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Comments Preparation of cells that actively transport VSV G protein has been most thoroughly characterized using CHO cells and CHO cytosol. However, there is considerable variability in transport using different cell lines or conditions. Where indicated in the procedures, measurement of transport using different marker proteins, or the use of different cell lines or cytosol preparations, will need to be optimized to obtain maximal efficiencies of transport. Extensive fragmentation and lysis of the ER (particularly in the case of strongly adherent cells) generally result in reduced transport. In particular, perforation conditions can lead to the release of soluble marker proteins from the ER during preparation of semiintact cells. In contrast, poor perforation leads to transport that is efficient, but cytosol independent. In the latter case, cytosol dependence can be enhanced by gently homogenizing the semiintact cells with a loose-fitting pestle in a glass Dounce (10-20 strokes) prior to washing.

[ 13] R e c o n s t i t u t i o n o f T r a n s p o r t f r o m E n d o p l a s m i c Reticulum to Golgi Complex Using Endoplasmic Reticulum-Enriched Membrane Fraction from Yeast B y LINDA J. WUESTEHUBE a n d R A N D Y W . SCHEKMAN

Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus may be monitored in vitro with gently lysed semiintact cells 1-a or with crude homogenates fractionated by differential centdfugation) ,4 A complete resolution of this reaction will require the purification of donor ER and acceptor Golgi membranes. Toward this end we have developed an in vitro assay that utilizes an ER-enriched membrane fraction to reconstitute ER-to-Golgi transport. This new assay allows a detailed analysis of the specific contributions of both membrane and soluble components to the transport process not possible in semiintact cells and crude homogenates.

t C. J. M. Beckers, D. S. Keller, and W. E, Balch, CellSO, 523 (1987). D. Baker, L. Hicke, M. Rexaeh, M. Sehleyer, and R. Schekman, Ce1154, 335 (1988). 3 H. Ruohola, A. K. Kabeenell, and S. Ferro-Novick, £ CellBiol. 107, 1465 (1988). 4 W. E. Balch, K. R, Wagner, and D. S. Keller, J. CellBiol. 104, 749 (1987).

METHODS IN ENZYMOLOGY,VOL. 219

~ t © 1992by AcademicPress,Inc. All~hts of~'producfionin any form reserved.

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P r e p a r a t i o n of SubceUular Fractions Three subcellular fractions are required to reconstitute ER-to-Golgi transport: sucrose-purified membranes, cytosol, and a high-speed pellet fraction. The protein concentration of all these fractions is determined in the presence of 1% (w/v) sodium dodecyl sulfate (SDS) by the procedure of Lowry et al.5 Fractions are divided into small aliquots, fast-frozen in liquid nitrogen, and stored at - 8 0 °. No significant loss in transport activity is detected in subcellular fractions stored for as long as 1 year.

Sucrose Density Gradient-Purified M e m b r a n e P r e p a r a t i o n Sucrose density gradient-purified membranes are prepared from osmotically lysed yeast spheroplasts. The method of lysis is important in the preparation of transport-competent membranes. We found the yield and specific activity of membranes prepared from osmotically lysed spheroplasts to be substantially higher than membranes prepared from glass bead-lysed cells. Formation of Yeast Spheroplasts. Wild-type yeast cultures are grown at 30 ° in YP medium [2% (w/v) Bacto-peptone, 1% (w/v) yeast extract] containing 2% (w/v) glucose to a density of 2 - 4 OD6o0 U/ml (1 OD~o U = 1 × 107 cells). We recommend using protease-deficient strains such as RSY445 (MA Tot ura3-52 trpl leu2 his4 pep4::URA3) or RSY607 (MA Tot, leu2-3,112 ura3-52 pep4::URA3). Cells are harvested by centrifugation (1000 g, 5 min, 24°), resuspended to 100 OD~oo U/ml in 10 m M dithiothreitol (DTT), 100 m M Tris-HC1, pH 9.4, and incubated for at least 10 min at 24 ° with gentle agitation. Cells are collected by centrifugation (1000 g, 5 min, 24 °) and resuspended to 100 OD6oo U/ml in 0.7 M sorbitol, 1.5% (w/v) Bacto-peptone, 0.75% (w/v) yeast extract, 0.5% (w/v) glucose, 10 m M Tris-HCl, pH 7.4. Lyticase 6 (fraction II, -60,000 U/ml, 1 0 - 2 0 U/ODr, oo U cells) is added and the cell suspension is incubated at 30 ° until the OD6oo of a 1 : 200 dilution in distilled H20 is less than 5% of the initial value. The effectiveness of the lyticase treatment is an important parameter in determining the degree of lysis achieved. Therefore, the OD6oo ofa 1 : 200 dilution in distilled H20 is monitored periodically during the incubation and additional lyticase is added if necessary. The total time of incubation with lyticase is usually no more than 1 hr. Lyticase-treated cells (spheroplasts) are collected by centrifugation in an HB-4 (Sorvall 50. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 6 j. Scott and R. Schekman, J. Bacteriol. 142~ 414 (1980).

