Q 1986 by The American Society of Biological Chemists, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 261, No. 31, Issue of November 5, pp, 14690-14696,1986

Printed in U.S.A.

ATP-coupled Transport of Vesicular Stomatitis Virus G Protein FUNCTIONAL BOUNDARIES OF SECRETORY COMPARTMENTS*

(Received for publication, May 2, 1986)

William E. BalchS and David S. Keller From the Department of Molecular Biophysics and Biochemistry, Yale Uniuersity, New Hauen, Connecticut 06510

The oligosaccharide processing intermediates of the vesicular stomatitis virus strain ts045 G protein were used to identify ATP- and temperature-sensitive steps in the constitutive pathway of protein transfer to the cell surface. In addition to the initial ATP-sensitive step required for export from the endoplasmic retieu- lum (Balch, W. E., Elliott, M. M., and Keller, D. S. (1986) J. Biol. Chem. 261, 14681-14689), two dis- tinct ATP-sensitive steps functionally dissect the Golgi into at least 3 compartments: a cis compartment con- taining the trimming enzyme mannosidase I, a medial compartment conferring resistance to endoglycosidase H, and a trans compartment containing terminal gly- cosyl transferases. A fourth ATP-sensitive step is re- quired for export of G protein from the trans Golgi to the cell surface. A high threshold of cellular ATP (70% of the control) was required for maximal rates of trans- port between Golgi compartments. Transport between compartments is inhibited at 40% of the normal cellu- lar ATP pool. Only a single temperature-sensitive step localized to the endoplasmic reticulum inhibited trans- port of ts045 G protein to the cell surface. The data suggest that ATP-sensitive steps punctuate transport of protein between compartmental boundaries of the secretory pathway.

The sequential morphologically (1 ,2) and biochemically (3) defined steps involved in the transport of protein along the secretory pathway provide ample evidence for the role of the ER’ and the Golgi in delivery of protein to the cell surface. At least 3 distinct compartments are now recognized as func- tional domains of the Golgi complex (4). In addition, pre- and post-Golgi elements (5 , 6) may be important way stations in intracellular trafficking of protein in the constitutive pathway of protein transport (7). Transport between cellular compart- ments is dissociative, implying the selective budding and fusion of carrier vesicles between compartmental boundaries (8-10). Both genetic (11) and biochemical (12) studies suggest that many cellular components are required for this process at different stages of the secretory pathway. The biochemical mechanisms which facilitate transport remain to be resolved.

One important component for transport between compart- ments of the exocytic pathway is ATP. Jamieson and Palade

* This work was supported by Research Grant GM-33301 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom all correspondence should be addressed: Dept. of Mo- lecular Biophysics and Biochemistry, Yale University School of Med- icine, 333 Cedar St., P. 0. Box 3333, New Haven, CT 06501.

The abbreviations used are: ER, endoplasmic reticulum; endo H, endoglycosidase H.

(13) used autoradiography and cell fractionation to estab- lished that ATP is required for export of protein from the ER in the pancreatic acinar cell. Cellular ATP is important in the assembly of secretory granules (14) and delivery of their content to the cell surface (13). Transport between secretory compartments in yeast is energy dependent (11). Transport of vesicular stomatitis virus G protein in vitro between com- partments of the Golgi requires ATP (46).

We have established the quantitative requirement for ATP during transport of protein at an early stage of the secretory pathway in uiuo (16). A remarkably high threshold of cellular ATP is required for an initial step in export of protein from the ER. The quantitative requirement for ATP in transfer of protein between the ensuing subcellular compartments is unknown. The temporal and stoichiometric requirements for these later ATP-sensitive steps in the constitutive pathway could provide insight into the mechanisms defining functional boundaries of exocytic compartments.

To elucidate the role of ATP in transport, we have taken advantage of the extensive body of knowledge of the transport of G protein, the surface glycoprotein of vesicular stomatitis virus (17). In the present work we use the thermoreversible export of vesicular stomatitis virus ts045 G protein, a mutant temperature-sensitive strain which is defective in the export of G protein from the ER when cells are grown at the restric- tive temperature but not the permissive temperature (18-24), to identify at least 3 new ATP-sensitive steps which are required for transport of G protein to the cell surface. These steps punctuate transfer a t compartmental boundaries which have previously been shown to be dissociative steps in trans- port of protein through the Golgi (9, 10). Based on the temporal location of these ATP-sensitive steps, the cellular location of mannosidase I is assigned to an early (cis) Golgi compartment.

