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Ectopic b-chain of ATP synthase is an apolipoprotein A-I receptor in ...

GluR2 and then again with b-actin. Levels of mRNA were densitized using ImageQuant(Molecular Devices). Blots for GluR1 and GluR2 revealed characteristic RNA species 30 ,and the main bands for GluR1 (5.2 kilobases) and GluR2 (5.9 and 3.9 kilobases) werequantified and normalized relative to b-actin.Received 16 May; accepted 11 October 2002; doi:10.1038/nature01249.1. Bouton, M. E. & Schwartzberg, D. Sources of relapse after extinction in pavlovian and instrumentallearning. Clin. Psych. Rev. 11, 123–140 (1991).2. Roberts, D. C. S. & Koob, G. F. Extinction and recovery of cocaine self-administration following6-hydroxydopamine lesions of the nucleus accumbens. Pharmacol. Biochem. Behav. 12, 781–787 (1980).3. Pettit, H. O., Ettenberg, A., Bloom, F. E. & Koob, G. F. Destruction of dopamine in the nucleusaccumbens selectively attenuates cocaine but not heroin self-administration in rats. Psychopharmacol.84, 167–173 (1984).4. Zito, K. A., Vickers, G. & Roberts, D. C. S. Disruption of cocaine and heroin self-administrationfollowing kainic acid lesions of the nucleus accumbens. Pharmacol. Biochem. Behav. 23, 1029–1036(1985).5. Carlezon, W. A. Jr, Devine, D. P. & Wise, R. A. Habit-forming actions of nomifensine in nucleusaccumbens. Psychopharmacol. 122, 194–197 (1995).6. Neve, R. L., Howe, J. R., Hong, S. & Kalb, R. G. Introduction of the glutamate receptor subunit 1 intomotor neurons in vitro and in vivo using a recombinant herpes simplex virus. Neuroscience 79,435–447 (1997).7. Kelz, M. B. et al. Expression of the transcription factor DFosB in the brain controls sensitivity tococaine. Nature 401, 272–276 (1999).8. Cornish, J. L., Duffy, P. & Kalivas, P. W. A role for nucleus accumbens glutamate transmission in therelapse to cocaine-seeking behavior. Neuroscience 93, 1359–1367 (1999).9. Cornish, J. & Kalivas, P. Glutamate transmission in the nucleus accumbens mediates relapse in cocaineaddiction. J. Neurosci. 20, RC89 (2000).10. Jaffe, J. H., Cascella, N. G., Kumor, K. M. & Sherer, M. A. Cocaine-induced cocaine craving.Psychopharmacology 97, 59–64 (1989).11. Robbins, S. J., Ehrman, R. N., Childress, A. R. & O’Brien, C. P. Relationships among physiological andself-report responses produced by cocaine-related cues. Addict. Behav. 22, 157–167 (1997).12. Sinha, R., Catapano, D. & O’Malley, S. Stress induced craving and stress response in cocainedependent individuals. Psychopharmacology 142, 343–351 (1999).13. White, F. J., Hu, X. T., Zhang, X. F. & Wolf, M. E. Repeated administration of cocaine or amphetaminealters neuronal responses to glutamate in the mesoaccumbens dopamine system. J. Pharmacol. Exp.Ther. 273, 445–454 (1995).14. Thomas, M., Beurrier, C., Bonci, A. & Malenka, R. Long-term depression in the nucleus accumbens: aneural correlate of behavioral sensitization to cocaine. Nature Neurosci. 4, 1217–1223 (2001).15. Lu, W., Chen, H., Xue, C. J. & Wolf, M. E. Repeated amphetamine administration alters the expressionof mRNA for AMPA receptor subunits in rat nucleus accumbens and prefrontal cortex. Synapse 26,269–280 (1997).16. Lu, W. & Wolf, M. E. Repeated amphetamine administration alters AMPA receptor subunit expressionin rat nucleus accumbens and medial prefrontal cortex. Synapse 32, 119–131 (1999).17. Churchill, L., Swanson, C. J., Urbina, M. & Kalivas, P. W. Repeated cocaine alters glutamate receptorsubunit levels in the nucleus accumbens and ventral tegmental area of rats that develop behavioralsensitization. J. Neurochem. 72, 2397–2403 (1999).18. Quinlan, E. M., Philpot, B. D., Huganir, R. L. & Bear, M. F. Rapid, experience-dependent expression ofsynaptic NMDA receptors in visual cortex in vivo. Nature Neurosci. 