[25]
DEFINING INTERMEDIATES IN E R - T O - G O L G I TRANSPORT
261
[25] U s e o f T w o - S t a g e I n c u b a t i o n s t o D e f i n e S e q u e n t i a l Intermediates in Endoplasmic Reticulum to Golgi Transport
By H. W. DAVmSON and W. E. BALCH Export of protein from the endoplasmic reticulum (ER) involves the formation (fission) of carrier vesicles from the ER, and their vectorial targeting and fusion to the cis-Golgi compartment. Each of these steps in protein transport is likely to require biochemically distinct components. In this chapter we describe an assay that efficiently reconstitutes the transport of vesicular stomatitis virus (VSV) G protein between the ER and the cis-Golgi compartment. To investigate the time at which individual components are required during the course of a single round of transport we utilize two-stage incubations. We also describe the principles related to the practice and interpretation of such assays. Vesicular Stomatitis Virus G Protein Transported as Synchronous Wave after T e m p e r a t u r e Shift Transport of VSV G protein between the ER and the cis-Golgi compartment involves a protocol that results in the export of a synchronous wave of protein from the ER. As illustrated in Fig. IA, transport between the ER and the Golgi proceeds through at least three distinctive kinetic phases. The first phase is a lag period (generally requiring 20 rain at 32 °). It is involved in vesicle formation and probably targeting. During this lag period no oligosaccharide processing of VSV G protein can be detected, indicating that the protein has not been delivered to the lumen of the cis-Golgi compartment. The lag period is followed by a period in which there is a progressive increase in the amount of processed VSV G protein, indicating delivery to the cis-Golgi compartment (Fig. IA). Because processing is virtually simultaneous with exposure of VSV G protein to the resident mannosidases and glycosidases of the cis-Golgi compartment, this step defines the progressive fusion of vesicles with the Golgi. Finally, there is a plateau after which no additional processing is observed, presumably measuring the maximum capacity of the assay to support transport between the ER and the cis-Golgi. To define the temporal requirements for protein factors involved in vesicle formation, targeting, and fusion, two different protocols can be employed. In the first protocol, inhibitors can be added during the time course of the reaction to prevent the function of one or more key compoMETHODS IN ENZYMOLOGY, VOL 219
Copyright© 1992by AcademicPress, Inc. All ~ghtsof reproduction in any form reserved.
262
IDENTIFICATION OF TRANSPORT INTERMEDIATES
A
I
sic c~osol ~lr
I
ATP NEM
I
I
,
o
At
~ ~
[2S]
to ice add ~ 9 0 m i n , inhibitor ;s~"
JJ" Time
~"
EGTA
IJ
g U-
Io
2Oll lag
6Oll processing
eo I pllteau
Time (At) (min)
B ER
Intermediate compartment
Golgi
;,o2,,, GTP'~ anti-rabl b NEM
I
H~G,u early
EGTA peptide
I I
I fate
FIG. 1. Transport of VSV G protein between endoplasmic reticulum and cis-Golgi compartment.
[25]
DEFINING INTERMEDIATESIN ER-TO-GOLGI TRANSPORT
263
nents during the course of transport. This is the method of choice for irreversible inhibitors. With reversible inhibitors, an additional approach may be applied in which a reversible inhibitor used to accumulate a transport intermediate is removed and the components required for further transport examined. These two protocols are described below. T i m e of Addition Experiments: Two-Stage Incubations to D e t e r m i n e Early vs Late Function in Transport In the first protocol, which we refer to as a "time of addition" experiment, specific or general inhibitors believed to block the function of one or more components are added to complete cocktail containing semiintact cells, cytosol, and ATP during the first stage at increasing times of incubation as illustrated in Fig. 1A [top flow diagram (At)]. After the addition of the inhibitor, incubations are continued in its presence in a second stage for the duration of the assay (generally a total time of 90 min). The second-stage incubation allows VSV G protein that was mobilized past the step sensitive to inhibition during the first stage to continue to the cis-Golgi compartment for processing in the second stage. Results obtained from such two-stage incubations will determine the potential role of the component(s) in vesicle formation (and possibly targeting) or fusion. If a component is required for vesicle