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DuPont, Wilmington, DE) swinging bucket rotor (10,500 g, 10 min, 4 °) through a cushion of 0.8 M sucrose, 1.5% (v/v) Ficoll 400, 20 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4 (1020 ml spheroplast suspension/15-ml cushion in a 40-ml polycarbonate tube). Alternatively, for large-scale preparations spheroplasts may be collected by centrifugation in a GSA fixed-angle rotor (1000 g, 5 rain, 4 °) and washed once or twice in 0.7 M sorbitol, 20 mM HEPES, pH 7.4. Spheroplasts may either be lysed directly or resuspended to 1000 ODeo0 U/ml in 0.7 M sorbitol, 20 mM HEPES, pH 7.4 and slowly frozen at - 8 0 ° in insulated containers for long-term storage prior to lysis. Osmotic" Lysis and Homogenization. Spheroplasts are resuspended to 100 OD6oo U/ml in lysis buffer [0.1 M sorbitol, 20 mM HEPES, pH 7.4, 50 mM potassium acetate, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.7 /tg/ml pepstatin, 0.5 /tg/ml leupeptin] at 4 ° and homogenized with a motordriven Potter-Elvehjem homogenizer (4-10 strokes). Care shouM be taken to ensure that the lysate is kept at temperatures < 4 ° at all times. The degree of lysis may be assessed by visualization of the lysate under a phase-contrast microscope. Intact spheroplasts, with characteristic bright halos resulting from the difference in refractive index between the spheroplasts and the surrounding buffer, are easily distinguished from the fragments oflysed spheroplasts. Greater than 95% lysis usually is obtained with this method. Differential Centrifugation and Sucrose Density Gradient Fractionation. The lysate is centrifuged (1,000 g, 10 min, 4 °) and the pellet and supernatant fractions are collected separately. Increased yields of membranes may be achieved by resuspending the low-speed pellet in lysis buffer to at least one-half the original volume of the lysate and repeating the Potter-Elvehjem homogenization and low speed centrifugation. The supernatant from the rehomogenized low-speed pellet then is combined with the previous supernatant fraction. Up to 30% additional membrane protein may be obtained by rehomogenization of the low-speed pellet. Rehomogenizations of subsequent low-speed pellets usually are not worthwhile. The membrane pellet is collected from the combined low-speed supernatants by centrifugation (27,000 g, 10 rain, 4 °) and resuspended to 5000 ODu~o U cell equivalent/ml in lysis buffer with gentle Dounce (Wheaton, Millville, NJ) homogenization (five strokes). Up to 0.5 ml of the membrane suspension is overlayed onto a 2.0-ml sucrose density step gradient (1.0 ml each of 1.5 M sucrose and 1.2 M sucrose in lysis buffer) and centrifuged in the SW50.1 swinging bucket rotor (100,000 g, 1 hr, 4 °). The membranes at the 1.2/1.5 M interface are collected and washed twice in reaction buffer (20 mM HEPES, pH 6.8, 150 mM potassium acetate,