2, 352–357 (1999).19. Heynen, A. J., Quinlan, E. M., Bae, D. C. & Bear, M. F. Bidirectional, activity-dependent regulation ofglutamate receptors in the adult hippocampus in vivo. Neuron 28, 527–536 (2000).20. Fabbricatore, A., Uzwiak, A., West, M. & Peoples, L. Comparisons of firing rates of rat nucleusaccumbens neurons during cocaine self-administration and extinction. Soc. Neurosci. Abstr. 24, 1736(1998).21. Hayashi, Y. et al. Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1and PDZ domain interaction. Science 287, 2262–2267 (2000).22. Shi, S. -H., Hayashi, Y., Esteban, J. A. & Malinow, R. Subunit-specific rules governing AMPA receptortrafficking to synapses in hippocampal pyramidal neurons. Cell 105, 331–343 (2001).23. Keys, A. S., Mark, G. P., Emre, N. & Meshul, C. K. Reduced glutamate immunolabeling in the nucleusaccumbens following extended withdrawal from self-administered cocaine. Synapse 30, 393–401 (1998).24. Bell, K., Duffy, P. & Kalivas, P. W. Context-specific enhancement of glutamate transmission by cocaine.Neuropsychopharmacology 23, 335–344 (2000).25. McLennan, H. The effect of decortication on the excitatory amino acid sensitivity of striatal neurones.Neurosci. Lett. 18, 313–316 (1980).26. Volkow, N. D. et al. Long-term frontal brain metabolic changes in cocaine abusers. Synapse 11,184–190 (1992).27. London, E. D., Bonson, K. R., Ernst, M. & Grant, S. Brain imaging studies of cocaine abuse:implications for medication development. Crit. Rev. Neurobiol. 13, 227–242 (1999).28. Volkow, N. D. & Fowler, J. S. Addiction, a disease of compulsion and drive: involvement of theorbitofrontal cortex. Cereb. Cortex 10, 318–325 (2000).29. Schmidt, E. F. et al. Extinction training regulates tyrosine hydroxylase during withdrawal from cocaineself-administration. J. Neurosci. 21, RC137 (2001).30. Guitart, X. et al. Regulation of ionotropic glutamate receptor subunits in different rat brain areas by apreferential j1 receptor ligand and potential atypical antipsychotic. Neuropsychopharmacol. 23,539–546 (2000).Acknowledgements This work was supported by US Public Health Service grants (NIDA), apostdoctoral National Research Service Award (to C.A.S.) and the Lydia Bryant TestProfessorship.Competing interests statement The authors declare that they have no competing financialinterests.Correspondence and requests for materials should be addressed to D.W.S.(e-mail: david.self@utsouthwestern.edu).letters to nature..............................................................Ectopic b-chain of ATP synthaseis an apolipoprotein A-I receptorin hepatic HDL endocytosisLaurent O. Martinez*, Sébastien Jacquet*, Jean-Pierre Esteve†,Corinne Rolland*, Elena Cabezón‡, Eric Champagne§, Thierry Pineauk,Valérie Georgeaud*, John E. Walker‡, François Tercé*, Xavier Collet*,Bertrand Perret* & Ronald Barbaras** Institut Fédératif de Recherche Claude de Preval, IFR 30, Institut National de laSanté et de la Recherche Médicale, Unité 563, Département Lipoprotéines, etMédiateurs Lipidiques and § Département Immunologie Moléculaire et Biologiedu Lymphocyte T, 31059, Toulouse cedex, France† Institut Fédératif de Recherche Louis Bugnard, IFR 31, Institut National de laSanté et de la Recherche Médicale, Unité 531, Biologie et Pathologie Digestive,Hôpital Rangueil, 31403, Toulouse cedex, France‡ Medical Research Council Dunn Human Nutrition Unit, Hills Road, CambridgeCB2 2XY, UKk Institut National de la Recherche Agronomique, Laboratoire de Pharmacologieet Toxicologie, 31931, Toulouse cedex 9, France.............................................................................................................................................................................The