EXPERIMENTAL PROCEDURES

Materials All materials were obtained as described previously (16).

Methods Transport Assay-Growth of cells, labeling with [35S]methionine,

and conditions for the transport assay were exactly as described previously (16). Samples were processed with endoglycosidase H (endo H) by resuspending the cell pellet for each time point in a buffer containing 1% sodium dodecyl sulfate, 50 mM Tris.HC1 (pH 6.8), and 15 mM dithiothreitol. Each sample was boiled for 5 min and diluted 1:20 with 0.1 M sodium acetate (pH 5.5) containing 1 mM phenylmethylsulfonyl fluoride. The samples were digested with 0.02 unit/ml of endo H for 24 h at 37 “C. Samples were processed for sodium dodecyl sulfate-gel electrophoresis using 10% polyacrylamide gels according to the method of Laemmli (25) . Gels were treated for autoradiographic enhancement (Enlightening,@ New England Nu- clear), dried, and autoradiographed for 1-4 days (XAR-5, Kodak). The fraction of the total G protein processed to endo H-resistant or

14690

ATP-coupled Golgi Transport 14691

complex forms (see "Results") was determined by densitometry of the exposed autoradiograph using a GS300 transmission densitometer (Hoefer Scientific Instruments) connected to an IBM-XT" pro- grammed with the GS350 integrating software (Hoefer Scientific Instruments).

Detection of Cell Surface G Protein-The amount of cell surface G protein for ea'ch time point in the transport assay was determined by incubating cells with 15 p1 of rabbit anti-G protein antiserum for 2 h at 4 "C in 100 pl of labeling medium. Cells were washed twice with 100 pl of labeling medium and resuspended in detergent buffer con- taining 50 mM Tris.HCI (pH 7.5), 250 mM NaCI, 1 mM NaZEDTA, 1% Triton X-100, and 1% sodium cholate. 25 pl of 10% Staphylococcus A (Pansorbin,@ Behring Diagnostics) which was prewashed twice with detergent buffer before use was added to the lysed cells. After incu- bation for 30 min at 4 "C, the Staphylococcus A cells were washed twice with 150 p1 of detergent buffer and once with distilled HZ0 before processing for sodium dodecyl sulfate-gel electrophoresis (25). Total G protein detected on the cell surface for each time point was determined by densitometry of the exposed autoradiograph as de- scribed above.

Depletion of Cellular ATP and ATP Determinution-Depletion of cellular ATP by incubation under an N, gas phase and determination of the cellular ATP content at each time point were carried out exactly as described previously (16).

RESULTS

Processing Can Be Used to Follow Transport of G Protein between Compartments of the Golgi-The transport of ts045 G protein from the ER to an early compartment of the Golgi was shown previously to be inhibited by incubation of cells at the restrictive temperature (40 "C) or by depletion of cellular ATP (16). Since export of ts045 G protein from the ER is thermoreversible (16, 26) we examined whether G protein is similarly sensitive to temperature and ATP during subsequent transport through the Golgi compartments to the cell surface. Identification of these steps would provide insight into the functional compartmentalization of the secretory pathway.

The oligosaccharide processing intermediates of G protein as it is transported through the Golgi have been extensively studied (3, 24, 27-29). After trimming by the mannosidase I to the Man5GlcNAcp structure upon arrival in an early Golgi compartment (29, 30) G protein is processed sequentially by N-acetylglucosamine transferase I (31, 32) and mannosidase I1 (28, 33-34) to form the GlcNAclMan3GlcNAc2 species which is resistant to the activity of endo H (35). The endo H- sensitive (band C in Fig. 1) and the various resistant forms of G protein (bands B + A in Fig. 1) can be distinguished by their unique mobility using SDS-gel electrophoresis. Both N- acetylglucosamine transferase I and mannosidase I1 have been localized morphologically to a medial Golgi region (36, 44). Thus, conversion of G protein from the endo H sensitive (band C in Fig. 1) to the endo H-resistant form (bunds B + A in Fig. 1) is a hallmark of arrival in a medial Golgi compart- ment.