formation, the component is likely to be required during the lag period. In this case, if the inhibitor is added at zero time in the first stage, vesicle formation will not occur and no processing will be observed during the second stage, because VSV G protein cannot exit the ER. However, after 20- 30 rain of incubation in the first stage in the absence of the inhibitor, a time period in which export from the ER is complete but little delivery to the Golgi can detected (Fig. 1), an early-acting component will no longer be required. At this time, addition of the inhibitor will have only a marginal effect on an early-acting component. Further incubation for 90 rain in the second stage will result in the efficient transport of VSV G protein to the cis-Golgi compartment. For early-acting components, when the total protein transported (that observed after a total of 90 min of incubation) is plotted for each time of addition of the inhibitor (A0, a curve is generated that shows no lag period and precedes the standard time course by 15- 25 min (Fig. 1; compare the early curve to the time course curve). Moreover, the total extent of transport observed after 90 min in the presence of inhibitor plateaus at a correspondingly earlier time point (Fig. 1, early curve). This idealized curve is diagnostic of components required for an early transport step. As shown in Fig. I B, using this protocol we have shown that formation of functional vesicles requires ATP and cytosol, requires the function of two
264
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[9.5]
known proteins, Rablb and N-ethylmaleimide-sensitive fusion protein (NSF), uncharacterized soluble and membrane-associated factors, and is sensitive to the general chemical inhibitor GTPyS. 1-9 In contrast to components required during vesicle formation in the lag period, components are also required for a late, vesicle fusion step. The activity of these components generates a different diagnostic curve when the time of addition experiment described above is performed. In this case, if an inhibitor blocks the step immediately preceding fusion (and processing by Golgi glycosidases and mannosidases is rapid relative to the fusion step), then addition of the inhibitor at each time point (At) in the first stage will stop the reaction abruptly, preventing further processing. Because inhibition of a late-acting component is equivalent to transferring the incubation to ice, further incubation for 90 rain during the second stage results in no additional processing. For late-acting components, when the total protein transported (that observed at 90 min) is plotted for each time of addition of the inhibitor (At), a curve is generated that is identical to the time course (Fig. 1A; compare late curve to the time course curve). In the extreme case, a membrane-permeant inhibitor may block the function of the Golgi processing enzymes. This possibility can be resolved by assaying the effect of the inhibitor on solubilized enzymes. Such inhibitors would obviously not provide useful information concerning transport components involved in late fusion steps. As illustrated in Fig. 1B, using this type of protocol we have shown that vesicle fusion requires Ca 2+ (0.1 #M), ATP, uncharacterized soluble and membrane-associated components, and is sensitive to a synthetic peptide analog homologous to the effector domains found in the rab gene family of small GTP-binding proteins.~-9 In the above protocol there are several variables that need to be considered. First, it is important to establish that processing in the Golgi compartment per se is not the rate-limiting step in the assay. If processing is slow relative to transport, the significance of early versus late becomes
t H. Plutner, A. D. Cox, R. Khosravi-Far, S. Pind, J. Bourne, R. Schwaninger, C. J. Der, and W. E. Balch, J. Cell Biol. 115, 31 (1991). 2 R. Schwaningcr, C. J. M. Beckers, and W. E. Balch, J. Biol. Chem. 266, 13055 (1991). H. Plutner, R. Schwaninger, S. Pind, and W. E. Balch, EMBO J. 9, 2375 (1990). 4 C. J. M. Beckers, H. Plutner, H. W. Davidson, and W. E. Balch, J. Biol. Chem. 265, 18298 (1990). C. J. M. Beckers, M. R. Block, B. S. Glick, J. E. Rothman, and W. E. Balch, Nature (London) 339, 397 (1989). 6 C. J. M. Beckers, and W. E. Balch, J. CellBiol. 108, 1245 (1989). W. E. Balch, J. Biol. Chem. 164, 16965 (1989). 8 C. J. M. Beckers, D. Keller, and W. E. Balch, CellSO, 523 (1987). 9 W. E. Balch, Curt. Opinions Cell Biol. 2, 634 (! 990).