effect of high-density lipoprotein (HDL) in protectingagainst atherosclerosis is usually attributed to its role in ‘reversecholesterol transport’ 1 . In this process, HDL particles mediatethe efflux and the transport of cholesterol from peripheral cells tothe liver for further metabolism and bile excretion. Thus, cellsurfacereceptors for HDL on hepatocytes are chief partners inthe regulation of cholesterol homeostasis 2 . A high-affinity HDLreceptor for apolipoprotein A-I (apoA-I) was previously identifiedon the surface of hepatocytes 3,4 . Here we show that thisreceptor is identical to the b-chain of ATP synthase, a principalprotein complex of the mitochondrial inner membrane. Differentexperimental approaches confirm this ectopic localization ofcomponents of the ATP synthase complex and the presence ofATP hydrolase activity at the hepatocyte cell surface. Receptorstimulation by apoA-I triggers the endocytosis of holo-HDLparticles (protein plus lipid) by a mechanism that dependsstrictly on the generation of ADP. We confirm this effect onendocytosis in perfused rat liver ex vivo by using a specificinhibitor of ATP synthase. Thus, membrane-bound ATP synthasehas a previously unsuspected role in modulating the concentrationsof extracellular ADP and is regulated by a principalplasma apolipoprotein.We previously showed that apoA-I that is not associated withlipids (hereafter called free apoA-I) interacts specifically with highaffinityHDL receptors (10 29 M) 3,4 , thereby representing a possibleligand for the affinity purification of HDL receptors. The passage ofsolubilized porcine liver plasma membrane proteins over immobilizedfree apoA-I (ref. 4) in surface plasmon resonance (Biacore)experiments indicated that this interaction was conserved (dissociationconstant, K d < 10 29 M; Fig. 1b, sensogram 1). Subsequently,several rounds of binding and desorption allowed thepurification and identification of a protein with a relative molecularmass of 50,000 (M r 50K; Fig. 1a, lane 3). Higher amounts of apoA-Iboundproteins were recovered by affinity chromatography (usingimmobilized free apoA-I; Fig. 1a, lane 2) and showed a fourfoldincrease in binding to free apoA-I immobilized on a sensor chip(Fig. 1b, sensogram 2). We removed the 50K band from the gel andmicrosequenced 50 pmol of protein by protease digestion, highperformanceliquid chromatography (HPLC) separation andEdman analysis. Unexpectedly, a peptide sequence derived fromthis protein was identical to a segment of the human b-chain of ATPsynthase.Mitochondrial ATP synthase has two main domains, F 1 and F o(ref. 5). The b-chain belongs to F 1 , a peripheral membrane proteinNATURE | VOL 421 | 2 JANUARY 2003 | www.nature.com/nature 75


letters to naturecomplex containing binding sites for ATP and ADP, and the catalyticsite for ATP synthesis or hydrolysis. F 1 is bound on the membrane byits interaction with F o , an integral membrane protein complex inmammalian mitochondria that contains a transmembrane channelfor protons 5–7 .The b-chain of ATP synthase is found mostly at the inner side ofthe mitochondrial membrane 5 , although studies have reported itspresence at the cell surface 8–10 . Immunofluorescence microscopywith an antibody against the b-chain or with an isotypic IgG2aconfirmed the presence of the ATP synthase b-chain on the surfaceof immortalized human hepatocytes (IHH cells 11 , Fig. 2a, g), butnot on the surface of Chinese hamster ovary (CHO) cells (Fig. 2f, i),which do not show apoA-I high-affinity sites (unpublished data).Notably, another principal protein of ATP synthase, the a-chain,was also detected on the hepatocyte cell