G protein is subsequently processed from the Glc- NAclMan3GlcNAcz form by the sequential action of N-ace- tylglucosamine transferase 11, galactosyl transferase, and sia-

Time 0 0 I O 20 30 45 60 60 Endo H - + + + + + + -

Sensitive - I A

C B

FIG. 1. Processing of ts045 G protein in the Golgi. Cells infected with ts045 were pulsed with [35S]methionine for 5 min at 40 "C as described under "Experimental Procedures" and incubated at 30 "C for the indicated time in the presence of excess unlabeled methionine. Transport was terminated by transfer to ice, and samples were processed in the presence (+) or absence (-) of endo H as described under "Experimental Procedures." The G proteins were analyzed by fluorography after SDS-gel electrophoresis (25).

lyl transferase to form the biantennary complex structure containing sialic acid (Sia) and galactose (SiazGa12Glc- NAc2Man3GlcNAc2) in Chinese hamster ovary cells (3, 28). The form of G protein which is observed to migrate signifi- cantly slower than the high mannose form found in the ER ( t = 0, minus endo H in Fig. 1) during gel electrophoresis has been previously demonstrated to contain galactoses or galac- tose and sialic acid (37) (band A in Fig. 1). Thus, the galactose- and sialic acid-containing (complex) endo H-resistant forms of G protein (band A in Fig. 1) can be readily distinguished from the earlier endo H-resistant processing intermediates (15) (band B in Fig. 1). Galactosyl transferase has been localized morphologically to the trans Golgi region (38). Ap- pearance of the complex form therefore provides a reliable marker for transport to the terminal Golgi compartment.

The distinct lag periods observed for the appearance of these two structural forms of ts045 G protein after shift to the permissive temperature is consistent with the temporal locations of the processing enzymes in the Golgi compart- ments (Fig. 2).

Transport through the Golgi Is Sensitive to ATP-To deter- mine whether processing or transport of G protein between these morphologically distinct compartments of the Golgi is temperature or ATP sensitive, cells infected with ts045 were incubated at the permissive temperature (30 "C) for 15 min to allow G protein to enter the Golgi. Cells were subsequently transferred to the restrictive temperature or incubated under an N2 gas phase to deplete cellular ATP. Processing of G protein to both the endo H-resistant and complex forms continued for 5-10 min before reaching an early plateau (Fig. 2).

The temporal locations of these steps along the exocytic pathway were identified by incubating infected cells for an increasing time at 30 "C prior to transfer to 40 "C or transfer to an N2 gas phase (Fig. 3). If a temperature- or ATP-sensitive step corresponds to the site previously identified for export of G protein from the ER (16), then incubation for even a brief period of time at the permissive temperature will result in a portion of G protein proceeding past the restrictive step. Processing proportional to the amount of G transferred be- yond this step will be observed after a shift to the restrictive conditions (the plateau value observed in Fig. 2). Alterna- tively, if additional temperature- or ATP-sensitive steps occur a t later times in the pathway, then a more extensive period of incubation at the permissive conditions will be required before G protein can be processed to the more mature forms at the restrictive conditions. The length of the incubation at the permissive temperature will define the cellular location of the block.

As shown in Fig. 3, additional temperature-sensitive steps do not intercede in the transport of G protein from the ER to the trans Golgi. Processing to the endo H-resistant (Fig. 3A, closed circles) and complex forms (Fig. 3B, closed circles) occurred with kinetics consistent with a single temperature- sensitive step defined by export from the ER (16). In contrast, the transport of G protein through the Golgi compartments was found to be sensitive to ATP. A short -3-4-min incuba- tion at the permissive temperature was required for the trans- fer of G protein through an ATP-sensitive step preceding the compartment conferring endo H resistance (medial Golgi) (Fig. 3A, open squares). A longer incubation (7-8 min) was required for transfer through an ATP-sensitive step preceding the trans Golgi as shown by the appearance of the complex structure (Fig. 3B, open squares). In each case, a 5-min time period was observed between transport through the ATP- sensitive step (Fig. 3, open squares) and processing of G

