[2]
T R A N S P O R T BETWEEN G O L G I C I S T E R N A E
5
[2] Transport between Golgi Cisternae B y CON J. M. BECK~RSand JAMES E. ROTHMAN
Introduction The transport of proteins between the different organelles or compartments that make up the secretory and endocytic pathways is thought to be mediated by small vesicles that bud off from one compartment and subsequently fuse with their target compartment. The actual biochemical reactions that occur during the budding of transport vesicles and their transfer to and fusion with their target compartment can now be analyzed using a number of cell-free systems. Three steps in the secretory pathway have been reconstituted to date: transport from the endoplasmic reticulum (ER) to the Golgi compartment, t-3 transport from the cis- to the medial-Golgi compartment, 4 and transport from the medial- to the trans-Golgi compartment. 5 In the endocytic pathway the internalization of proteins from the plasma membrane in coated vesicles has been reconstituted,6 as well as the fusion between endosomes. 7,8 In addition, Goda and Pfeffer9 have established a cell-free system to study recycling of proteins between endosomes and the trans-Golgi network. The cell-free system used to reconstitute cis- to medial-Golgi transport has provided us with most of the information to date regarding the biochemical pathway of vesicular transport and the specific cofactors involved. In this system, transport of the vesicular stomatitis virus glycoprorein (VSVG protein) is detected between the cis compartment of a mutant Golgi preparation (donor membranes) and the medial compartment of a wild-type Golgi preparation (acceptor membranes). Vesicular stomatitis virus G protein is used as a model protein because it is synthesized in large quantities in VSV-infected cells and because it is known to be transported along the secretory pathway to the plasma membrane in a fashion indistinguishable from normal cellular plasma membrane glycoproteins. The donor Golgi membranes are prepared from VSV-infected Chinese hamster C. J. M. Beckers,D. S. Keller,and W. E. Balch,Cell 50, 523 (1987). 2D. Baker,L. Hicke,M. Rexaeh,M. Sehleyer,and R. Schekman,Cell 54, 335 (1988). 3H. Ruohola,A. K. Kabcenell,and S. Ferro-Novick,J. CellBiol. 107, 1465 (1988). 4 W. E. Balch,W. G. Dunphy,W. A. Braeil,and J. E. Rothman,Cell 39, 405 (1984). s j. E. Rothman,J. Biol. Chem. 262, 12505 (1987). 6E. Smythe,M. Pypaert,J. Lueoeq,and G. Warren,J. Cell Biol. 108, 843 (1989). 7R. Diaz, L. S. Mayorga,and P. Stahl, J. Biol. Chem. 263, 6093 (1988). 8W. A. BraeU,Proc. Natl. Acad. Sci. USA 84, 1137 (1987). 9y. Godaand S. R. Pfeffer,Cell 55, 309 (1988). METHODS IN ENZYMOLOGY, VOL. 219
~ t © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
6
RECONSTITUTION IN CELL-FREE EXTRACTS
[2]
ovary (CHO) 15B cells. ~° These lack the medial-Golgi enzyme Nacetylglucosamine (GlcNAc) transferase I, which transfers GlcNAc from UDPGIcNAc onto the N-linked oligosaccharides found in VSV G protein. The acceptor Golgl membranes are prepared from uninfected wild-type CHO cells that do contain GlcNAc transferase I in their medial-Golgi compartment. Vesicular transport between the two Golgl populations can now be detected because GlcNAc will be incorporated into the N-linked oligosaccharides of VSV G protein as soon as it arrives in the acceptor compartment. Ifa transport reaction is therefore performed in the presence of UDP[3H]GIcNAc, transport of VSV G protein between the donor and acceptor Golgi membranes can be measured by simply determining the incorporation of [3H]GlcNAc in VSV G protein. The incorporation of [3H]GlcNAc, and therefore the transport of VSV G protein between the two Golgi membrane populations, was found to require intact membranes, ATP, and soluble and membrane-associated proteins? This system has contributed in a significant way to our understanding of vesicular transport. Not only has it made possible the identification and purification of a number of proteins that are required for transport, H-~6 it has also made possible the identification, for the first time, of a number of possible reaction intermediates in vesicular transport. ~7-~9 Methods
Materials All chemicals are obtained, unless indicated otherwise, from Sigma Chemical Company (St. Louis, MO). UDP[3H]GlcNAc (5-25 Ci/mmol) is purchased from New England Nuclear (Boston, MA). The rabbit antimouse immunoglobulin G (IgG) (whole molecule; Cat. No. 0111-0082) is obtained from Cappel (Organon Teknika, West Chester, PA) and reconsti1oC. Gottlieb, J. Baenziger, and S. Kornfeld, J. Biol. Chem. 250, 3303 (1975). al M. R. Block, B. S. Glick, C. A. Wilcox, F. T. Wieland, and J. E. Rothman, Proc. Natl. Acad. Sci. USA 85, 7852 (1988). 12 D. O. Clary and J. E. Rothman, J. Biol. Chem. 265, 10109 (1990). 13B. S. Glick and J. E. Rothman, Nature (London) 326, 309 (1987). 14 M. G. Waters, T. Seratini, and J. E. Rothman, Nature (London) 349, 248 (1991). is M. G. Waters and J. E. Rothman, J. CellBiol. in press (1992). ~6p. j. Weidman, P. Melancon, M. R. Block, and J. E. Rothman, J. Cell Biol. 108, 1589 (1989). 17 V. Malhotra, L. Orci, B. S. Glick, M. R. Block, and J. E. Rothman, Cell54, 211 (1988). is L. Orci, B. S. Glick, and J. E. Rothman, Cell46, 171 (1986). 19j. E. Rothman, Nature (London) 355, 409 (1992).