surface (Fig. 2d). As acontrol, a typical mitochondrial protein, the subunit I of cytochromeoxidase (COX-I), was not detected on the cell surface(Fig. 2e), but was visualized after permeabilization of the cells(Fig. 2h). After preincubating the cells with apoA-I, confocalimmunofluorescence microscopy with an antibody against apoA-Iidentified a specific cell-surface signal (Fig. 2b) that was superimposed(Fig. 2c) on that of the b-chain of ATP synthase (Fig. 2a).This confirmed colocalization of the b-chain of ATP synthase andapoA-I binding at the hepatocyte cell surface.Fluorescence-assisted flow cytometry experiments on intact cells(selected as cells excluding propidium iodide) confirmed the cellsurfacelocalization of the b-chain specifically on HepG2 (Fig. 3a,curve 5) but not CHO (Fig. 3c, curve 5) cells. The selectivity of theresponse to the b-chain antibody was clearly shown by the muchlower signal obtained with isotypic immunglobulin-g (IgG), anexcess of purified F 1 -ATPase (as a competitor) or an antibodyagainst COX-I (Fig. 3, curves 1–3, respectively).The presence ofthe a-chain at the cell surface was confirmed using a specificantibody (data not shown), indicating that the whole F 1 -ATPasedomain might be present on the cell surface. Incubation of hepatocyteswith an excess of free apoA-I reduced the immunoreactivity ofthe b-chain antibody over threefold, confirming the cell-surfaceinteraction of free apoA-I with the b-chain of ATP synthase (Fig. 3aand b, curve 4).The association between apoA-I and the b-chain was verified bytwo types of competition experiment (ref. 3 and SupplementaryFig. 1): first, the b-chain antibody (Supplementary Fig. 1a) and thecorresponding Fab fragments (data not shown) completely inhibitedthe binding of 125 I-labelled free apoA-I, as well as unlabelledligand; second, the same antibody or Fab fragments inhibited thebinding of 125 I-labelled HDL 3 (the most abundant subfraction ofHDL, which binds to the high- and low-affinity binding sitesthrough apoA-I; ref. 3) on inverted purified mitochondria (ref. 12and Supplementary Fig. 1b), in which the F 1 fraction of ATPsynthase is exposed on the outside and so avoids interference withthe cell-surface low-affinity HDL-binding sites.The presence of ATP synthase at the cell surface of lymphocytes 8and human endothelial cells 9,10,13 has been reported. In lymphocytes,the interaction of ATP synthase with angiostatin suggestedthat it might have a role in angiogenesis. In addition, the a- andb-chains of ATP synthase have been identified as a receptor for apo-E-enriched HDL 14,15 , although the presence of this protein on thecell surface was not demonstrated nor was a role proposed in thosestudies. Our data clearly show the presence of the b-chain, probablyas a F 1 complex, on the cell surface of HepG2, IHH cells and primaryhuman hepatocytes 16 , but not on epithelial CHO cells 8 .We measured the functional activity of cell-surface ATP synthaseby adding either ADP plus 32 P, or [a- 32 P]ATP, to HepG2 cells, andidentifying and quantifying the nucleotides generated in cultureFigure 1 Affinity purification of the free apoA-I receptor. a, Total solubilized porcine liverplasma membrane proteins (lane 1) were purified either by apoA-I affinity chromatography(lane 2) or by micro-recovery on an apoA-I BI sensor chip (lane 3). Eluted proteins wereresolved by SDS–PAGE and silver staining (lanes 1 and 2 contain 5 mg of protein, lane 3contains 0.1 mg of protein). b, Biacore sensograms of the interaction of total solubilizedporcine liver plasma membrane proteins injected either directly (curve 1), or afterpurification by apoA-I affinity chromatography (curve 2), with an apoA-I-bound sensorchip. Curves