Dec 2, 1993 - Peter J. Peters$, Richard D. Klausner, and. Julie G. Donaldsonll ..... Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz. J. (1992) J. Cell.
Communication
THE JOURNAL OF BIOUXICAL CHEMISTRY Vol. 269, No. 5, Issue of February 4, pp. 3135-3138, 1994 Printed an U.S.A.
Hydrolysis of the ARF-bound G T P is proposed to be coupled the to the release of bothARF-GDPandcoatomerfrom membrane (7). Pharmacologic reagents that perturb the activation/ inactivation cycle of GTPases have proved tobe valuable tools for dissecting their functions. Addition of the nonhydrolyzable (Received for publication, November 1, 1993, and in revised form, December 2, 1993) GTP analogue GTPyS, which persistently activates GTPases, of ARF and results in enhancedandirreversiblebinding Stephanie B. Teal$, Victor W. Hsu, coatomer to Golgi membranes (8-10). In contrast, addition of Peter J. Peters$, RichardD. Klausner, and the activation ofARF catalyzed the by brefeldinA(BFA1 inhibits Julie G. Donaldsonll Golgi-associated nucleotide exchange proteinand prevents the From the Cell Biology a n d Metabolism Branch, binding ofARF and, therefore, coatomer to membrane (8,9,11). National Instituteof Child Health and Human While it is tempting to conclude that ARF is the sole GTP bindDevelopment, National Institutes of Health, ing protein required for coatomer binding, previous experiments Bethesda, Maryland 20892 do not exclude a requirement for other GTP binding proteins The Ras-relatedproteinADP-ribosylationfactor 1 known to be associated with the Golgi membrane (12-14). is a low molecular weightGTP binding protein, To determine whether ARFlis both the sole, GTP-requiring which in its GTP state supports the binding of coatomer, component for coatomer binding andthe sole target of BFA in a cytosolic coat protein complex, toGolgi membranes. this process, we constructeda point mutation in h u m a n A R F l To create an "active" A R F , we constructeda point muta- that was predicted to inhibitthe rate of GTP hydrolysis, creattion inARF1,Q711, which was predicted to slow the rate ing a persistently activeARF1. Characterizationof this m u t a n t of GTP hydrolysis. We demonstrate that 6711, in conAFtF is presented below and confirms that ARFl is the key trast to wild typeARF1, exhibits a =-fold increase in the half-life of ARF-GTP and is able to promote stable BFA-sensitive component required for coat protein binding to of Golgi membranes. coatomer binding to Golgi membranes in the presence
A n Activating Mutationin ARFl Stabilizes Coatomer Binding to Golgi Membranes*
(bl)
GTP in vitro. Additionally, Q71I is able to support the EXPERIMENTALPROCEDURES binding of a significant amount of coatomer to memPurification ofARE: Golgi Membranes,and Cytosol-Wild type ARFl branes in the absence of added nucleotides, effectively and themutantARF1, Q711, were co-expressedin Escherichia coli with bypassing the brefeldinA (BFA)-sensitive exchange ac- N-myristoyltransferase and isolated as previously described (15, 16). tivity. Furthermore, transfectionof cells withQ711, but Golgi-enrichedmembrane fractions from rat liver (17) or Chinese hamnot -1, renders the Golgi association of coatomer re-ster ovary cells (18) were obtained as described. Cytosol was isolated sistant to the effectsof BFA in vivo. These observations from bovine brains (19). Nucleotide Exchange Assays-Nucleotide exchange was assessed by provide compelling evidence that ARFl is a necessary measuring nucleotide binding to ARF andor ratliver Golgi membranes GTP binding protein that regulates the reversible bindusing a filter binding assay as described previously (4). Incubations ing of coat proteins to Golgi membranes and that the effects ofBFA on this process in living cells must be a were carried out in a 0.1-ml reaction volume containing 25 m HepesKOH, pH 7.0, 25 m KC1, 2.5 m MgC12, 0.2 M sucrose, 1 m dithioconsequence of BFA's inhibition of guanine nucleotide threitol, 1 m~ ATP (SigmaA2383),2-5 pg of recombinant ARF protein, exchange ontoARF1. and 4-6 pg of Golgi membranes and [a-32P1GTPor [y-32PlGTP(1 m, 0.7-1.4 Ci mmol-') at 30 "C for the indicated times. The reaction was stopped by the addition of cold