[2]
TRANSPORT BETWEEN GOLGI CISTERNAE
7
tuted by the addition of 2 ml water. The hybridoma secreting the 8G5 monoclonal anti-VSV G protein antibody z° is obtained from Dr. Binks Wattenberg (Upjohn, Kalamazoo, MI). The monoclonal antibody is purified from ascites with protein A-Sepharose (Pharmacia, Piscataway, N J) using standard procedures, z~ General Procedures
Protein concentrations are determined using the BCA protein assay reagent (Pierce, Rockford, IL) with bovine serum albumin (BSA) as a standard. All pH values mentioned are determined at room temperature and all sucrose concentrations are expressed as the ratio of grams of sucrose per total mass of solution (w/w). Ce//s Chinese hamster ovary wild-type (wt) and CHO 15B cells to are grown in a-modified minimum essential medium (aMEM) with 10% (v/v) fetal bovine serum (FCS; Gemini Bioproducts, Calabasas, CA). L cells and BHK21 cells are grown in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal bovine serum and 1 m M glutamine. Virus
To obtain and maintain stocks of VSV (Indiana serotype, San Juan isolate) with sufficiently high titers, it is in our experience necessary to limit the number of times the virus is passed. To this end we usually start a virus preparation by three rounds of plaque purification of VSV on mouse L cells exactly as described by Bergmann3 z A single plaque (3- to 5-ram diameter) is used to infect a single 10-cm plate of BHK21 cells (80-90% confluent) in 10 ml medium. After more than 90% of the cells have died and become detached from the plate (12- 24 hr), the medium is harvested, cell debris removed by centrifugation (10 min at 800 g, 4°), and frozen in liquid nitrogen in 0.5-ml aliquots. A suitable aliquot of this "primary stock" [10 ml at 0.1-1 × 108 plaque-forming units (pfu)/ml] is used to infect four 15-cm plates (15 ml medium/plate) of BHK21 cells at 0.1 pfu/ cell. After 90% of the cells have become detached from the plate, the medium is collected, clarified, and frozen as before. An aliquot of this "secondary stock" (60 ml at 0.5- 1 × 109 pfu/ml) is used to infect twenty 20 L. Lefrancois and D. S. Lyles, Virology 121, 157 (1982). 2~ E. Harlow and D. Lane, "Antibodies: A Laboratory Manual." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1988. z2 j. E. Bergmann, Methods Cell Biol. 32B, 85 (1989).
8
RECONSTITUTION IN CELL-FREE EXTRACTS
[2]
15-cm plates of BHK21 cells at 0.1 pfu/cell. After 90% of the cells have become detached the medium is collected, clarified, and frozen. This virus stock (300 ml at 0.5- 1 X 109 pfu/ml) is used to infect CHO 15B cells for the preparation of donor membranes.
Preparation of Donor Homogenate Chinese hamster ovary 15B cells, grown on 15-era plates until they are about 90% confluent (2.5-3 × l0 T cells/plate), are infected with VSV at 10 pfu/cen in serum-free otMEM (5 ml/plate), containing actinomycin D (10/zg/ml) and 25 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-KOH (pH 7.2). After 1 hr at 37 °, during which the plates are rocked every 10 rain, complete growth medium is added (10 ml/plate) and the incubation continued at 37 ° for an additional 2.5 hr. Cells can be harvested either by mild trypsinization or by scraping with a rubber policeman. In either case the medium is poured off and the plates are washed twice with 10 ml Tris-buffered saline (TBS). Each plate is briefly rinsed with 5 ml of TBS containing 0.05% (w/v) trypsin and 2 m M ethylenediaminetetraacetic acid (EDTA) and subsequently incubated for 5 - 10 rain at room temperature. The cells are washed off the plates in 5 ml ice-cold a M E M with 10% FCS (to inhibit the trypsin) by vigorous pipetting and collected in 50-ml conical centrifuge tubes on ice. Cells are pelleted (10 min at 800 g, 4°), washed once in TBS, and once in homogenization buffer (HB) (250 m M sucrose in 10 m M Tris-HC1, pH 7.4). The final pellet (2.5- 3 ml/forty 15-cm plates) is resuspended after addition of 4 vol homogenization buffer and homogenized by passing the cell suspension 6 - 10 times through a ball-bearing homogenizer.4 The extent of cell breakage can easily be monitored by examining a small aliquot of the homogenate by light microscopy. The resulting homogenate can be stored frozen in liquid nitrogen up to 6 months without a loss in activity.