[25]
DEFINING INTERMEDIATES IN ER-TO-GOLGI TRANSPORT
265
difficult to interpret because even a late-acting fusion protein would yield an apparent early phenotype in such two-stage incubations. As indicated previously, processing of VSV G protein by a-1,2-mannosidase I (Mann I) in the cis-Golgi compartment" or by N-acetylglucosamine transferase I (Tr I) in the medial-Golgi compartment is not rate limiting. 1° Second, it is important determine the half-time required for the function of an inhibitor. Some inhibitors, such as N-ethylmaleimide (NEM; a sulfhydryl alkylating reagent), are chemical reagents that can modify proteins during incubation on ice. These work very rapidly (hi2 of seconds). Pretreatment on ice has the added advantage that it allows the investigator to eliminate any variables related to kinetics of inhibition per se during incubation at temperatures that support transport. On the other hand, some inhibitors have a slow half-time [such as GTPTS (tl/z of minutes)] and will not inhibit on ice. These inhibitors block transport and may serve as competitive or noncompetitive substrates through incorporation into one or more key components during the cycling of transport components. For example, GTPTS efficiently competes for GTP, blocking transport at the step presumably requiting GTP hydrolysis.6 The half-time of inhibition will strongly influence the observed slope and point of origin of the time of addition curves. Determination of the half-time of inhibition for some inhibitors is easily accomplished by two-stage incubations. In this case, a complete reaction cocktail is incubated in the presence of the inhibitor from time zero. After increasing time (At), the inhibitor is washed out by pelleting of the semiintact cells (5 see at 10,000 g in a microfuge), followed by a subsequent incubation of the cells in the second stage for 90 min in complete cocktail lacking the inhibitor. A parallel incubation in the absence of the inhibitor serves to control for variables other than those directly affected by the inhibitor. Determination of the half-time required for inhibition is applicable principally to inhibitors that are irreversible and can be readily washed out. In instances where it is not possible to obtain a q/z of inhibition, interpretation of the participation of a component in an early or late step is more difficult. In general, time of addition experiments have been useful for determination of the temporal role of nucleotides through use of nucleotide analogs, and the temporal role of cytosolic and membrane components through the use of either the chemical inhibitors described above, 2-6 neutralizing antibodies that inhibit components of the transport machinery, z or augmentation of incubations depleted of known components by addition of the purified protein. 5 Because many of the components required for
~oW. E. Balch, W. G. Dunphy, W. A. Braell, and J. E. Rothman, Cell39, 405 (1984).
266
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[25]
vesicular trafficking are likely to function in the context of molecular complexes that undergo maturation and/or recycling for subsequent rounds of transport, these experiments are likely to inform the investigator of only one step in which a particular protein may participate in a complete round of vesicle fission and fusion. It is also important to emphasize that while the idealized curves shown above serve to delineate components potentially participating in the early vesicle formation vs late vesicle fusion steps, curves of intermediate values are likely to be obtained. These may reflect the properties of the inhibitors per se (such as efficiency of inhibition and tie2 of inhibition) or may reflect the participation of a component in an intermediate step in transport, such as targeting. The latter step is presently a difficult step to identify in semiintact cells, although current evidence tentatively suggests that targeting occurs rapidly relative to fusion.4 Two-Stage Assays to Accumulate Transport Intermediates A second approach that is complementary to the above experiments and provides a more defined role for individual components is to accumulate VSV G protein in transport intermediates. This approach can be used to study either the components required to form the intermediate or those required for its targeting and/or fusion to the cis-Golgi compartment. To identify components required to form a transport intermediate, semiintact cells are incubated in a first stage in the presence of a reversible inhibitor that is believed to block a later step in transport. Included in this first-stage incubation are the components being tested for their role in formation of the intermediate. Subsequently, the inhibitor is removed (by a brief pelleting of semiintact cells as described above) and the cells supplemented with a complete cocktail containing all the necessary components to support vesicle delivery to the cis-Golgi compartment. If a critical component required for vesicle formation is deleted in the first stage, no transport (processing of VSV G protein) will be observed in the second stage. Alternatively, to identify components required for fusion of a transport intermediate to the cis-Golgi compartment, a transport intermediate is again generated in the first stage by incubation in the presence of the reversible inhibitor and a completecocktail to ensure intermediate formation. In the second stage, cells are pelleted and reincubated in a cocktail containing components being tested for their putative role in later transport steps. If a particular component is required for delivery to the cisGolgi compartment, its absence in the second-stage incubation would preclude vesicle fusion and processing of VSV G protein.