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ature and ATP. Cells infected with ts045 were pulsed with [35S] methionine for 5 min at 40 "C and incubated for 15 min at 30 "C in the presence of excess unlabeled methionine. Cells were subsequently tranferred to 40 "C (0) or incubated under an N2 gas phase at 30 "C (0) for the indicated time ( t ) . Transport was terminated by transfer of samples to ice. All samples were processed with endo H as described under "Experimental Procedures" and were analyzed using SDS-gel electrophoresis (25). The fraction of total G protein in the endo H- resistant (Panel A ) or complex (panelB) structures during incubation was quantitated by densitometry of the exposed autoradiograph.

protein in the control (Fig. 3, open circles). A similar result was obtained for wild-type G protein (data not shown). The kinetics of transit through the ATP-sensitive step exactly parallel the kinetics of processing, thus ensuring that proc- essing is not the rate-limiting step in the present analysis.

Since transfer of G protein from the ER to the compartment containing mannosidase I occurs through an initial tempera- ture-sensitive ATP-coupled step (16), the two distinct ATP- sensitive temperature-insensitive transport steps discovered here suggest that at least three compartments comprise the temporal and functional organization of the Golgi.

Inhibition of Transport by Depletion of Cellular ATP Is Reversible-To establish that transport, not processing, was inhibited by ATP depletion, cells were first incubated for 15 min to transport G protein to the Golgi (Fig. 4, open circles). Some of the cells were subsequently incubated under an N2 gas phase at 30 "C for 20 min (Fig. 4, open squares). Since

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sensitive to ATP. Cells infected with ts045 were pulsed with [35S] methionine for 5 min at 40 "C and incubated in the presence of excess unlabeled methionine at 30 "C. Cells were either transferred to ice at time t (O), transferred to 40 "C at time t and incubated for an additional 15 min (O), or transferred to an N2 gas phase at 30 "C at time t and incubated for an additional 15 min (0). Cells were proc- essed as described in the legend to Fig. 2. Panel A presents the fraction of total G protein found in the endo H-resistant structure during incubation. Panel B presents the fraction of total G protein found in the complex structure during incubation.

processing of G protein is sequential, transport in the absence of processing would prevent recovery of G protein in the fully processed form upon return to the permissive conditions. When cells were reincubated in the presence of ATP, trans- port of G protein resumed with a brief lag (Fig. 4, open squares). G protein was completely recovered in the fully processed form. These results suggest that G protein transport was arrested in intermediate Golgi compartments, the lag reflecting the amount of time required to regain cellular ATP (16).

In contrast to the above results, when cells containing G protein in the Golgi were incubated at the restrictive temper- ature for 20 min (Fig. 4, closed circles), an extensive lag was observed before processing resumed upon reincubation at the permissive temperature (Fig. 4, closed circles). 95% of the endo H-resistant form of G protein was recovered after extended incubation at the permissive temperature (Fig. 44, closed circles). Only 60-70% of the G protein was recovered in the complex structure (Fig. 4B, closed circles). These results are

ATP-coupled Golgi Transport 14693

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sensitive to ATP. Cells infected with ts045 were pulsed with [35S] methionine for 5 min at 40 "C and incubated in the presence of excess unlabeled methionine at 30 "C. At 15 min, a portion of the cells was incubated under an N, gas phase at 30 "C for 15 min prior to reincubation in the presence of O2 (0). Alternatively, at 15 min a portion of the cells was incubated at 40 "C for 15 min prior to reincubation at 30 "C (0). In the control incubation (0) at 30 "C transport was terminated at the indicated time ( t ) . Cells were proc- essed as described in the legend to Fig. 2. Panel A is the fraction of total G protein processed to the endo H-resistant structure during incubation. Panel B is the fraction of total G protein processed to the complex structure during incubation.

consistent with the interpretation that processing (terminal glycosylation) of ts045 G protein is sensitive to incubation at the restrictive temperature. In contrast, incubation of wild- type G protein at 40 "C does not inhibit formation of the complex structure (data not shown). In comparison to the rapid recovery of transport by addition of O2 to ATP-depleted cells, the lag observed for initiation of processing after a temperature block may reflect in part the requirement for transport of G protein from the initial temperature-sensitive site in the ER.