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5 mM magnesium acetate, 250 m M sorbitol) by centrifugation (27,000 g, 10 min, 4°). Sucrose-purified membranes are resuspended to approximately 5 mg/ml in reaction buffer with gentle Dounce homogenization. Approximately 1.5 mg sucrose-purified membranes per 1000 OD6oo U cells is obtained with this procedure. Cytosol Preparation The cytosol fraction is prepared using glass bead breakage of whole cells. Wild-type yeast cultures are grown at 30 o in YP medium containing 2% (w/v) glucose to late log phase (10- 18 OD6ooU/ml). Cells are harvested by centrifugation (3000 g, 5 min, 4 o), washed twice in reaction buffer, and resuspended to 2000 OD6oo U/ml in reaction buffer containing 1 mM DTT, 1 mM ATP, 100 pM GTP, 0.5 mM phenylmethylsulfonyl fluoride. Approximately 1 g acid-washed glass beads (425 -600 pm) per I000 OD6oo U of cells is added and the cells are lysed in 10 cycles by 30-sec periods of agitation on a VWR Vortexer 2 (San Francisco, CA) at full speed, spaced by l-rain intervals on ice. For large preparations (>-40,000 OD6oo U cells), a 100-ml bead beater chamber (Biospecs Products, Bartlesville, OK) may be used. The extent of lysis is monitored by phase-contrast microscopy as described above. Lysis efficiency is generally 50- 60% using this procedure. The lysate is clarified by centrifugation (27,000 g, 10 rain, 4 °) and the supernatant fraction further clarified by centrifugation in a Beckman (Palo Alto, CA) TLA 100.3 rotor (300,000 g, 1 hr, 4°). The high-speed supernatant (cytosol) is collected, carefully avoiding the pellet and the lipid layer on top of the centrifuge tube. If desired, small molecules may be removed by filtration on a Sephadcx G-25 column equilibrated in reaction buffer. Approximately 15 rag cytosol (7- 10 mg protein/ml) is obtained per 1000 OD6oo U cells. High-Speed Pellet Preparation The high-speed pellet (HSP) fraction may be prepared either from osmotically lysed spheroplasts or from glass bead-lysed cells. The specific transport activity of HSP fractions prepared from osmotically lysed spheroplasts may be somewhat higher than HSP fractions prepared from beadlysed cells, but the yield is comparable regardless of the method of lysis used. Because the HSP and membrane fractions have different sedimentation properties, it is convenient to prepare these fractions from the same preparation of osmotically lysed spheroplasts. It also is possible to prepare the HSP fraction from the glass bead-lysed cells used in the preparation of cytosol; however, this method precludes treatment of the lysate with

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RNase, which we have found to be a critical step in obtaining high specific activity HSP fractions. Yeast cells are either glass bead lysed (as described for cytosol preparation) or spheroplasts are osmotically lysed and homogenized (as described for membrane preparation) in lysis buffer at a density of 100-300 OD6oo U/ml. The lysate is clarified by centrifugation (27,000 g, 10 rain, 4 °) and the supernatant is collected. RNase A (from a 10 mg/ml stock solution) is added to a final concentration of 100 pg/ml and the lysate is incubated with gentle agitation either at 30 ° for 40 min or on ice for 1 hr. In large preparations of-> 4000 OD6oo U cells, a visible white precipitate will form during this incubation. The RNase-treated lysate is clarified by centrifugation (27,000 g, 10 min, 4 °) and the supernatant is collected. If the supernatant is cloudy, then the RNase treatment and centrifugation is repeated. The HSP fraction is collected by centrifugation in a Beckman Ti45 rotor (100,000 g, 1 hr, 4 °) and then is washed into reaction buffer by centrifugation in a Beckman TLA 100.3 rotor (300,000 g, 20 rain, 4°). To collect the HSP fraction, the supernatant is removed by aspiration and the clear pellet is scraped from the bottom of the centrifuge tube with a metal spatula and transferred to a Dounce homogenizer on ice. Residual amounts of HSP fraction remaining in the bottom of the tube are recovered more easily if allowed to incubate on ice in a small volume of reaction buffer for 5 - 10 min prior to resuspension. The HSP fraction is resuspended to approximately 20 mg/ml in reaction buffer with gentle Dounce homogenization. Approximately 1.5 mg HSP fraction/1000 OD~o U cells are obtained. It should be noted that the transport activity of the HSP fraction is resistant to several cycles of freezing and thawing but is not stable at 4 °. Therefore, it is best to work quickly when preparing this fraction and to freeze it as soon as possible. E n r i c h e d Endoplasmic Reticulum T r a n s p o r t Assay Design of Endoplasmic Reticulum to Golgi Transport Assay Transport of prepro-ot-factor, precursor of the secreted mating pheromone a-factor, has been reconstituted in vitro using ER-enriched membranes from yeast (Fig. 1). We chose prepro-a-factor as a substrate because it is translocated efficiently postranslationally into yeast membranes 7-9 and its transport in vivo ~°-~2and in vitro 2,3has been well characterized. The 7 j. Rothblatt and D. Meyer, EMBO 3". 5, 1543 (1986). s M. G. Waters and G. Blobel, J. CellBiol. 102, 1543 (1986). 9 W. Hansen, P. D. Garcia, and P. Walter, Cell 45, 397 (1986). 1oD. Julius, R. Schekman, and J. Thorner, Cell 36, 309 (1984).

