Extracted Soybean Phospholipid. This phospholipid is prepared from soybean asolectin (Associated Concentrates, Woodside, New York) as described by Kagawa and Racker, ~6 except that the acetone wash procedure is repeated three times. Five grams of the acetone-insoluble phospholipid pellet is dissolved in 50 ml of anhydrous ether, and 125 ml of absolute ethanol are added with vigorous stirring. Stirring is continued for 2 hr under nitrogen. After centrifugation (700 g for 10 min) the supernatant is evaporated under reduced pressure and the residue of phospholipid is dissolved in 10 ml of chloroform-methanol (4: 1, v/v) and stored at - 2 0 °. Phospholipid prepared by this procedure is composed primarily of phosphatidylcholine and phosphatidylethanolamine. 17 Liposomes are prepared as follows. An aliquot of the phospholipid is dried with a stream of nitrogen and lyophilized for 2 hr to remove the last traces of organic solvent. The dried phospholipid is dispersed in 40 mM triethanolamine-HCl, pH 8.1, 0.02% NaN3 by vortex mixing. Liposomes are prepared from the dispersed phospholipid by sonication (under nitrogen) in a Branson 220 sonic bath until a pale opalescent solution is produced. Stock solutions of liposomes are routinely stored at - 8 0 ° and resonicated before use. Assay Procedure A stock substrate mixture sufficient for assaying the desired number of samples is premixed and stored on ice. For instance, for the experiment illustrated in Fig. 1 (12 assay tubes) 350/xl of substrate mix, containing 1.0% Triton X-100, 40 m M triethanolamine-HCl, pH 8.1, 5.0 mM soybean phospholipid, and 2. I0 × 10 7 cpm of pituitary translation products, was prepared by mixing 174 ~1 of [35S]Met-labeled pituitary translation products, containing 1.21 × 105 trichloroacetic acid-precipitable counts per minute, 17.5/xl of 20% Triton X-100, 97.8/zl of distilled water, 14/xl of 1.0 M triethanolamine-HC1, pH 8.1, and 46.7 /zl of 37.5 mM soybean phospholipid, which had been dispersed by brief sonication (approximately 10 sec) in a Branson B-220 sonic bath before use. Each assay tube 15 R. J. Mans and G. D. Novelli, Arch. Biochem. Biophys. 94, 48 (1971). 16 y . Kagawa and E. Racker, J. Biol. Chem. 246, 5477 (1971). 17 C. Miller and E. Racker, J. Membr. Biol. 26, 319 (1976).
[62]
SIGNAL PEPTIDASE ASSAY
FIG. 1. Quantitation of the translocation-independent assay for signal peptidase. The indicated amounts (0-25/~1) of a sample of delipidated signal peptidase were assayed in the presence of 2.5 mM ethanol-extracted soybean phospholipid. The [35S]prolactin produced by the cleavage reaction was separated from other labeled proteins by electrophoresis in 15% polyacrylamide gels containing 0.1% SDS and detected by autoradiography. The amount of [35S]prolactin produced was quantitated by excising and counting the portion of the gel containing the prolactin band. Radioactivity in the prolactin region of control samples (no enzyme) was subtracted from each experimental sample. Bars represent the range of duplicate samples.
50 4C
,"? O
3c
x E ~¢J
789
2(i
Signal Peptidase (ju[)
receives 25/~l of this substrate mix and 25/zl of a solution containing the indicated amount of delipidated signal peptidase in 1% Triton X-100, 40 mM triethanolamine-HCl, pH 8.1. After 90 min of incubation at 25°, the reaction is terminated by the addition of 25/zl of concentrated (3 ×) gel 50,
A
40
'?_o 30 E
~- 20
O
I0
5
[0
15
20
25
Signal Peplidase (#1)
FIG. 2. The concentration of [35S]preprolactin in the assay is rate limiting. The indicated amounts (0-25/~l) of a sample of delipidated signal peptidase were assayed in the presence of 2.5 mM ethanol-extracted soybean phospholipid. Each assay tube received either 1.5 x 10~ cpm of translation products (0 0) or 0.75 x l06 cpm of translation products ( × - - x ) . The [3~S]prolactin produced by the cleavage reaction was separated from other labeled proteins by electrophoresis in 15% polyacrylamide gels containing 0.1% SDS and detected by autoradiography (panel B, see next page). The amount of [35S]prolactin produced was quantitated by excising and counting the portion of the gel containing the prolactin band. The percentage of [35S]preprolactin converted to [35S]prolactin was determined by excising and counting the [3~S]preprolactin band as well as the [35S]prolactin band. By this method it was determined that 15.1% of the total available [~SS]preprolactin in the sample receiving 25/xl of signal peptidase and 1.5 × 106 cpm of translation products was processed to [35S]prolactin. PL, prolactin; pPL, preprolactin.