Preparation of Acceptor Homogenate Chinese hamster ovary wt cells used for acceptor preparations are usually grown in suspension in spinner flasks (Bellco, Vineland, NJ). In a typical preparation 3 liters of cell suspension (at 4 - 6 X 105 cells/ml) are harvested by centrifugation (10 min at 800 g and 4°), washed twice with TBS, and once with HB. The cell pellet ( 3 - 4 ml/3-1iter culture) is resuspended after addition of 4 vol HB and homogenized as described for the preparation of the donor homogenate. The acceptor homogenate can also be stored frozen in liquid nitrogen.
[2]
TRANSPORT BETWEEN GOLGI CISTERNAE
9
Preparation of Donor and Acceptor Golgi Membrane Fractions by Sucrose Density Centrifugation To 12 ml of either donor or acceptor homogenate, add 11 ml of 62% sucrose (w/w) in 10 m M Tris-HC1, pH 7.4 and 230°#1 100 m M EDTA, pH 7.4. After vigorous mixing, the final sucrose concentration is checked by refractometry and should be 37%. If necessary the sucrose concentration can be adjusted by addition of either 10 mMTris-HC1, pH 7.4, or 62% sucrose in 10 m M Tris-HCl, pH 7.4. The mixture is placed in the bottom of a polycarbonate SW28 tube (Beckman, Palo Alto, CA) and overlaid with 15 ml 35% (w/w) sucrose in I0 m M Tris-HC1, pH 7.4, and 9 ml 29% (w/w) sucrose in 10 m M Tris-HC1, pH 7.4. After centrifugation of the gradients for 2.5 hr at 25,000 rpm and 4 ° in an SW28 rotor, the membranes at the 35- 29% interface are recovered by puncturing the side wall of the tube with a syringe. From 12 ml of homogenate one usually obtains 1.5- 2 ml of the Golgi-enriched membrane fraction at a protein concentration of about 0.5- 1 mg/ml. After the membranes are frozen in liquid nitrogen in suitable aliquots, they can be stored at - 80 ° up to 1 year without a detectable loss of activity.
Preparation of Chinese Hamster Ovary Cytosol The high-speed supernatant or cytosol can be prepared by centrifuging homogenates from either CHO wt or 15B cell for 60 min at 55,000 rpm at 4 ° in an SW55 rotor. The supernatant is recovered without disturbing either the pellets or the whitish lipid floating on top, and is subsequently desalted on a P-6DG (Bio-Rad, Richmond, CA) column (2.5 × 50 cm) equilibrated in 50 m M KC1, 25 m M Tris-HCl, pH 7.4, 0.5 m M dithiothreitol (DTT). The void volume fractions containing the bulk of the protein are pooled, frozen in aliquots in liquid nitrogen, and stored at - 8 0 °.
Assay Conditions A standard transport reaction contains the following components: HEPES-KOH, 25 raM, pH 7.4 KC1, 25 11134 Magnesium acetate, 2.5 m M ATP, 50 p M UTP, 250 # M Creatine phosphate (CP), 2 m M Rabbit muscle creatine phosphokinase (CPK), 7.3 IU/ml UDP[3H]GIcNAc, 0.4/23//(0.5/2Ci)
l0
RECONSTITUTION IN CELL-FREE EXTRACTS
[2]
Donor membranes Acceptor membranes Cytosol This is prepared by mixing in a disposable glass tube [10 X 75 mm, e.g., Fisher Scientific (Pittsburgh, PA) 14-962-10A] on ice (per 50-/zl reaction): 5/tl of a 10X concentrated reaction buffer (250 mM HEPES-KOH, pH 7.4, 250 mM KC1, 25 mM magnesium acetate), 5 #l of an "ATP mix" [prepared fresh daily by mixing 5/tl CPK (1600 IU/ml, stored at -80°), 25/A 200 mM CP, 5/ll l0 mM ATP (neutralized with NaOH), 10/zl 100 mM UTP (neutralized with NaOH), and 65/tl water], 5/L1(0.1/tCi//d) UDP[3H]GIcNAc (prepared fresh by gently drying down the appropriate amount of the ethanolic stock under a stream of nitrogen gas), and finally the appropriate amount of water. To this mixture were added gel-filtered cytosol and freshly thawed donor and acceptor membranes. Although the optimal amounts of the latter three components can vary between preparations, good results are usually obtained using 5/tl of gel-filtered cytosol and 5/tl of both donor and acceptor membranes. Transport is initiated by transfer of the reaction to 37 ° and stopped by returning it to ice. Immunoprecipitation of Labeled Vesicular Stomatitis Virus G Protein The labeled VSV G protein is immunoprecipitated with a combination of a monoclonal anti-VSV G protein antibody 8G5 2° and a polyclonal rabbit anti-mouse-IgG antiserum (to form the actual immune complexes). Again, the exact amounts and ratio of the monoclonal and polyclonal antibodies needed to obtain an optimal immunoprecipitation of the VSV G protein will depend on the specific preparations used, although in general we find that 1/tl of the monoclonal antibody (at 1.3- 1.7 mg/ml protein) and 2/A of the polyclonal antiserum per transport reaction are sufficient. Immune complexes are preformed by preincubating (per reaction) 1/tl monoclonal anti-VSV G protein antibody and 2/tl of the rabbit anti-mouse IgG antiserum for 5-10 min at room temperature, followed by the addition of 50/tl stop buffer [1% (v/v) Triton X-100, 1% (w/v) sodium cholate, 5 mM EDTA, 250 mMNaCI in 50 mMTris-HCl, pH 7.5]. This is added to a transport reaction, mixed, and allowed to incubate for at least 1 hr at room temperature. The immune complexes are collected on glass microfiber filters (Cat. No. 934-AH; Whatman, Clifton, NJ) by vacuum filtration. To prevent nonspecific adsorption of proteins the filters are preblocked for 5 min in a 2.5% (w/v) solution of nonfat dry milk (Carnation) in detergent buffer (DB) [1% (v/v) Triton X-100, 5 mM EDTA, 250 mM NaC1 in 50 mM Tris-HC1, pH 7.5]. Each filter is first rinsed with 3 ml DB. The immuno-