buffer, the mixture was filtered on BAS5 The reversible bindingof cytosolic coat proteins (coatomer) tonitrocellulose filters and washed with five 2-ml volumesof wash buffer, and the amount of radionucleotidetrapped to the filter was measured as Golgi membranes is believed to functionin the maintenance of described (4). The amount of membrane-catalyzed nucleotide bound to structure and regulation of membrane trafficin the Golgi com- ARF was calculated by subtracting the nucleotide bound after incubaplex (1). The assembly of coatomer onto Golgi membrane re- tions ofARF alone and Golgi membranes alone fromthe amount bound quires GTP and a small GTP binding protein, ADP-ribosylationafter incubations of ARF plus Golgi membranes (4). Coatomer Binding Assay-Membranes from Chinese hamster ovary factor 1 (ARF1)l (2, 3). The ability of ARFl to mediate the assembly of coatomer onto Golgi membranes is believed to re- cells (8 pg of protein) and ARFl or Q71I (10 pg of protein each) were incubated under conditions described above for nucleotide exchange quire the activation of ARFl by a membrane nucleotide ex- assays with and without activators prior to the addition of cytosol (600 change protein (4-6) which results in the association of ARF1- pg of protein) and further incubation to allow for coatomer binding. The G T P with the membrane followed by coatomer binding (2, 3). membranes and bound material were pelleted at 14,000 x g, and the amount of p-COP associated with the pellet was analyzed by SDSpolyacrylamide gelelectrophoresisand immunoblot, and quantitated by * The costs of publication of this article were defrayed in partby the PhosphorImager (Molecular Dynamics)(2). The background amount of payment of page charges. This article must therefore be hereby marked p-COP binding to membranes that occurred in the absence of added "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to ARF was subtracted from values obtained with ARFl and 6711. indicate this fact. Dansfection of Cells and Immunofluorescence Assays-ARF1 and $ Supported by the Howard Hughes Medical Institute-National Insti6711 containing the hemagglutinin epitope at the C terminus were tutes of Health Research Scholars Program. subcloned into the expression vector PCDLSRa(20). COS-1 cells grown 5 VisitingFellowfrom the Department of CellBiology,Medical on coverslips were transiently transfected by the calcium phosphate School, University of Utrecht, The Netherlands. ll To whom correspondence should be addressed: CBMB, Bldg. 18Tl precipitation method and were analyzed 40 h later to assess sensitivity 101, NIH,Bethesda, MD 20892. Tel.: 301-402-0360; Fax: 301-402-0078. to BFA. For immunofluorescence, cells on coverslips were fixed in 2% The abbreviations used are: ARF, ADP ribosylation factor; BFA, formaldehyde and double labeledwith a monoclonal antibody to the HA brefeldin A , GTPyS, guanosine 5'-0-(3-thiotriphosphate); COP, epitope (12CA5) to detect transfected HA-tagged ARF protein and a coatomer protein; HA, influenza hemagglutinin. polyclonal anti-p-COP peptide (EAGE) antiserum as described (9).
3135
3136
ARFl and Golgi Membrane Coat Proteins A
0
10
20
B
30
0
I
I
I
10
20
30
Time (rnin)
Time (min)
i
.1
1
1000 10
100
[BFA] uM
0
D
2
4
6
6
1
0
1
2
Time (rnin)
FIG.1. Golgi membrane-catalyzed GTP exchange and hydrolysis onto ARFl and Q7lI. A and B , comparison between [or-32P]GTP/GDP (open squares) and [y-3zPlGTP(solid squares) bound to A R F l (A) or Q71I ( B ) after incubation ofARF and membranes for the indicated times at 30 "C. C , nucleotide binding associated with AFtFl (open squares) and 6711 (solid squares) after incubation of A R F l and 6711 with Golgi membranes in the presence of 1 w [ C ~ - ~ ~ P I for G T20 P min a t 30 "C in the presence of increasing amounts of BFA. D,hydrolysis of [ys2P]GTP prebound ontoA R F l (open squares) and Q71I (solid sqwres) after incubation ofARF with membranes in the presence of [y32P]GTPfor 20 min followed by the addition of 100 w unlabeled GTP; the loss of y-phosphate bound to ARF was assayed with time after addition of unlabeled GTP. All experiments were repeated at least 3 times. For C and D,the mean & S.E. of triplicate samples is shown.