790
[62]
TARGETING: SELECTED TECHNIQUES
0
5
10
15
25
_pPL .........PL
B FIG. 2B.
[62]
SIGNAL PEPTIDASE ASSAY
791
electrophoresis sample buffer containing 8% (w/v) SDS, 60 mM dithiothreitol, 12 mM EDTA, 48% sucrose, 0.24 M Tris-HCl, pH 8.8, and 0.04% Bromphenol Blue. The samples are heated in a boiling water bath for 3 min, cooled to room temperature, and alkylated by the addition of 10 /zl of 1.0 M iodoacetamide, followed by a 20-min incubation at 37°. If the Bromphenol blue indicator dye in any of the samples turns yellow owing to hydrolytic release of HI from iodoacetamide, the sample pH is restored to the correct range by adding 1.0 M Tris base (1-2/A) until the indicator dye turns blue. [35S]Met-prolactin is separated from [35S]Met-preprolactin and other labeled translation products by slab gel electrophoresis in SDS. 18Our gels contain 15% acrylamide and 0.4% bisacrylamide. They are 1.2 mm thick and are cast between 26.5 x 35.5 cm glass plates. Each gel contains 20 sample wells (1.5 cm deep × 1.0 cm wide). Each sample (85/zl) is loaded into a single well. Wells not receiving a sample are loaded with 85 ~1 of sample buffer (2.7% SDS, 4 m M EDTA, 0.08 M Tris-HC1, pH 8.8, 14% sucrose, 0.013% Bromphenol Blue). Gels are run at 23 mA for 15 hr. The gels are fixed, and free [35S]methionine is removed by washing each gel three times (1 hr per wash) in 500 ml of a solution containing 50% methanol, 10% acetic acid, and 5% glycerol. The washed gels are dried onto Whatman 3 MM filter paper. The border of filter paper around the dried gel is marked in several places with radioactive ink (approximately 25,000 cpm of 35S per microliter) and an autoradiograph of the dried gel is produced by exposing the gel to an 11 x 14 in sheet of Kodak X-RP-1 X-ray film for 2 days. The amount of [35S]prolactin produced in the assay is quantitated either by directly counting the portion of the gel containing the prolactin band or by densitometry of the autoradiograph. For direct quantitation, the autoradiograph is placed on a light box and the dried gel is positioned over it, so that the ink spots on the border of the dried gel correspond exactly with their replicas on the autoradiograph. The portion of the gel corresponding to the prolactin band (see Fig. 2B) on the underlying autoradiograph is traced in pencil and excised with scissors. The prolactin band from each lane is deposited into a 20-ml scintillation vial, hydrated with 100 ~1 of distilled water for approximately 5 rain, and heated with 1.0 ml of 90% NCS tissue solubilizer (Amersham Corp., Arlington Heights, Illinois) at 55° for 6 hr. Samples are cooled to room temperature, acidified by the addition of 50 ~1 of glacial acetic acid, and counted in 10 ml of Aquasol-2 (New England Nuclear, Boston, Massachusetts). Samples that receive no enzyme, but are otherwise identical to the experimental samples, are used to control for background radioactivity. 18 U. K. Laemmli, Nature (London) 227, 680 (1970).
792
TARGETING:
SELECTED TECHNIQUES
[62]
-0.5
I00
I
80
-0.4
60
-0.3
4O
•0,2 -~ o
20
.0.1
% x
g Q
E
2b
40
6'o
go
160
Time (mini
FIG. 3. Production of [35S]prolactinas a function of time of reaction. Duplicate aliquots (50 tzl each) of a reaction mixture (700/zl) containing delipidated signal peptidase (350 ~1), bovine pituitary translation products (2.08 x 107 cpm), 1.0% Triton X-100, 2.0 mg/ml of ethanol-extracted soybean phospholipid, and 40 mM triethanolamine-HC1,pH 8.1, were removed at the indicated times and assayed for the productionof [3~S]prolactin.The amount of [35S]prolactin produced was determined by densitometry of a 2-day autoradiograph (x x) or by excising and counting the portion of the gel containing the prolactin band (O 0). Bars represent the range of duplicate samples. Radioactivity in the prolactin region of control samples (generally about 20,000-30,000 cpm) is subtracted from the radioactivity of each experimental sample. Alternatively, the assay is quantitated by densitometrically scanning each lane of the autoradiograph at 540 nm on a Gelman DCD-16 densitometer. The relative amount of processed prolactin produced is proportional to the area under the prolactin peak (or to the maximum absorbance of the prolactin peak, provided that the peak widths at half height are identical, which is usually the case). Both methods of quantitating the assay yield comparable results (see Fig. 3). Characteristics of the Signal Peptidase Reaction Utilizing a qualitative assay for signal peptidase, we have previously shown that signal peptidase can be inactivated by delipidation and that activity can be restored to the delipidated enzyme by readdition of phospholipid, s However, although phospholipid was required for signal peptidase activity, excess phospholipid proved to be inhibitory: Furthermore, the optimal phospholipid concentration was shown to increase with increasing detergent concentration. (In these previous experiments a mixture of detergents, sodium deoxycholate, and Nikkol, was used. The deoxycholate concentration remained constant while the Nikkol concentration was varied.)