[26]
USEOF sec MUTANTS
267
Summary Identification of the temporal requirement for components through the use of two-stage incubations is valuable in dissecting the overall transport reaction into steps relevant to vesicle fission and those related to vesicle fusion. In the context of semiintact mammalian cells in which a functional vesicle intermediate has not bccn detected, components playing a role in targeting arc presently difficult to identify. However, the two-stage incubations arc particularly powerful when either the donor or acccptor compartments can be manipulated independently, as is the case for intra-Golgi transport using enriched Golgi fractions or in the case of ER-to-Golgi transport in perforated yeast, in which a vesicle intermediate can be physically isolated.
[26]
U s e o f s e c M u t a n t s t o D e f i n e I n t e r m e d i a t e s in Protein Transport from Endoplasmic Reticulum B y MICHAEL F. REXACH and RANDY W. SCHEKMAN
Introduction
Vesicle-mediated protein transport from the endoplasmic rcticulum (ER) to the Golgi apparatus requires as many as 25 "transport proteins," which orchestratethe intermediate stagesof vesiclebudding, targeting,and fusion. The dissection of intermediates in this process is facilitatedby the availabilityof specificinhibitors:point mutations in any of the genes that encode a transport protein may render the genc product temperature sensitive for function, resultingin a conditional block at an intcrrncdiatc stage in ER-to-Golgi protein transport. Electron microscopic analysis of Saccharomyces cerevisiae strains that carry point mutations in sec ~ and yptl 2 genes showed a differentialrequirement for the gcne products in the formation or consumption of 60-nm vesicles.3,4Arc these vesiclesintermediates in the transfer of proteins from the E R to the Golgi apparatus? Which intermediate stage of vesicleformation is blocked, the generation of the vesicle or its scission from the E R membrane? What stage of vesicle consumption isblocked, the targetingof the vesicleto the Golgi membrane 1 p. Novick, C. Field, and R. Schekman, Cell 21,205 (1980). 2 D. GaUwitz, C. Donath, and C. Sander, Nature (London) 306, 704 (1983). 3 C. Kaiser and R. Schekman, Cell 61, 723 (1990). 4j. necker, T. Tan, H. Trepte, and D. Gallwitz, EMBOJ. 10, 785 (1991).
METHODS
IN E N Z Y M O L O G Y ,
VOL. 219
Copyright© 1992 by Academic Press,Inc. All rightsof reproductionin any form reserved.
DEFINING INTERMEDIATES IN E R - T O - G O L G I TRANSPORT
261
[25] U s e o f T w o - S t a g e I n c u b a t i o n s t o D e f i n e S e q u e n t i a l Intermediates in Endoplasmic Reticulum to Golgi Transport
By H. W. DAVmSON and W. E. BALCH Export of protein from the endoplasmic reticulum (ER) involves the formation (fission) of carrier vesicles from the ER, and their vectorial targeting and fusion to the cis-Golgi compartment. Each of these steps in protein transport is likely to require biochemically distinct components. In this chapter we describe an assay that efficiently reconstitutes the transport of vesicular stomatitis virus (VSV) G protein between the ER and the cis-Golgi compartment. To investigate the time at which individual components are required during the course of a single round of transport we utilize two-stage incubations. We also describe the principles related to the practice and interpretation of such assays. Vesicular Stomatitis Virus G Protein Transported as Synchronous Wave after T e m p e r a t u r e Shift Transport of VSV G protein between the ER and the cis-Golgi compartment involves a protocol that results in the export of a synchronous wave of protein from the ER. As illustrated in Fig. IA, transport between the ER and the Golgi proceeds through at least three distinctive kinetic phases. The first phase is a lag period (generally requiring 20 rain at 32 °). It is involved in vesicle formation and probably targeting. During this lag period no oligosaccharide processing of VSV G protein can be detected, indicating that the protein has not been delivered to the lumen of the cis-Golgi compartment. The lag period is followed by a period in which there is a progressive increase in the amount of processed VSV G protein, indicating delivery to the cis-Golgi compartment (Fig. IA). Because processing is virtually simultaneous with exposure of VSV G protein to the resident mannosidases and glycosidases of the cis-Golgi compartment, this step defines the progressive fusion of vesicles with the Golgi. Finally, there is a plateau after which no additional processing is observed, presumably measuring the maximum capacity of the assay to support transport between the ER and the cis-Golgi. To define the temporal requirements for protein factors involved in vesicle formation, targeting, and fusion, two different protocols can be employed. In the first protocol, inhibitors can be added during the time course of the reaction to prevent the function of one or more key compoMETHODS IN ENZYMOLOGY, VOL 219
Copyright© 1992by AcademicPress, Inc. All ~ghtsof reproduction in any form reserved.