Transport between the trans Golgi and the Cell Surface Is Sensitiue to ATP-The time course for transport of G protein to the cell surface is shown in Fig. 5 (open circles). To determine whether an ATP-sensitive site intercedes between the Golgi and the cell surface, cells were incubated for 30 min at 30 "C. Cells were subsequently transferred to an N2 gas phase at 30 "C. Transport to the cell surface in the presence of NZ reached an early plateau after an additional 5-10 min of incubation (Fig. 5, closed circles). Only the fully processed

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Time (t), min FIG. 5. Transport of G protein to the cell surface is sensitive

to ATP. Cells infected with ts045 were pulsed for 5 min with [35S] methionine at 40 "C and incubated in the presence of excess unlabeled methionine for the indicated time t (0). At 30 min, a portion of the cells was incubated at 40 "C (0) or under an N2 gas phase at 30 "C (0) for time t . Transport was terminated by transfer to ice, and cell surface G protein was determined as described under "Experimental Procedures." Each time point is reported as the fraction of the G protein found on the cell surface relative to the control value at 60 min of incubation (0).

complex form was detected on the surface (data not shown). In contrast to ATP inhibition, when cells were shifted to

40 "C after incubation for 30 min at 30 "C, the appearance of G protein on the cell surface rapidly peaked (Fig. 5, open squares). Further incubation resulted in loss of G protein from the cell surface. This result was not observed during incuba- tion under N2. Loss of G protein was not due to budding of virus from the cell surface at 40 "C (data not shown). 40% of the total G protein found on the cell surface at the peak time point (Fig. 5, open squares, 45 min) was endo H resistant, but not processed to the complex form (data not shown). These results are consistent with the previous observation that terminal glycosylation of ts045 G protein is sensitive to in- cubation at the restrictive temperature. Thus, late steps in processing, not transport, are sensitive to temperature after export of G protein from the ER.

To define the temporal location of the ATP-sensitive step(s) regulating transport to the cell surface, cells infected with ts045 were incubated for increasing time at the permis- sive temperature prior to incubation under an N2 gas phase. A third ATP-sensitive step at 15 min preceded transport of G protein to the cell surface (Fig. 6A, open circles). It was distinct from the two earlier sites observed for transport between compartments of the Golgi (Fig. 3). A lag of -10 min was observed between the time of transit through this step and arrival on the cell surface (Fig. 6A, closed circles). An identical ATP-sensitive step was required for delivery of G protein to the cell surface in ts045-infected 15B cells (Fig. 6B).Transport of wild-type G protein at 37 "C is similarly transferred through a late ATP-sensitive step prior to delivery to the cell surface (data not shown). Only the initial temper- ature-sensitive ER export step (16) impaired delivery of ts045 G protein to the cell surface (Fig. 6, open squares).

Export from the trans Golgi-To establish that the third

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G protein to the cell surface. Cells infected with ts045 were pulsed with [35SS]methionine for 5 min at 40 "C and incubated in the presence of excess unlabeled methionine at 30 "C. At each time point ( t ) samples were either transferred to ice (O), incubated a t 40 "C for up to 30 min (U), or incubated under an N? gas phase at 30 "C for 30 min (0). Cell surface G protein was determined as described under "Experimental Procedures." The value reported for incubations at 40 "C (m) was the maximum amount of cell surface G protein detected between 10 and 30 min of incubation (see Fig. 5). Each time point is reported as the fraction of the G protein found on the cell surface relative to the control value at 60 min of incubation (0). Panel A transport of G protein in wild-type cells. Panel B, transport of G protein in clone 15B cells.

ATP-sensitive step corresponds to export from the Golgi, cells were incubated for 2.5 h a t 20 "C to accumulate G protein in the trans Golgi tubular complex (39). When cells were shifted from 20 to 30 "C, G protein rapidly (tnh = 10 min) appeared on the cell surface in the complex form (Fig. 7, closed circles), a time period substantially less than that required for trans- port from the ER to the cell surface (tlh = 40 min, Fig. 6). When cells were transferred directly from 20 "C to an N2 gas phase a t 30 "C and incubated for an additional 45 min (Fig. 7, open circles, t = O), less than 30% of the total G protein transported in the control (Fig. 7, closed circles, t = 40) was recovered on the cell surface a t reduced cellular ATP. These