[2]
TRANSPORT BETWEEN GOLGI C1STERNAE
11
precipitation reaction is then diluted with 3 ml DB and rapidly filtered. The tube is rinsed once with 3 ml DB, which is again filtered. Finally each filter is washed rapidly with two 3-ml aliquots of DB and subsequently dried under a heat lamp. The filters are transferred to scintillation vials, scintillation fluid (Scintiverse BD, Cat. No. SX18-4; Fisher Scientific) is added, and the samples are counted in a liquid scintillation counter.
Titration of acceptor membranes
Titration of donor membranes 12 co
10
10
X
8-t
01
~
6
r-
4-
e~
> if)
0
lag donor
4 ~g acceptor
Titration of CHO cytosol
Requirements for in vitro transport
1
2
3
0
4
2
6
e-
"o $
P
15
0
z
N
5
-1~
i:l .
0 0
.
20
.
40
.
.
60
~tg eytosol
80
~u
0 J. 100
8
? g
FIG. I. All incubations were performed for 60 rain at 37 ° as described in text. (A) Donor Golgi membranes were added at the amounts indicated to otherwise complete transport reactions, containing saturating amounts of cytosol ( 100 #g protein) and acceptor membranes (5 #g protein). (B) Increasing amounts of acceptor Golgi membranes were added to reactions containing saturating amounts of donor membranes (5 #g protein) and cytosol. (C) Increasing amounts of cytosol were added to transport incubations containing saturating amounts of donor and acceptor Golgi membranes. (D) Standard transoort reactions were carried out but, where indicated, the reaction was left on ice, or was performed in the absence of the ATP mix, cytosol, donor, acceptor, or UDP[3HIGIcNAc.
12
RECONSTITUTION IN CELL-FREE EXTRACTS
[3]
Demonstration of Requirements for in Vitro Transport The cell-free transport system described above has been extensively studied over the years and has allowed the characterization of multiple factors, some better defined than others, that are required for transport. 19 The general requirements for vesicular transport that one would have predicted from in vivo observations in fact turn out to be essential for transport in vitro: transport occurs only at physiological temperatures, and requires both ATP and cytosolic factors in addition to Golgi membranes. The donor and acceptor Golgi membranes and UDp[3H]GlcNAc are specific to the cell-free system used and are essential only to actually detect transport. Experiments demonstrating the different requirements for transport in the cell-free system are shown in Fig. 1.
[3] Detection of Endocytic Vesicle Fusion in Vitro, Using Assay Based on Avidin-Biotin Association Reaction By
WILLIAM A. BRAELL
Principle The detection of the fusion of endocytic vesicles in a cell-free system generally involves an assay in which two probes are incorporated into two different preparations of endocytic vesicles, isolated from separate populations of cells. These probes are configured to generate a detectable signal if they contact each other when the two populations of vesicles are permitted to fuse together in vitro. The use of a detection scheme relying on the avidin-biotin association reaction for such a measurement possesses unique advantages. First, the association of avidin with biotin is rapid and extremely strong (Ka 10-1~), which makes the association effectively irreversible.I Thus, the signal will develop immediately on vesicle fusion, and will remain even if the product vesicles are subsequently lysed and the reacted probes are isolated. Moreover, any nondetectable biotinylated proteins added extravesicularly will act as scavengers for any avidin outside the vesicles, because the irreversibility of the association reaction precludes the scavenged avidin from subsequently detaching and reacting with the detectable biotinylated protein. This ensures that the only detectable signal will result from avidin-biotin associations occurring within the interior of the fused vesicles. Second, the association reaction is insensitive to variaN. M. Green,
Biochem.J.
89, 585 (1963).
METHODS IN ENZYMOLOGY, VOL. 219
Copyright© 1992by AcademicPress,inc. All rightsof reproductionin any formreserved.