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psS]methionine-labeled a-factor precursor

+ ER-enriched membranes ATP 10 °

Translocation into ER addition of N-linked core carbohydrate structures

+ high-speed pellet

cytosol ATP

20-300

Transport to Golgi Complex addition of outer chain carbohydrate structures

FIG. 1. Diagram of the enriched ER transport assay. In the first stage of the reaction, a-factor precursor labeled with [35S]methionine is translocated into the endoplasmic reticulum, where it acquires N-linked core carbohydrate structures (V). In the second stage, ER-enriched membranes containing core-glycosylated a-factor precursor are incubated with a high-speed pellet (HSP) fraction, cytosol, and energy at 20-30 °. Arrival of the a-factor precursor in the Golgi complex is marked by the addition of outer chain carbohydrate structures (O). Transport efficiency is measured as the fraction of core-glycosylated a-factor precursor that receives outer chain modifications.

in vitro assay requires sucrose-purified membranes as a source of translocation- and transport-competent ER, cytosol, and a high-speed pellet fraction prepared as described above. The reaction in addition requires in vitro translated [35S]methionine-labeled a-factor precursor, GDP-

1~ R. S. Fuller, R. E. Sterne, andJ. Thorner, Annu. Rev. Physiol. 50, 345 (1988). 12 A. J. Brake, R. S. Fuller, and J. Thorner, in "Neuropeptides and Their Receptors" (T. W. Schwartz, L. M. Hilstead, and J. F. Rehfeld, eds.), p. 197. Munksgaard, Copenhagen, 1990.

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mannose, and an ATP-regenerating system. Preparation of in vitro translated [35S]methionine-labeled a-factor precursor is described in detail by Baker and Schekman.13 Transport is determined in vitro by measuring the amount of [aSS]methionine-labeled core-glycosylated a-factor precursor originating in the ER that has received outer chain carbohydrate in the Golgi apparatus. On translocation into the ER, the a-factor precursor acquires N-linked core carbohydrate and can be precipitated with concanavalin A, a lectin that recognizes mannose-containing oligosaccharides, a-Factor precursor that is transported to the Golgi apparatus acquires outer chain carbohydrate t4,15 and is identified by precipitation with anti-(al---,6)-mannose serum. This antiserum was produced in response to mnnl mnn2 yeast strains 16as described by Ballou. 17 Reagents Sucrose-purified membranes: 2-20/tg protein ( - 3 - 5 mg/ml) Cytosol: 100- 120/tg protein ( - 10- 15 mg/ml) High-speed pellet: 20-40/tg protein ( - 2 0 rng/ml) [35S]Methionine-labeled prepro-a-factor: - 100,000 cpm//d ATP mixture (10×): 10 mM ATP (equine muscle disodium salt; Sigma, St. Louis, MO), 400raM creatine phosphate (disodium salt; Boehringer Mannheim, Indianapolis, IN), 2 mg/ml creatine phosphokinase (type 1 from rabbit muscle; Sigma) in reaction buffer GDP-mannose (10 raM) (type 1 sodium salt from yeast; Sigma) in reaction buffer Reaction buffer: 20 mM HEPES, pH 6.8, 150 mM potassium acetate, 5 mM magnesium acetate, 250 mM sorbitol Concanavalian A-Sepharose (Sigma) Antiserum specific for (al--*6)-linked mannose Protein A-Sepharose CL-4B (Pharmacia, Piscataway, NJ) Translocation (First Stage) Sucrose-purified membranes (2-20/tg) are mixed with 3-6/tl [35S]methionine-labeled prepro-~-factor, 1.2/d 10× ATP mixture, and 13 D, Baker and R. Schekman, Methods CelIBioL 31, 127 (1989). 14 B. Esmon, P. Novick, and R. Schckman, Cell 25, 451 (1981). t5 C. E. Ballou, in "The Molecular Biology of the Yeast Saccharomyces" (J. N. Strathern, E. Jones, and J. Broach, eds.), pp. 335-360. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982. 16 W. C. Rashke, K. A. Kern, C. Antalis, and C. E. Ballou, J. Biol. Chem. 248, 4660 (1973). 17 C. Ballou, J. BioL Chem. 245, 1197 (1970).