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face is ATP sensitive. Cells infected with ts045 were pulsed for 5 min with [35S]methionine at 40 "C and incubated for 2.5 h at 20 "C in the presence of excess unlabeled methionine. At each time point (t) cells were either transferred to ice (O), incubated at 40 "C for 15 min (O), or incubated under an N2 gas phase for 30 min (0). Cell surface G protein was determined as described under "Experimental Procedures." Each time point is reported as the fraction of the G protein detected on the cell surface relative to the control value at 40 min of incubation (0).

data suggest that a major fraction of G protein resides in a compartment in which processing is substantially complete, but export is ATP sensitive. When cells were transferred from 20 to 30 "C and incubated for -10 min prior to ATP depletion (Fig. 7, open circles), the major fraction of the total G protein observed in the control incubation was recovered on the cell surface. Transport to the cell surface was not inhibited by transfer of cells from 20 "C to the restrictive temperature (Fig. 7 , open squares).

ATP-sensitive Steps Require a High Threshold of Cellular ATP to Maintain Maximum Transport-Transport of G pro- tein from the ER to the Golgi was previously shown to be highly sensitive to the cellular ATP pool (16). The ATP dependence of transport between Golgi compartments was examined at reduced steady state levels of ATP (16). Cells were incubated for 15 min to populate the Golgi as before but subsequently incubated for 10 min under an N, gas phase in the absence of glucose to deplete cellular ATP pools (16) and accumulate G protein at an ATP-sensitive step. After 10 min under N P , vials were supplemented with increasing concentra- tions of glucose, and the extent of G protein converted to the endo H-resistant structure was determined after an additional 20 min of incubation (Fig. 8, open squares). An ATP pool of at least 70% of the control value was required for optimal rates of transport between Golgi compartments. Transport was totally inhibited at 40% of the normal level.

The ATP dependence of transport from the trans Golgi to the cell surface was examined using an identical procedure (Fig. 8, closed circles). Transport was similarly sensitive to reduced cellular ATP pools when G protein transport was reinitiated by the addition of glucose to ATP-depleted cells.

ATP-coupled Golgi Transport 14695 n

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Golgi compartments and between the Golgi and the cell sur- face at steady state ATP. 0, cells infected with ts045 were pulsed with [35S]methionine for 5 min at 40 "C in minus glucose labeling medium (16) and incubated for 15 min at 30 'C in the presence of excess unlabeled methionine. Cells were incubated for 15 min at 30 "C and subsequently incubated for 10 min at 30 "C under an N, gas phase to deplete cellular ATP (16). After 10 min under N,, increasing concentrations of glucose were added to each vial, and incubation continued for an additional 20 min. G protein transported between Golgi compartments at steady state ATP was determined by subtract- ing the extent of G protein processed to the endo H-resistant form observed at 10 min from that observed at 30 min of incubation under N,. 0, G protein transported to the cell surface at steady state ATP was determined in an identical fashion to that described above with the exception that cells were preincubated at 30 "C for 30 min before incubation under N2. Extent of transport and the steady state cellular ATP for each condition is reported as the fraction observed in the 0, control.

DISCUSSION

ATP-sensitive Punctuation Steps Define the Functional Boundaries of Golgi Compartments-Extending the observa- tions presented in the previous paper (16) we took advantage of the thermoreversibility of G protein export from the ER to document three unique ATP-sensitive stages in the transport of G protein to the cell surface: two between compartments of the Golgi and a third between the trans Golgi and the cell surface. Assignment of the temporal location of the ATP- sensitive steps is accurate only if inhibition of transport is rapid upon incubation under Nz. Experiments similar to those used previously (16) to establish this point were not useful for these later compartments. The temporal assignment for each of these steps must be interpreted as the minimum transport time to arrive at the new ATP-sensitive site. Based on the kinetics of processing, each stage could be defined by a two-step transfer, an initial ATP step followed by an ATP- insensitive step. The quantitative requirement for ATP in the transport of G protein between Golgi compartments was similar to that observed for export of G protein from the ER (16).