[62]
SIGNAL PEPT1DASE ASSAY
793
In designing a quantitative assay for signal peptidase we chose to use Triton X-100 as the detergent, because it is both readily available and compatible with most protein purification techniques. The optimal concentration of ethanol-extracted soybean phospholipid to be used in an assay containing 1.0% Triton X-100 was determined to be 2.5 m M (i.e., approximately 2.0 mg/ml; see Fig. 4); consequently, we maintain a final phospholipid concentration of 2.5 mM in all assays. Samples containing endogenous phospholipid, e.g., detergent-solubilized RER or EDTAstripped RER, represent an unresolved problem, because the relative efficacies of soybean phospholipid and endogenous RER phospholipid have not as yet been determined. However, since the phospholipid optimum is broad (Fig. 4), a small amount of additional endogenous RER phospholipid probably does not significantly affect signal peptidase activity. The concentration of [35S]preprolactin in the assay can be estimated from the specific activity of the [35S]methionine used, the number of methionines in preprolactin, and proportion of the total radioactivity in [35S]preprolactin (assumed to be -50%, see Fig. 2B), to be on the order of 1 nM. This low substrate concentration has several consequences for the signal peptidase assay. First, since the substrate concentration is below 20-
/
IO --
x
L0-
t
E
o~
/ 0-
-4 Phospholipid(raM) FIG. 4. Determination of the optimal concentration of phospholipid for expression of signal peptidase activity. Canine pancreatic signal peptidase was prepared by deoxycholate extraction of rough microsomes and delipidated by gel filtration chromatography in Sepharose CL-6B equilibrated with 1.0% Triton X-100. Signal peptidase was assayed, in duplicate, by the translocation-independent assay, using [35S]preprolactin as substrate. Assays contained 1.0% Triton X-100, 1.5 x 106 cpm [3%]Met-labeled bovine pituitary translation products, 40 mM triethanolamine-HC1, pH 8.1, and the indicated concentration of ethanol-extracted soybean phospholipid, in a total volume of 50 tzl. The [3%]prolactin produced by the cleavage reaction was quantitated by excising and counting the portion of the gel containing the prolactin band (0 . 0). Bars represent the range of duplicate samples.
794
TARGETING: SELECTED TECHNIQUES
[62]
that required to saturate signal peptidase, any decrease in the number of substrate counts per minute in the assay results in a proportional decrease in the number of product counts per minute accumulated. In Fig. 2A, it is shown that a twofold decrease in the amount of [asS]Met-labeled translation products added to the assay results in a similar decrease in the number of counts per minute of [35S]prolactin produced. This situation, wherein the rate of reaction and the amount of product accumulated are directly proportional to the substrate concentration, occurs only when the substrate concentration is much less than the Michaelis constant (i.e., IS] ~ Km). A second consequence of the limiting amount of substrate in the assay is that the rate of reaction decreases as the substrate is utilized; therefore the accumulation of ['S]prolactin as a function of reaction time deviates from linearity (see Fig. 3). However, since only a small fraction (approximately 15%, see legend to Fig. 2) of the available [35S]preprolactin is converted to [35S]prolactin by 25/xl of signal peptidase during a 90-min incubation, the deviation from linearity is tolerably small. Consequently, we incubate samples for 90 min in order to increase the total [35S]prolactin accumulation and enhance the sensitivity of the assay. As shown in Figs. 1 and 2, this longer incubation time does not seriously affect the linearity of the assay over the range of enzyme concentrations employed. Finally, the substrate-limiting conditions of the assay make it imperative that each assay tube receive an identical number of substrate counts per minute. When utilizing a single preparation of bovine pituitary translation product this represents no problem, since when the substrate concentration is much less than Km, the Michaelis-Menten equation reduces to a form in which the fraction of substrate converted to product is independent of the initial substrate concentration. Practically, this means that the results obtained with a particular preparation of translation products should not change as the substrate decays. On the other hand, the results obtained with two different preparations of translation products cannot be compared since the proportion of radioactivity in [35S]preprolactin may change (e.g., owing to the use of different preparations of pituitary RNA). Any change in the proportion of radioactivity in [35S]preprolactin will affect the results obtained by altering the number of counts per minute of [35S]preprolactin added to the assay. Problems of this sort can be avoided by comparing only signal peptidase activities obtained in a particular experiment with a single preparation of bovine pituitary translation products.