RESULTS AND DISCUSSION
To create a persistently active ARF1,we selected a point mutation based upon the homology of ARFl protein sequence domains with Ras (21). In Ras the conserved glutamine at position 61 isrequired for efficient GTP hydrolysis;mutations at this position stabilize the Ras-GTP state, resulting ina persistently activated protein (22, 23). The analogous position in ARFl is glutamine 71, and this was changed t o an isoleucine. This mutant, called Q711, was co-expressed in E. coli with N-myristoyltransferase in order to produce the biologically active N-myristoylated form of ARF (15). The recombinant protein was isolated and its activity monitored in nucleotide exchange, GTP hydrolysis, and coatomer binding assays in uitro, as well as in transfected cells. Isolated Golgi membranes catalyzed nucleotide exchange onto both ARFl and Q71I with similar kinetics using a-labeled L3'P1GTP (Fig. 1, A and B , open squares). This exchange activity was inhibited for both ARFl and Q71I in a dose-dependent manner byBFA (Fig. 1C). During the membrane-catalyzed nucleotide exchange assay, GTP is bound to ARF and is then hydrolyzed to GDP (4,7). Thus, using a-labeled [32P]GTP,total exchange events onto ARF are recorded even if the ARF-bound GTP has been hydrolyzed to GDP. When y-labeled [32P]GTP was used in the exchange assay to assess the amount of GTP exchanged onto ARF that was not hydrolyzed (Fig. 1,A and B , solid squares), a larger fraction of nucleotide bound to Q71I remained as GTP as compared with ARF1. To further assess this distinction, we measured therate of hydrolysis of
FIG.2. Nucleotide requirements for preactivation of Golgi membranes by ARFl and Q71I. Golgi membranes and A R F l (A) or Q71I ( B ) were preincubated with no activator (open bars), 10 w GTP (hatched bars), or 10 GTPyS (solid bars) for 5 min at 37 "C, followed
by the addition of BFA to all samples (300w).Then, cytosol was added, and samples were incubated for an additional 5 min to allow coatomer binding. To some samples, during the first incubation BFA was added before the addition of activators to inhibit activation (BFAbefore).After incubation the amount of p C 0 P bound to membranes was measured as described under "Experimental Procedures."
[y-32PlGTPthat had been preloaded onto ARFl and Q71I by monitoring the loss of ARF-bound radioactivity after the addition of unlabeled GTP. GTP that was bound to ARF was hydrolyzed with a t Mof less than 2 min for ARFl and approximately 5 min for Q71I (Fig. lD). The addition ofBFA, in place of unlabeled GTP, yielded similar results. Thus, while there was no observed change in the BFA-sensitive nucleotide exchange onto Q711, there was a 2-3-fold decrease in the rate of membrane-dependent GTP hydrolysis associated with the Q71I mutation in vitro.