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ENRICHED ER TRANSPORT ASSAY

13 1

reaction buffer in a final volume of 12 #1. The reaction mixture is incubated at 10 ° for 30 min and then is diluted at least 1 : 5 in reaction buffer. Membranes are collected by centrifugation (27,000 g, 10 min, 4°), washed once, and resuspended to 1 mg/ml in reaction buffer. Up to eighteen 12-#1 reactions usually are combined in a single 1.5-ml microfuge tube for this first-stage reaction and then aliquoted into separate tubes for the secondstage reaction.

Transport (Second Stage) Membranes (2-20 pg) containing translocated [35S]methionine-labeled pro-a-factor from the first-stage reaction are mixed with cytosol (100120/~g), HSP (20-40#g), 5#1 10X ATP mixture, 5/~1 10raM GDPmannose, and reaction buffer in a final volume of 50 #1. After incubation for 2 hr at 20 °, the reaction is terminated by addition of 50 #1 2% (w/v) SDS and heated for 5 rain at 95 °. The amount of cytosol and HSP added should be titrated for each preparation to obtain optimal transport efficiency. As a control for background, 5/tl 100 U]ml apyrase (grade VII; Sigma) may be added in place of 10× ATP mixture.

Concanavalin A Precipitation and Immunoprecipitation The total amount of core-glycosylated [35S]methionine-labeled pro-afactor is quantified by precipitation with concanavalin A-Sepharose. The terminated reaction mixture (up to 100 pl), 30 pl 20% (v/v) concanavalin A-Sepharose, and 1 ml 500 m M NaCI, 1% (v/v) Triton X-100, 20 m M Tris-HC1, pH 7.5 are combined in a 1.5-ml microfuge tube and incubated with continuous rotation for 2 hr at room temperature or overnight at 4°. The precipitates are collected by centrifugation (12,000 g, 1 rain, 20 °) and washed once with 1 ml of 150 m M NaC1, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 15 rnM Tris-HC1, pH 7.5, twice with 1 ml of 2 M urea, ~.00 m M NaC1, 1% (v/v) Triton X-100, 100 rnM Tris-HC1, pH 7.5, once with 1 ml of 500 mMNaC1, 1% (v/v) Triton X-100, 20 m M Tris-HC1, pH 7.5, and once with l ml of 50 m M NaCI, 10 mMTris-HCl, pH 7.5. The amount of core-glycosylated [~SS]methionine-labeled pro-a-factor that has received outer chain modifications is quantified by precipitation with antiserum specific for (al---,6)-linked mannose and protein ASepharose. The amount of antiserum added to precipitate all of the outer chain-modified pro-a-factor in a reaction should be titrated for each batch of antiserum. Generally, 5 pl 20% (v/v) protein A-Sepharose is added per 1 pl of antiserum. The terminated reaction mixture (up to 100 #1), an optimal amount (X #1) of antiserum specific for (al--,6)-linked mannose, 5Xkd 20% (v/v) protein A-Sepharose, and 1 ml 150 m M NaC1, 1% (v/v)

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Triton X-100, 15 m M Tris-HC1, pH 7.5, are combined in a 1.5-ml microfuge tube. All incubations and washes are carried out as described above. The washed precipitates are analyzed by scintillation counting. Samples are resuspended in 150 ~1 2% (w/v) SDS, heated at 95" for 5 rain, and then transferred to scintillation vials. Five milliliters of scintillant (Universol; ICN Biomedicals, Costa Mesa, CA) is added and samples are counted in a scintillation counter.

Determination of Transport Efficiency The efficiency of transport is expressed as the percentage of coreglycosylated [35S]methionine-labeled pro-a-factor that has received outer chain modifications. Transport efficiency is determined for each reaction by the following equation: counts per minute (cpm) precipitated with antiserum specific for (al--,6)-linked mannose divided by cpm precipitated with concanavalin A-Sepharose times 100.