T R A N S P O R T BETWEEN G O L G I C I S T E R N A E
5
[2] Transport between Golgi Cisternae B y CON J. M. BECK~RSand JAMES E. ROTHMAN
Introduction The transport of proteins between the different organelles or compartments that make up the secretory and endocytic pathways is thought to be mediated by small vesicles that bud off from one compartment and subsequently fuse with their target compartment. The actual biochemical reactions that occur during the budding of transport vesicles and their transfer to and fusion with their target compartment can now be analyzed using a number of cell-free systems. Three steps in the secretory pathway have been reconstituted to date: transport from the endoplasmic reticulum (ER) to the Golgi compartment, t-3 transport from the cis- to the medial-Golgi compartment, 4 and transport from the medial- to the trans-Golgi compartment. 5 In the endocytic pathway the internalization of proteins from the plasma membrane in coated vesicles has been reconstituted,6 as well as the fusion between endosomes. 7,8 In addition, Goda and Pfeffer9 have established a cell-free system to study recycling of proteins between endosomes and the trans-Golgi network. The cell-free system used to reconstitute cis- to medial-Golgi transport has provided us with most of the information to date regarding the biochemical pathway of vesicular transport and the specific cofactors involved. In this system, transport of the vesicular stomatitis virus glycoprorein (VSVG protein) is detected between the cis compartment of a mutant Golgi preparation (donor membranes) and the medial compartment of a wild-type Golgi preparation (acceptor membranes). Vesicular stomatitis virus G protein is used as a model protein because it is synthesized in large quantities in VSV-infected cells and because it is known to be transported along the secretory pathway to the plasma membrane in a fashion indistinguishable from normal cellular plasma membrane glycoproteins. The donor Golgi membranes are prepared from VSV-infected Chinese hamster C. J. M. Beckers,D. S. Keller,and W. E. Balch,Cell 50, 523 (1987). 2D. Baker,L. Hicke,M. Rexaeh,M. Sehleyer,and R. Schekman,Cell 54, 335 (1988). 3H. Ruohola,A. K. Kabcenell,and S. Ferro-Novick,J. CellBiol. 107, 1465 (1988). 4 W. E. Balch,W. G. Dunphy,W. A. Braeil,and J. E. Rothman,Cell 39, 405 (1984). s j. E. Rothman,J. Biol. Chem. 262, 12505 (1987). 6E. Smythe,M. Pypaert,J. Lueoeq,and G. Warren,J. Cell Biol. 108, 843 (1989). 7R. Diaz, L. S. Mayorga,and P. Stahl, J. Biol. Chem. 263, 6093 (1988). 8W. A. BraeU,Proc. Natl. Acad. Sci. USA 84, 1137 (1987). 9y. Godaand S. R. Pfeffer,Cell 55, 309 (1988). METHODS IN ENZYMOLOGY, VOL. 219
~ t © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
6
RECONSTITUTION IN CELL-FREE EXTRACTS
[2]
ovary (CHO) 15B cells. ~° These lack the medial-Golgi enzyme Nacetylglucosamine (GlcNAc) transferase I, which transfers GlcNAc from UDPGIcNAc onto the N-linked oligosaccharides found in VSV G protein. The acceptor Golgl membranes are prepared from uninfected wild-type CHO cells that do contain GlcNAc transferase I in their medial-Golgi compartment. Vesicular transport between the two Golgl populations can now be detected because GlcNAc will be incorporated into the N-linked oligosaccharides of VSV G protein as soon as it arrives in the acceptor compartment. Ifa transport reaction is therefore performed in the presence of UDP[3H]GIcNAc, transport of VSV G protein between the donor and acceptor Golgi membranes can be measured by simply determining the incorporation of [3H]GlcNAc in VSV G protein. The incorporation of [3H]GlcNAc, and therefore the transport of VSV G protein between the two Golgi membrane populations, was found to require intact membranes, ATP, and soluble and membrane-associated proteins? This system has contributed in a significant way to our understanding of vesicular transport. Not only has it made possible the identification and purification of a number of proteins that are required for transport, H-~6 it has also made possible the identification, for the first time, of a number of possible reaction intermediates in vesicular transport. ~7-~9 Methods
Materials All chemicals are obtained, unless indicated otherwise, from Sigma Chemical Company (St. Louis, MO). UDP[3H]GlcNAc (5-25 Ci/mmol) is purchased from New England Nuclear (Boston, MA). The rabbit antimouse immunoglobulin G (IgG) (whole molecule; Cat. No. 0111-0082) is obtained from Cappel (Organon Teknika, West Chester, PA) and reconsti1oC. Gottlieb, J. Baenziger, and S. Kornfeld, J. Biol. Chem. 250, 3303 (1975). al M. R. Block, B. S. Glick, C. A. Wilcox, F. T. Wieland, and J. E. Rothman, Proc. Natl. Acad. Sci. USA 85, 7852 (1988). 12 D. O. Clary and J. E. Rothman, J. Biol. Chem. 265, 10109 (1990). 13B. S. Glick and J. E. Rothman, Nature (London) 326, 309 (1987). 14 M. G. Waters, T. Seratini, and J. E. Rothman, Nature (London) 349, 248 (1991). is M. G. Waters and J. E. Rothman, J. CellBiol. in press (1992). ~6p. j. Weidman, P. Melancon, M. R. Block, and J. E. Rothman, J. Cell Biol. 108, 1589 (1989). 17 V. Malhotra, L. Orci, B. S. Glick, M. R. Block, and J. E. Rothman, Cell54, 211 (1988). is L. Orci, B. S. Glick, and J. E. Rothman, Cell46, 171 (1986). 19j. E. Rothman, Nature (London) 355, 409 (1992).