262
IDENTIFICATION OF TRANSPORT INTERMEDIATES
A
I
sic c~osol ~lr
I
ATP NEM
I
I
,
o
At
~ ~
[2S]
to ice add ~ 9 0 m i n , inhibitor ;s~"
JJ" Time
~"
EGTA
IJ
g U-
Io
2Oll lag
6Oll processing
eo I pllteau
Time (At) (min)
B ER
Intermediate compartment
Golgi
;,o2,,, GTP'~ anti-rabl b NEM
I
H~G,u early
EGTA peptide
I I
I fate
FIG. 1. Transport of VSV G protein between endoplasmic reticulum and cis-Golgi compartment.
[25]
DEFINING INTERMEDIATESIN ER-TO-GOLGI TRANSPORT
263
nents during the course of transport. This is the method of choice for irreversible inhibitors. With reversible inhibitors, an additional approach may be applied in which a reversible inhibitor used to accumulate a transport intermediate is removed and the components required for further transport examined. These two protocols are described below. T i m e of Addition Experiments: Two-Stage Incubations to D e t e r m i n e Early vs Late Function in Transport In the first protocol, which we refer to as a "time of addition" experiment, specific or general inhibitors believed to block the function of one or more components are added to complete cocktail containing semiintact cells, cytosol, and ATP during the first stage at increasing times of incubation as illustrated in Fig. 1A [top flow diagram (At)]. After the addition of the inhibitor, incubations are continued in its presence in a second stage for the duration of the assay (generally a total time of 90 min). The second-stage incubation allows VSV G protein that was mobilized past the step sensitive to inhibition during the first stage to continue to the cis-Golgi compartment for processing in the second stage. Results obtained from such two-stage incubations will determine the potential role of the component(s) in vesicle formation (and possibly targeting) or fusion. If a component is required for vesicle formation, the component is likely to be required during the lag period. In this case, if the inhibitor is added at zero time in the first stage, vesicle formation will not occur and no processing will be observed during the second stage, because VSV G protein cannot exit the ER. However, after 20- 30 rain of incubation in the first stage in the absence of the inhibitor, a time period in which export from the ER is complete but little delivery to the Golgi can detected (Fig. 1), an early-acting component will no longer be required. At this time, addition of the inhibitor will have only a marginal effect on an early-acting component. Further incubation for 90 rain in the second stage will result in the efficient transport of VSV G protein to the cis-Golgi compartment. For early-acting components, when the total protein transported (that observed after a total of 90 min of incubation) is plotted for each time of addition of the inhibitor (A0, a curve is generated that shows no lag period and precedes the standard time course by 15- 25 min (Fig. 1; compare the early curve to the time course curve). Moreover, the total extent of transport observed after 90 min in the presence of inhibitor plateaus at a correspondingly earlier time point (Fig. 1, early curve). This idealized curve is diagnostic of components required for an early transport step. As shown in Fig. I B, using this protocol we have shown that formation of functional vesicles requires ATP and cytosol, requires the function of two
264
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[9.5]
known proteins, Rablb and N-ethylmaleimide-sensitive fusion protein (NSF), uncharacterized soluble and membrane-associated factors, and is sensitive to the general chemical inhibitor GTPyS. 1-9 In contrast to components required during vesicle formation in the lag period, components are also required for a late, vesicle fusion step. The activity of these components generates a different diagnostic curve when the time of addition experiment described above is performed. In this case, if an inhibitor blocks the step immediately preceding fusion (and processing by Golgi glycosidases and mannosidases is rapid relative to the fusion step), then addition of the inhibitor at each time point (At) in the first stage will stop the reaction abruptly, preventing further processing. Because inhibition of a late-acting component is equivalent to transferring the incubation to ice, further incubation for 90 rain during the second stage results in no additional processing. For late-acting components, when the total protein transported (that observed at 90 min) is plotted for each time of addition of the inhibitor (At), a curve is generated that is identical to the time course (Fig. 1A; compare late curve to the time course curve). In the extreme case, a membrane-permeant inhibitor may block the function of the Golgi processing enzymes. This possibility can be resolved by assaying the effect of the inhibitor on solubilized enzymes. Such inhibitors would obviously not provide useful information concerning transport components involved in late fusion steps. As illustrated in Fig. 1B, using this type of protocol we have shown that vesicle fusion requires Ca 2+ (0.1 #M), ATP, uncharacterized soluble and membrane-associated components, and is sensitive to a synthetic peptide analog homologous to the effector domains found in the rab gene family of small GTP-binding proteins.