25

20

"i .-~-.__~ 0

•

0

20

40 Time

60

80

100

120

(rain)

FIG. 2. Time course of ER-Golgi transport in vitro. ER-enriched membranes containing core-glycosylated 3sS-iabeled a-factor precursor were combined with high-speed pellet (HSP), eytosol, and either an ATP-regenerating system (II) or 10 U/ml apyrase (13) on ice. The reaction mixtures were incubated at 20* and aliquots were terminated at the indicated times. Reaction products were precipitated with concanavalin A-Sepharose (core-glycosylated afactor precursor) or with anti-(or l---,6)-mannose serum and protein A-Sepharose (outer chain glycosy!ated a-factor precursor, indicative of arrival in the Golgi complex) and processed for scintillation counting. Percentage transport equals anti-(al--,6)-mannose-precipitable cpm divided by concanavalin A-precipitable cpm times 100.

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Features of Enriched Endoplasmic Reticulum Transport Assay The enriched ER transport assay shares many basic features with previously developed in vitro assays that use semiintact yeast cells prepared by freeze-thaw2 and osmotic 3 lysis. Both semiintact and enriched ER assays utilize [35S]methionine-labeled prepro-a-factor as a substrate to measure transport from the ER to the Golgi apparatus (Fig. 1). The in vitro assays have properties expected of intercompartmental protein transport: transport requires energy, is stimulated by addition of cytosolic protein, and is time and temperature dependent (Figs. 2 and 3), Unlike the in vitro assay with freeze-thaw lysed semiintact cells, the enriched ER transport assay shows a marked dependence on the addition of a HSP fraction for transport activity. Sucrose density gradient purification of membranes appears to be a critical step in obtaining a substantial HSP dependence in this assay. Stimulation of the transport reaction by the HSP fraction is increased from - 2 - to 3-fold to > 20-fold after density gradient purification of membranes (Fig. 4). The HSP and cytosol fractions provide different limiting factors because these fractions act synergistically rather than additively in transport (Fig. 3). Proteins are required because HSP or cytosol fractions treated

20

0 O. m

15

I-

10

5,

0 HSP Cytosol ATP

+ + +

-

+

+

+ +

+

+ -

FIG. 3. The high-speed pellet (HSP) and cytosol fractions act synergistically to promote ER-Golgi transport. Endoplasmic reticuhim-enriched membranes containing coreglycosylated 35S-labeled a-factor precursor were prepared and aliquoted to tubes containing HSP, cytosol, and either an ATP-rcgcncrating system of l0 U/ml apyrasc as indicated. Samples were incubated for 2 hr at 20 °. Reaction products were precipitated and analyzed as described in the legend to Fig. 2.

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25"

20o

o. a-

.a

15"

I10-

5"

0 HSP Cytosol

+ + + Membranes before sucrose gradient purification

-

+

+

+

Membranes after sucrose gradient purification

FIG. 4. Sucrose density gradient-purified membranes show an increased dependence on the high-speed pellet (HSP) fraction for ER-Golgi transport in vitro. 3~S-Labeled a-factor precursor was translocated into ER membranes isolated by differential centrifugation and/or sedimentation on sucrose density gradients as described in the text. Transport efficiency was measured after incubation of these membranes with cytosol and an ATP-regenerating system in the presence or absence of HSP as indicated.

with heat or with trypsin fail to stimulate transport while control treatments with trypsin inhibitor have little effect (L. Wuestehube, unpublished observations, 1989). The factors associated with the HSP are unique to this subcellular fraction. Addition of low-speed pellet (1000 g, 10 min, 4 °) or medium-speed pellet (10,000 g, 10 min, 4*) fractions does not replace the HSP in the transport assay (Fig. 5). Applications and Future Directions The development of an enriched ER in vitro assay represents a methodological advance in defining the molecular requirements for ER-toGolgi transport. We have used this assay to demonstrate a direct role in transport for Yptlp, ~s a small GTP-binding protein that is required for transport of secretory proteins from the ER to the Golgi apparatus in vivo.~9,2° Antibody fragments directed against Ypt 1p inhibit transport and is D. Baker, L. J. Wuestehube, R. Schekman, D. Botstein, and N. Segev, Proc. Natl. Acad. Sci. USA 87, 355 (1990). 19N. Segev, J. Mulholland, and D. Botstein, Ce1152, 915 (1988). 20 H. D. Schmitt, M. Puzicha, and D. Gallwitz, Cell53, 635 (1988).