[2]
TRANSPORT BETWEEN GOLGI CISTERNAE
7
tuted by the addition of 2 ml water. The hybridoma secreting the 8G5 monoclonal anti-VSV G protein antibody z° is obtained from Dr. Binks Wattenberg (Upjohn, Kalamazoo, MI). The monoclonal antibody is purified from ascites with protein A-Sepharose (Pharmacia, Piscataway, N J) using standard procedures, z~ General Procedures
Protein concentrations are determined using the BCA protein assay reagent (Pierce, Rockford, IL) with bovine serum albumin (BSA) as a standard. All pH values mentioned are determined at room temperature and all sucrose concentrations are expressed as the ratio of grams of sucrose per total mass of solution (w/w). Ce//s Chinese hamster ovary wild-type (wt) and CHO 15B cells to are grown in a-modified minimum essential medium (aMEM) with 10% (v/v) fetal bovine serum (FCS; Gemini Bioproducts, Calabasas, CA). L cells and BHK21 cells are grown in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal bovine serum and 1 m M glutamine. Virus
To obtain and maintain stocks of VSV (Indiana serotype, San Juan isolate) with sufficiently high titers, it is in our experience necessary to limit the number of times the virus is passed. To this end we usually start a virus preparation by three rounds of plaque purification of VSV on mouse L cells exactly as described by Bergmann3 z A single plaque (3- to 5-ram diameter) is used to infect a single 10-cm plate of BHK21 cells (80-90% confluent) in 10 ml medium. After more than 90% of the cells have died and become detached from the plate (12- 24 hr), the medium is harvested, cell debris removed by centrifugation (10 min at 800 g, 4°), and frozen in liquid nitrogen in 0.5-ml aliquots. A suitable aliquot of this "primary stock" [10 ml at 0.1-1 × 108 plaque-forming units (pfu)/ml] is used to infect four 15-cm plates (15 ml medium/plate) of BHK21 cells at 0.1 pfu/ cell. After 90% of the cells have become detached from the plate, the medium is collected, clarified, and frozen as before. An aliquot of this "secondary stock" (60 ml at 0.5- 1 × 109 pfu/ml) is used to infect twenty 20 L. Lefrancois and D. S. Lyles, Virology 121, 157 (1982). 2~ E. Harlow and D. Lane, "Antibodies: A Laboratory Manual." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1988. z2 j. E. Bergmann, Methods Cell Biol. 32B, 85 (1989).
8
RECONSTITUTION IN CELL-FREE EXTRACTS
[2]
15-cm plates of BHK21 cells at 0.1 pfu/cell. After 90% of the cells have become detached the medium is collected, clarified, and frozen. This virus stock (300 ml at 0.5- 1 X 109 pfu/ml) is used to infect CHO 15B cells for the preparation of donor membranes.
Preparation of Donor Homogenate Chinese hamster ovary 15B cells, grown on 15-era plates until they are about 90% confluent (2.5-3 × l0 T cells/plate), are infected with VSV at 10 pfu/cen in serum-free otMEM (5 ml/plate), containing actinomycin D (10/zg/ml) and 25 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-KOH (pH 7.2). After 1 hr at 37 °, during which the plates are rocked every 10 rain, complete growth medium is added (10 ml/plate) and the incubation continued at 37 ° for an additional 2.5 hr. Cells can be harvested either by mild trypsinization or by scraping with a rubber policeman. In either case the medium is poured off and the plates are washed twice with 10 ml Tris-buffered saline (TBS). Each plate is briefly rinsed with 5 ml of TBS containing 0.05% (w/v) trypsin and 2 m M ethylenediaminetetraacetic acid (EDTA) and subsequently incubated for 5 - 10 rain at room temperature. The cells are washed off the plates in 5 ml ice-cold a M E M with 10% FCS (to inhibit the trypsin) by vigorous pipetting and collected in 50-ml conical centrifuge tubes on ice. Cells are pelleted (10 min at 800 g, 4°), washed once in TBS, and once in homogenization buffer (HB) (250 m M sucrose in 10 m M Tris-HC1, pH 7.4). The final pellet (2.5- 3 ml/forty 15-cm plates) is resuspended after addition of 4 vol homogenization buffer and homogenized by passing the cell suspension 6 - 10 times through a ball-bearing homogenizer.4 The extent of cell breakage can easily be monitored by examining a small aliquot of the homogenate by light microscopy. The resulting homogenate can be stored frozen in liquid nitrogen up to 6 months without a loss in activity.