~-9 In the above protocol there are several variables that need to be considered. First, it is important to establish that processing in the Golgi compartment per se is not the rate-limiting step in the assay. If processing is slow relative to transport, the significance of early versus late becomes
t H. Plutner, A. D. Cox, R. Khosravi-Far, S. Pind, J. Bourne, R. Schwaninger, C. J. Der, and W. E. Balch, J. Cell Biol. 115, 31 (1991). 2 R. Schwaningcr, C. J. M. Beckers, and W. E. Balch, J. Biol. Chem. 266, 13055 (1991). H. Plutner, R. Schwaninger, S. Pind, and W. E. Balch, EMBO J. 9, 2375 (1990). 4 C. J. M. Beckers, H. Plutner, H. W. Davidson, and W. E. Balch, J. Biol. Chem. 265, 18298 (1990). C. J. M. Beckers, M. R. Block, B. S. Glick, J. E. Rothman, and W. E. Balch, Nature (London) 339, 397 (1989). 6 C. J. M. Beckers, and W. E. Balch, J. CellBiol. 108, 1245 (1989). W. E. Balch, J. Biol. Chem. 164, 16965 (1989). 8 C. J. M. Beckers, D. Keller, and W. E. Balch, CellSO, 523 (1987). 9 W. E. Balch, Curt. Opinions Cell Biol. 2, 634 (! 990).
[25]
DEFINING INTERMEDIATES IN ER-TO-GOLGI TRANSPORT
265
difficult to interpret because even a late-acting fusion protein would yield an apparent early phenotype in such two-stage incubations. As indicated previously, processing of VSV G protein by a-1,2-mannosidase I (Mann I) in the cis-Golgi compartment" or by N-acetylglucosamine transferase I (Tr I) in the medial-Golgi compartment is not rate limiting. 1° Second, it is important determine the half-time required for the function of an inhibitor. Some inhibitors, such as N-ethylmaleimide (NEM; a sulfhydryl alkylating reagent), are chemical reagents that can modify proteins during incubation on ice. These work very rapidly (hi2 of seconds). Pretreatment on ice has the added advantage that it allows the investigator to eliminate any variables related to kinetics of inhibition per se during incubation at temperatures that support transport. On the other hand, some inhibitors have a slow half-time [such as GTPTS (tl/z of minutes)] and will not inhibit on ice. These inhibitors block transport and may serve as competitive or noncompetitive substrates through incorporation into one or more key components during the cycling of transport components. For example, GTPTS efficiently competes for GTP, blocking transport at the step presumably requiting GTP hydrolysis.6 The half-time of inhibition will strongly influence the observed slope and point of origin of the time of addition curves. Determination of the half-time of inhibition for some inhibitors is easily accomplished by two-stage incubations. In this case, a complete reaction cocktail is incubated in the presence of the inhibitor from time zero. After increasing time (At), the inhibitor is washed out by pelleting of the semiintact cells (5 see at 10,000 g in a microfuge), followed by a subsequent incubation of the cells in the second stage for 90 min in complete cocktail lacking the inhibitor. A parallel incubation in the absence of the inhibitor serves to control for variables other than those directly affected by the inhibitor. Determination of the half-time required for inhibition is applicable principally to inhibitors that are irreversible and can be readily washed out. In instances where it is not possible to obtain a q/z of inhibition, interpretation of the participation of a component in an early or late step is more difficult. In general, time of addition experiments have been useful for determination of the temporal role of nucleotides through use of nucleotide analogs, and the temporal role of cytosolic and membrane components through the use of either the chemical inhibitors described above, 2-6 neutralizing antibodies that inhibit components of the transport machinery, z or augmentation of incubations depleted of known components by addition of the purified protein. 5 Because many of the components required for
~oW. E. Balch, W. G. Dunphy, W. A. Braell, and J. E. Rothman, Cell39, 405 (1984).