ENRICHED ER TRANSPORT ASSAY

[13]

13 5

25

20 0

o. ¢"

15

L_

I-,10'

5

0 Protein(pg):

lo 20 50 loo

Cell Fraction:

LSP

dtN I0 20 50 I00

MSP

I0 20 50 i00

HSP

FIG. 5. Factors required for ER-Golgi transport in vitro are associated uniquely with the HSP fraction. Endoplasmic reticulum-enriched membranes containing core-glycosylated 3~Slabeled s-factor precursor were prepared and aliquoted to tubes containing cytosol, ATPregenerating system, and increasing amounts of cell fractions as indicated. Cell fractions were isolated from homogenized yeast spheroplasts by sequential sedimentation at 4 ° at 1000 g for l0 rain (LSP), 10,000 g for 10 rain (MSP), and 100,000 g for 1 hr (HSP). Transport efficiency was measured after incubation at 20* for 2 hr.

subcellular fractions prepared from the yptl-1 mutant strain are defective in the enriched ER transport assay. Further analysis has shown that Ypt lp acts after the formation of ER-derived transport vesicles.2~,22The function of other gene products could similarly be determined by inhibiting transport with specific antibodies or by reproducing a conditional defect in vitro for other temperature-sensitive mutant strains in this assay. The enriched ER transport assay offers more flexibility than assays using semiintact cells in that membrane fractions from different sources can readily be mixed. We have taken advantage of this to demonstrate the biochemical interaction of Secl2p and Sarlp, two proteins that are required for transport in vivo 23'24 and whose genes display genetic interactions. 24 Transport in vitro is inhibited by the addition of membranes 21 M. Rexach and R. Schekman, J. Cell Biol. 114, 219 (1991). 22 N. Segev, Science 252, 1553 (1991). 23 p. Novick, C. Field, and R. Schekman, Cell 21, 205 0980). 24 A. Nakano and M, Muramatsu, J. Cell BioL 109, 2677 (1989).

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containing elevated levels of Secl2p, and is restored by the addition of purified Sarlp. 25 Independent studies in which a temperature-sensitive defect in the sec12-4 mutant is restored by the addition of bacterially expressed Sarlp 26 confirm these results. The enriched ER transport assay thus authentically reproduces in vivo protein transport and can be used to detect specific, physiologically relevant biochemical interactions. Finally, it is possible to dissect the enriched ER transport assay into specific subreactions. The formation of ER-derived transport vesicles from sucrose-purified membranes can be determined by measuring [35S]methionine-labeled core-glycosylated a-factor precursor released into a protease-protected compartment that sediments after centrifugation at 100,000 g at 4 ° for 1 hr. 27 It is therefore possible to distinguish the specific biochemical requirements for vesicle formation from the set of reactions that compose transport from the ER to the Golgi apparatus. The development of the enriched ER transport assay represents an advance toward the reconstitution of ER-to-Golgi transport in a fully resolved system. This assay provides an efficient means to identify membrane and soluble components essential for intracellular membrane transport. The use of this assay in analyzing the biochemical requirements of transport in vitro will aid in defining the molecular details of transport in vivo.

Acknowledgments We would like to thank Dr. Jftrg Sommer for helpful discussions throughout the course of this work. We are grateful to Dr. Linda Silveira for advice on RNase treatment of yeast cell lysates and to Kristina Whitfield for critically reading the manuscript. This work was supported by grants from the National Institutes of Health (National Institute of General Medical Sciences) and the Howard Hughes Medical Institute. L Wuestehube was supported by Fellowship DRG-991 of the Damon Runyon-Walter WincheU Cancer Fund and by a fellowship from the American Cancer Society, California Division.

25 C. d'Enfert, L. J. Wuestehube, T. Lila, and R. Schekman, J. CellBiol. 114, 663 (1991). 26 T. Oka, S. Nishikawa, and A. Nakano, J. CellBiol. 114, 671 (1991). 27 L. J. Wuestehube and R. Schekman, J. CellBiol. 111, 325a (1990).