Preparation of Acceptor Homogenate Chinese hamster ovary wt cells used for acceptor preparations are usually grown in suspension in spinner flasks (Bellco, Vineland, NJ). In a typical preparation 3 liters of cell suspension (at 4 - 6 X 105 cells/ml) are harvested by centrifugation (10 min at 800 g and 4°), washed twice with TBS, and once with HB. The cell pellet ( 3 - 4 ml/3-1iter culture) is resuspended after addition of 4 vol HB and homogenized as described for the preparation of the donor homogenate. The acceptor homogenate can also be stored frozen in liquid nitrogen.
[2]
TRANSPORT BETWEEN GOLGI CISTERNAE
9
Preparation of Donor and Acceptor Golgi Membrane Fractions by Sucrose Density Centrifugation To 12 ml of either donor or acceptor homogenate, add 11 ml of 62% sucrose (w/w) in 10 m M Tris-HC1, pH 7.4 and 230°#1 100 m M EDTA, pH 7.4. After vigorous mixing, the final sucrose concentration is checked by refractometry and should be 37%. If necessary the sucrose concentration can be adjusted by addition of either 10 mMTris-HC1, pH 7.4, or 62% sucrose in 10 m M Tris-HCl, pH 7.4. The mixture is placed in the bottom of a polycarbonate SW28 tube (Beckman, Palo Alto, CA) and overlaid with 15 ml 35% (w/w) sucrose in I0 m M Tris-HC1, pH 7.4, and 9 ml 29% (w/w) sucrose in 10 m M Tris-HC1, pH 7.4. After centrifugation of the gradients for 2.5 hr at 25,000 rpm and 4 ° in an SW28 rotor, the membranes at the 35- 29% interface are recovered by puncturing the side wall of the tube with a syringe. From 12 ml of homogenate one usually obtains 1.5- 2 ml of the Golgi-enriched membrane fraction at a protein concentration of about 0.5- 1 mg/ml. After the membranes are frozen in liquid nitrogen in suitable aliquots, they can be stored at - 80 ° up to 1 year without a detectable loss of activity.
Preparation of Chinese Hamster Ovary Cytosol The high-speed supernatant or cytosol can be prepared by centrifuging homogenates from either CHO wt or 15B cell for 60 min at 55,000 rpm at 4 ° in an SW55 rotor. The supernatant is recovered without disturbing either the pellets or the whitish lipid floating on top, and is subsequently desalted on a P-6DG (Bio-Rad, Richmond, CA) column (2.5 × 50 cm) equilibrated in 50 m M KC1, 25 m M Tris-HCl, pH 7.4, 0.5 m M dithiothreitol (DTT). The void volume fractions containing the bulk of the protein are pooled, frozen in aliquots in liquid nitrogen, and stored at - 8 0 °.
Assay Conditions A standard transport reaction contains the following components: HEPES-KOH, 25 raM, pH 7.4 KC1, 25 11134 Magnesium acetate, 2.5 m M ATP, 50 p M UTP, 250 # M Creatine phosphate (CP), 2 m M Rabbit muscle creatine phosphokinase (CPK), 7.3 IU/ml UDP[3H]GIcNAc, 0.4/23//(0.5/2Ci)
l0
RECONSTITUTION IN CELL-FREE EXTRACTS
[2]
Donor membranes Acceptor membranes Cytosol This is prepared by mixing in a disposable glass tube [10 X 75 mm, e.g., Fisher Scientific (Pittsburgh, PA) 14-962-10A] on ice (per 50-/zl reaction): 5/tl of a 10X concentrated reaction buffer (250 mM HEPES-KOH, pH 7.4, 250 mM KC1, 25 mM magnesium acetate), 5 #l of an "ATP mix" [prepared fresh daily by mixing 5/tl CPK (1600 IU/ml, stored at -80°), 25/A 200 mM CP, 5/ll l0 mM ATP (neutralized with NaOH), 10/zl 100 mM UTP (neutralized with NaOH), and 65/tl water], 5/L1(0.1/tCi//d) UDP[3H]GIcNAc (prepared fresh by gently drying down the appropriate amount of the ethanolic stock under a stream of nitrogen gas), and finally the appropriate amount of water. To this mixture were added gel-filtered cytosol and freshly thawed donor and acceptor membranes. Although the optimal amounts of the latter three components can vary between preparations, good results are usually obtained using 5/tl of gel-filtered cytosol and 5/tl of both donor and acceptor membranes. Transport is initiated by transfer of the reaction to 37 ° and stopped by returning it to ice. Immunoprecipitation of Labeled Vesicular Stomatitis Virus G Protein The labeled VSV G protein is immunoprecipitated with a combination of a monoclonal anti-VSV G protein antibody 8G5 2° and a polyclonal rabbit anti-mouse-IgG antiserum (to form the actual immune complexes). Again, the exact amounts and ratio of the monoclonal and polyclonal antibodies needed to obtain an optimal immunoprecipitation of the VSV G protein will depend on the specific preparations used, although in general we find that 1/tl of the monoclonal antibody (at 1.3- 1.7 mg/ml protein) and 2/A of the polyclonal antiserum per transport reaction are sufficient. Immune complexes are preformed by preincubating (per reaction) 1/tl monoclonal anti-VSV G protein antibody and 2/tl of the rabbit anti-mouse IgG antiserum for 5-10 min at room temperature, followed by the addition of 50/tl stop buffer [1% (v/v) Triton X-100, 1% (w/v) sodium cholate, 5 mM EDTA, 250 mMNaCI in 50 mMTris-HCl, pH 7.5]. This is added to a transport reaction, mixed, and allowed to incubate for at least 1 hr at room temperature. The immune complexes are collected on glass microfiber filters (Cat. No. 934-AH; Whatman, Clifton, NJ) by vacuum filtration. To prevent nonspecific adsorption of proteins the filters are preblocked for 5 min in a 2.5% (w/v) solution of nonfat dry milk (Carnation) in detergent buffer (DB) [1% (v/v) Triton X-100, 5 mM EDTA, 250 mM NaC1 in 50 mM Tris-HC1, pH 7.5]. Each filter is first rinsed with 3 ml DB. The immuno-