3137
A R F l and Golgi Membrane Coat Proteins Q71I
Untreated
I
p-COP
ARFl
p-COP
1
n ~3. .6711 confers BFA resistance onto the membrane association of the coatomer protein &COP. COS-I cells transfected with HA-tagged ARFl or 6711 were eithernot treated or treated with BFA (10 pdml)for 10 min at 37 “C prior to double labeling with antibodiesto the HA epitope and B-COP. Incubation of Golgi membranes with ARFl in thepresence of cytosol, effects indistinguishable from those seen in untransGTPyS is sufficient to preactivate the membranes, making fected cells. The effects of BFA on cells transfected with Q71I them competent for binding of subsequently addedcoatomer in were quite different. The distributionof neither theintroduced the absence of free nucleotide (2). While BFA inhibits the initial Q71I nor p-COP changed in response to the addition of the activation step, it has no effect on subsequent coatomer binding drug. Even afterextended incubations of 1h in thepresence of to such “preactivated” membranes. Importantly, this preactiva- 10 pg/ml BFA, @COP and Q71I remained co-localized to perition is not observed with hydrolyzable GTP. The relative sta- nuclear structures. Other markers of the Golgi complex also bility of GTP bound to Q71I suggested that the mutant might, remained in these p-COP-labeled structures (not shown). The in contrast, preactivate the membrane for coatomer binding presence of the HA epitope had no effect on the phenotype since with GTP alone. Indeed, incubation of Q71I with Golgi mem- transfection of untagged ARFs resulted in similar effects (not branes in thepresence of GTP allowed maximal levels of bind- shown). Myristoylation of the proteins was required for their ing of the coatomer protein p C 0 P to occur during a second biological function, since cells transfected with ARF sequences incubation in the presence of coatomer (Fig. 2 B ) . The level of that contained an additional mutation a t position 2 from a P C O P binding achievedwas nearly that achieved with GTPyS. glycine to an alanineabolished myristoylation and Golgi localIn contrast, as observed previously ( 3 , preactivation of Golgi ization for both ARFl andQ71I and eliminated BFA protection membranes by wild type ARFl was only observed if GTPyS was in cells transfected with Q71I. The BFA-resistant phenotype of included in the firstincubation (Fig. 2A 1. Q71I cannot be explained by overexpression of ARF proteins Another difference between Q71I and wild type ARFl was per se since it was observed in all cells expressing variable the significant level of BFA-resistant activation of membranes amounts of Q71I and never observed in cells overexpressing by Q71I that wasobserved in theabsence of any addednucleo- wild type ARF1. tide (Fig. 2 B , open bars). This activity of Q71I increased with Our expectation in creating Q71I was that this mutation in longer incubation but wasnever observed with ARF1. Possible ARF, analogous to QSlL in Ras, would inhibit hydrolysis of explanations for this observation are that the preparation of GTP bound to the protein, creating a persistently active GTPQ71I contains “activated” ARF that effectively bypasses any ARF. If the inhibition of hydrolysis was sufficiently strong, BFA-sensitive step in the membrane. Thus, 6711 appears to resistance to the effects of BFA on coatomer binding would be have the characteristicsof both a longer lived, persistent ARF- expected. We measured, however, only a 2-%fold increase in GTP as well as a constitutively active ARF1. the half-life of GTP bound to Q71I in ourin vitroassays, which Preincubation of permeabilized cells with GTPyS blocks the would predict a delay in, butnot the complete inhibition of, the effects of subsequent addition of BFA on coatomer dissociation effects of BFA on coatomer binding that was observed in cells from the Golgi apparatus (8).Since Q71I has the characteristic transfected with Q71I. Although it is possible that thehalf-life of acting in vitro like ARF-GTPyS in the absence of added of Q71I-GTP in vivo may be longer, studies with the analogous GTPyS, we could now test whether the expression of this “ac- mutation inRab3A have shown that therates of GTP hydrolytive” ARFl in cells would confer resistance to BFA’s effects on sis as measured in vivo are actually similarfor the wild type coatomer association with the Golgi apparatus. COS-1 cells and mutant proteins, and these authorscaution that the analwere transfected with either ARFl or Q71I containing a n HA ogy made to the observation with Ras mutations may not alepitope at the carboxyl terminus so that the transfected protein ways be valid (24). This raises the possibility that this mutation could be immunologically detected. In cells transfected with in ARF1, while it does affect to some extent GTP hydrolysis, either ARFl or Q711, the HA tagged ARFs were localized to a may also result in a conformational change in theprotein that perinuclear region, which co-localized, by immunofluorescence, effectively makes it“active” regardless of the nucleotide bound. with the coatomer component p-COP (Fig. 3)and Golgi resident In this regard, it might be significant that the site of this markers (notshown). p-COP localization in untransfectedcells mutation is adjacent to a critical glycine (at position 70 in was indistinguishablefrom that in the transfected cells. When ARF1, 60 in Ras), which is believed to make contact with the BFA was added to cells transfected with wild type ARF1, both y-phosphate and be involved in the GDP-GTP-induced conforARFl and p-COP rapidly became diffusely distributed to the mational change in theprotein (21).