266
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[25]
vesicular trafficking are likely to function in the context of molecular complexes that undergo maturation and/or recycling for subsequent rounds of transport, these experiments are likely to inform the investigator of only one step in which a particular protein may participate in a complete round of vesicle fission and fusion. It is also important to emphasize that while the idealized curves shown above serve to delineate components potentially participating in the early vesicle formation vs late vesicle fusion steps, curves of intermediate values are likely to be obtained. These may reflect the properties of the inhibitors per se (such as efficiency of inhibition and tie2 of inhibition) or may reflect the participation of a component in an intermediate step in transport, such as targeting. The latter step is presently a difficult step to identify in semiintact cells, although current evidence tentatively suggests that targeting occurs rapidly relative to fusion.4 Two-Stage Assays to Accumulate Transport Intermediates A second approach that is complementary to the above experiments and provides a more defined role for individual components is to accumulate VSV G protein in transport intermediates. This approach can be used to study either the components required to form the intermediate or those required for its targeting and/or fusion to the cis-Golgi compartment. To identify components required to form a transport intermediate, semiintact cells are incubated in a first stage in the presence of a reversible inhibitor that is believed to block a later step in transport. Included in this first-stage incubation are the components being tested for their role in formation of the intermediate. Subsequently, the inhibitor is removed (by a brief pelleting of semiintact cells as described above) and the cells supplemented with a complete cocktail containing all the necessary components to support vesicle delivery to the cis-Golgi compartment. If a critical component required for vesicle formation is deleted in the first stage, no transport (processing of VSV G protein) will be observed in the second stage. Alternatively, to identify components required for fusion of a transport intermediate to the cis-Golgi compartment, a transport intermediate is again generated in the first stage by incubation in the presence of the reversible inhibitor and a completecocktail to ensure intermediate formation. In the second stage, cells are pelleted and reincubated in a cocktail containing components being tested for their putative role in later transport steps. If a particular component is required for delivery to the cisGolgi compartment, its absence in the second-stage incubation would preclude vesicle fusion and processing of VSV G protein.
[26]
USEOF sec MUTANTS
267
Summary Identification of the temporal requirement for components through the use of two-stage incubations is valuable in dissecting the overall transport reaction into steps relevant to vesicle fission and those related to vesicle fusion. In the context of semiintact mammalian cells in which a functional vesicle intermediate has not bccn detected, components playing a role in targeting arc presently difficult to identify. However, the two-stage incubations arc particularly powerful when either the donor or acccptor compartments can be manipulated independently, as is the case for intra-Golgi transport using enriched Golgi fractions or in the case of ER-to-Golgi transport in perforated yeast, in which a vesicle intermediate can be physically isolated.
[26]
U s e o f s e c M u t a n t s t o D e f i n e I n t e r m e d i a t e s in Protein Transport from Endoplasmic Reticulum B y MICHAEL F. REXACH and RANDY W. SCHEKMAN
Introduction
Vesicle-mediated protein transport from the endoplasmic rcticulum (ER) to the Golgi apparatus requires as many as 25 "transport proteins," which orchestratethe intermediate stagesof vesiclebudding, targeting,and fusion. The dissection of intermediates in this process is facilitatedby the availabilityof specificinhibitors:point mutations in any of the genes that encode a transport protein may render the genc product temperature sensitive for function, resultingin a conditional block at an intcrrncdiatc stage in ER-to-Golgi protein transport. Electron microscopic analysis of Saccharomyces cerevisiae strains that carry point mutations in sec ~ and yptl 2 genes showed a differentialrequirement for the gcne products in the formation or consumption of 60-nm vesicles.3,4Arc these vesiclesintermediates in the transfer of proteins from the E R to the Golgi apparatus? Which intermediate stage of vesicleformation is blocked, the generation of the vesicle or its scission from the E R membrane? What stage of vesicle consumption isblocked, the targetingof the vesicleto the Golgi membrane 1 p. Novick, C. Field, and R. Schekman, Cell 21,205 (1980). 2 D. GaUwitz, C. Donath, and C. Sander, Nature (London) 306, 704 (1983). 3 C. Kaiser and R. Schekman, Cell 61, 723 (1990). 4j. necker, T. Tan, H. Trepte, and D. Gallwitz, EMBOJ. 10, 785 (1991).
METHODS
IN E N Z Y M O L O G Y ,
VOL. 219
Copyright© 1992 by Academic Press,Inc. All rightsof reproductionin any form reserved.