[2]
TRANSPORT BETWEEN GOLGI C1STERNAE
11
precipitation reaction is then diluted with 3 ml DB and rapidly filtered. The tube is rinsed once with 3 ml DB, which is again filtered. Finally each filter is washed rapidly with two 3-ml aliquots of DB and subsequently dried under a heat lamp. The filters are transferred to scintillation vials, scintillation fluid (Scintiverse BD, Cat. No. SX18-4; Fisher Scientific) is added, and the samples are counted in a liquid scintillation counter.
Titration of acceptor membranes
Titration of donor membranes 12 co
10
10
X
8-t
01
~
6
r-
4-
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0
lag donor
4 ~g acceptor
Titration of CHO cytosol
Requirements for in vitro transport
1
2
3
0
4
2
6
e-
"o $
P
15
0
z
N
5
-1~
i:l .
0 0
.
20
.
40
.
.
60
~tg eytosol
80
~u
0 J. 100
8
? g
FIG. I. All incubations were performed for 60 rain at 37 ° as described in text. (A) Donor Golgi membranes were added at the amounts indicated to otherwise complete transport reactions, containing saturating amounts of cytosol ( 100 #g protein) and acceptor membranes (5 #g protein). (B) Increasing amounts of acceptor Golgi membranes were added to reactions containing saturating amounts of donor membranes (5 #g protein) and cytosol. (C) Increasing amounts of cytosol were added to transport incubations containing saturating amounts of donor and acceptor Golgi membranes. (D) Standard transoort reactions were carried out but, where indicated, the reaction was left on ice, or was performed in the absence of the ATP mix, cytosol, donor, acceptor, or UDP[3HIGIcNAc.
12
RECONSTITUTION IN CELL-FREE EXTRACTS
[3]
Demonstration of Requirements for in Vitro Transport The cell-free transport system described above has been extensively studied over the years and has allowed the characterization of multiple factors, some better defined than others, that are required for transport. 19 The general requirements for vesicular transport that one would have predicted from in vivo observations in fact turn out to be essential for transport in vitro: transport occurs only at physiological temperatures, and requires both ATP and cytosolic factors in addition to Golgi membranes. The donor and acceptor Golgi membranes and UDp[3H]GlcNAc are specific to the cell-free system used and are essential only to actually detect transport. Experiments demonstrating the different requirements for transport in the cell-free system are shown in Fig. 1.
[3] Detection of Endocytic Vesicle Fusion in Vitro, Using Assay Based on Avidin-Biotin Association Reaction By
WILLIAM A. BRAELL
Principle The detection of the fusion of endocytic vesicles in a cell-free system generally involves an assay in which two probes are incorporated into two different preparations of endocytic vesicles, isolated from separate populations of cells. These probes are configured to generate a detectable signal if they contact each other when the two populations of vesicles are permitted to fuse together in vitro. The use of a detection scheme relying on the avidin-biotin association reaction for such a measurement possesses unique advantages. First, the association of avidin with biotin is rapid and extremely strong (Ka 10-1~), which makes the association effectively irreversible.I Thus, the signal will develop immediately on vesicle fusion, and will remain even if the product vesicles are subsequently lysed and the reacted probes are isolated. Moreover, any nondetectable biotinylated proteins added extravesicularly will act as scavengers for any avidin outside the vesicles, because the irreversibility of the association reaction precludes the scavenged avidin from subsequently detaching and reacting with the detectable biotinylated protein. This ensures that the only detectable signal will result from avidin-biotin associations occurring within the interior of the fused vesicles. Second, the association reaction is insensitive to variaN. M. Green,
Biochem.J.
89, 585 (1963).
METHODS IN ENZYMOLOGY, VOL. 219
Copyright© 1992by AcademicPress,inc. All rightsof reproductionin any formreserved.