ARFl and Membrane Golgi
3138
Regardless of the mechanism, the effect of expressing this activated ARF is comparable with selectively activating ARF with GTPyS, rendering the cell resistant to the effects of BFA on both coat assembly and redistribution of Golgi membrane into the endoplasmic reticulum. Furthermore, these results demonstrate that the only process inhibited byBFA in the ARF-coatomer binding reactionis the nucleotide exchange activity. ARF thus becomes one of the few small GTPases for which an effector function is identifie4 its cycle of activation and inactivation regulates the binding of coatomer to Golgi membranes. Acknowledgments-We thank J. Bonifacino, A. Finegold, and C. Ooi for critical reading of the manuscript. REFERENCES 1. Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz. J. (1992) J. Cell Bwl. 118, 1071-1080 2. Donaldson, J. G., Cassel, D., K a h n , R. A,, and Klausner, R.D. (1992) Proc. Natl. Acad. Sci. U. S.A. 89, 6408-6412 3. Palmer, D. J., Helms, J. B., Beckers, C. J. M., Orci, L., and Kothman, J. E. (1993) J. Biol. Chem. 288, 12083-12089 4. Donaldson, J. G., Finazzi, D., and Klausner, R. D. (1992)Nature 380,350-352 5. Helms J. B., and Rothman, J. E. (1992)Nature 380,352-354 6. Randazzo, P. A., Yang, Y. C., Rulka, C., and Kahn, R. A. (1993) J. Biol. Chem. 288,9555-9563
Coat Proteins 7. Helms, J. B., Palmer, D. J.. and Rothman, J. E. (1993) J. Cell Bwl. 121, 751-760 8. Donaldson, J. G., Lippincott-Schwartz, J., and Klausner, R. D. (1991) J. Cell Biol. 112, 579-588 9. Donaldson, J. G., Kahn, R. A,, Lippincott-Schwartz, J., and Klausner, R.D. (1991) Science 264, 1197-1199 10. Serafini, T.,Orci, L., Amherdt, M., Brunner, M., Kahn, R. A,, and Rothman, J. E. (1991) Cell 67.239-253 11. Orci, L., Tagaya, M.; Amherdt, M., Perrelet, A,, Donaldson, J. G., LippincottSchwartz, J., Klausner, R. D., and Rothman, J. E.(1991) Cell 64,1183-1195 12. Ercolani, L.,Stow, J. L., Boyle, J. F.. Holtzman, E. J.. Lin, H., Grove,J. R., and Ausiello, D. A. (1990)F'roc. Natl. Acad. Sci. U. S.A. 87, 46354639 13. Goud, B., Zahraoui, A., Tavitian, A,, and Saraste, J. (1990) Nature 346,553556 14. Ktistakis, N.T.,Linder, M. E., and Roth, M. G. (1992)Nature 356,344-346 Devine, 15. Duronio, R. J.,Jackson-Machelski, E., Heuckeroth, R. O., Olins, P. 0.. S.,Gordon, J. I. (1990) Proc. C. S.,Yonemoto, W., Slice, L. W., Taylor, S. and Natl. Acad. Sci. U. S. A. 87, 15W1510 16. Weias, O., Holden, J., Rulka, C., and K a h n , R. A. (1989) J. Biol. Chem. 264, 21066-21072 17. Seratini T.,and Rothman, J. E.(1992) Methods Enzymol. 219,286-300 18. Balch, W. E., Glick, B. S.,and Rothman, J. E.(1984) Cell 39,525-536 19. Malhotra, V.,Seraiini, T., Orci, L., Shepherd, J. C., and Rothman, J. E.(1989) Cell 68, 329-336 20. Takebe, Y., Seiki, M., Fqjisawa, J.-I., Hoy, P., Yokota, K, Ami, K-I., Yoshida, M., and A m i , N.(1988) Mol. Cell. Biol. 8, 466-472 21. Bourne, H. R., Sanders, D. A,, and McCormick, F. (1991)Nature 349,117-127 22. Barbacid, M. A. (1987)Annu. Rev. Biochem. 66,779-827 23. Patel. G., MacDonald, M. J., Khosravi-Far, R., Hisaka, M.M., and Der, C. J. (1992) Oncogene 7,283-288 24. Bmndyk, W. H., McKiernan, C. J., Burstein, E. S.,and Macara, I. G . (1993)J. Biol. Chem. 288,9410-9415 ~
~