The compartmentalized organization of the Golgi proposed by Griffiths et al. (40) has been confirmed and extended on the basis of fractionation (41, 42), in vivo localization of processing enzymes (36, 38, 43, 44), and more recently, iden- tification of boundaries which define the vectorial transport of protein in vivo between Golgi elements (9, 10) (for a recent review see Ref. 4). The ATP-sensitive steps identified here punctuate G protein transport, providing a new functional definition for these compartmental boundaries. In particular, the second ATP-sensitive step inhibiting conversion of G

protein to the early endo H-resistant forms provides the first functional evidence that mannosidase I is located in a distinct compartment between the ER and the medial Golgi compart- ment containing N-acetylglucosamine transferase I (42) and mannosidase I1 (44). These data support the current "concen- sus" model for a 3-compartment structure of the Golgi (4): a cis compartment containing the trimming enzyme mannosi- dase I, a medial compartment containing the processing activ- ities conferring endo H resistance, and a trans compartment containing terminal glycosyl transferases (Fig. 9).

Temperature-sensitive Transport-Only one temperature- sensitive step was detected for transport of ts045 G protein to the cell surface. This step was identical to the ATP-coupled temperature-sensitive step observed for the export of G pro- tein from the ER of clone 15B cells (16), providing an impor- tant internal control to establish the distinct cellular locations of the additional ATP-sensitive steps in the wild-type Chinese hamster ovary cell line. In addition, these data suggest that the initial ATP-sensitive ER export step may differ from later ATP-sensitive steps for transfer of G protein through the Golgi to the cell surface.

While transport through the Golgi to the cell surface was not inhibited at the restrictive temperature, terminal glyco- sylation of ts045 G protein was found to be sensitive. G protein transported from the Golgi to the cell surface at the restrictive temperature was endo H resistant, but not processed to the complex form. In addition, incubation at the restrictive tem- perature led to an unusual loss of cell surface ts045 G protein not observed at 30 "C or under Nz. Since ts045 G protein in mature virus is irreversibly aggregated after incubation at the restrictive temperature (45), a related conformational change could alter oligosaccharide processing and susceptibility to internalization or proteolysis at the restrictive temperature.

Role of ATP in Transport-Transport along the secretory pathway occurs by the budding and fusion of carrier vesicles (1, 8, 9, 10, 46). The requirement for ATP should reflect one or more steps in the formation or targeting of these carrier vesicles. Recent evidence has suggested that ATP is required for internalization of receptor-ligand complexes through coated pits from the cell surface (47, 48). Although reversal of inhibition of transport by addition of glucose clearly sup- ports the conclusion that we are looking at an effect related to energy, it is presently unclear whether ATP per se or a secondary product generated as a consequence of ATP deple- tion is responsible for the effects observed here.

What are the targets for inhibition by ATP depletion? One possibility is based on the observation that ionophores such as monensin disrupt trafficking through the Golgi (49). ATP- dependent proton pumps are important for proper function

ER GOLGI CELL

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FIG. 9. ATP-sensitive sites in the exocytic pathway.

14696 ATP-coupled Golgi Transport

of the endosomal pathway (50). ATP depletion may function- ally disrupt a critical ion gradient maintained by these en- zymes leading to a rapid inhibition of transfer. If so, similar activities have yet to be demonstrated to be important for export from the ER.

A second possibility is that components of the transport machinery are sensitive to ATP depletion. Genetic analysis of the yeast secretory pathway indicates that at least 23 gene products are required for transport of all protein to the cell surface (11). Transport of G protein in vitro between com- partments of the Golgi requires ATP and both membrane- bound and soluble cytoplasmic components (46). The bio- chemical pathway which regulates the recycling of these com- ponents in vivo for multiple rounds of budding remains to be identified. It has recently been observed in the pancreatic acinar cell that cellular ATP depletion results in a rapid loss of transport vesicles between ER and Golgi and at the rims of the Golgi, and accumulation of fibrous aggregates and empty cagelike structures in the cytoplasm (51). Since an initial step in transport of protein at each stage is coupled to cellular ATP, inhibition of vesicle formation through disrup- tion of the normal recycling of components of the exocytic machinery at reduced ATP (possibly through reversible phos- phorylation steps) may be the key to understanding the strik- ing ATP dependence, a line of investigation we are currently pursuing.

Acknowledgment-We wish to thank Dr. S. Schmid for helpful discussion and critical reading of the manuscript.

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