represent the resonance units as a function of time.76Figure 2 Immunofluorescence localization of the b- and a-chains of ATP synthase withapoA-I on the surface of hepatocytes. a–c, Colocalization of the b-subunit of ATPsynthase (green, a) and apoA-I (red, b) on intact IHH cells is apparent in the merged image(yellow, c). d, Localization of the a-chain of ATP synthase by immunofluorescence usingan antibody specific for the a-chain on intact IHH cells. e, h, As a control, an antibodyagainst COX-I, another typical mitochondria protein, shows that COX-I is not present onintact (e) or but is present on permeabilized (h) IHH cells. f, The b-subunit of ATP synthaseis undetectable on intact CHO cells. g, i, Control experiments for antibody efficacy usingisotypic purified mouse IgG (IgG2a) in IHH (g) and CHO (i) cells.NATURE | VOL 421 | 2 JANUARY 2003 | www.nature.com/nature


letters to naturemedia (Methods). Notably, no synthesis of [ 32 P]ATP from ADP plus32 P could be detected for up to 10 min either without or with freeapoA-I (Supplementary Fig. 2a and b). By contrast, a hydrolysisactivity was measured within 10 min (the generation of [a- 32 P]ADPfrom [a- 32 P]ATP), which was increased markedly (up to 79%) inthe presence of apoA-I (Supplementary Fig. 2c and d). The purifiedIF 1 protein, a natural inhibitor of mitochondrial F 1 -ATPase thatinteracts with the b-subunit to inhibit the ATP hydrolysisactivity 17,18 , could reduce both the basal (48% decrease as comparedwith the control) and the free apoA-I-stimulated hydrolysis activity(Supplementary Fig. 2e and f). Because ATP hydrolase activity is amain feature of a whole F 1 -ATPase complex 5 , our data indicatethat on the plasma membrane of hepatocytes there is a completeF 1 -ATPase that functions as an ATP hydrolase and can be stimulatedby free apoA-I.We previously suggested that the ability of HDL or apoA-I to bindto high-affinity sites on hepatocytes might stimulate the internalizationof HDL through low-affinity sites 19 . Indeed, whereas HDL 2enriched in triglyceride (TG-HDL 2 ) binds to only low-affinitybinding sites, remnant HDL 2 (a particle generated after the actionof hepatic lipase on TG-HDL 2 ) binds to both low- and high-affinitybinding sites and is internalized faster and to a greater extent thanis wild-type TG-HDL 2 (ref. 19). Here we found that free apoA-I(0.1–20 mgml 21 ) stimulated the internalization of 125 I-labelledTG-HDL 2 by HepG2 or IHH cells, with maximum internalizationoccurring between 5 and 15 min of incubation (data not shown).Notably, 100 nM ADP (the only compound produced by ATPsynthase at the hepatocyte cell surface) stimulated the internalizationof TG-HDL 2 by 30% (Fig. 4a, column 2), similar to the amountstimulated by free apoA-I (Fig. 4a, column 1). The effect of botheffectors was not additive (Fig. 4a, column 3), suggesting that theyaffect a similar endocytotic pathway. By contrast, 100 nM ATPstimulated only a small increase in TG-HDL 2 internalization(Fig. 4a, column 1). In addition, apyrase (E.C. 3.6.1.5), whichhydrolyzes both ATP and ADP, completely abolished both thebasal and the apoA-I- stimulated endocytosis of TG-HDL 2(Fig. 4a, column 5). 2MeS-ADP, a non-hydrolysable analogue ofADP, showed a stimulatory effect at 10–100 nM, followed by aninhibition of endocytosis at higher concentrations (1–10 mM). Bycontrast, ATP-gS, a non-hydrolysable analogue of ATP, had a weakeffect (Fig. 4c). Together, these data indicate that there may be aspecific ADP-dependent pathway for HDL endocytosis. Addition ofepidermal growth factor (EGF), which induces endocytosis of theEGF receptor (EGFR), did not stimulate HDL internalization(Fig. 4a, column 6), indicating that HDL processing is not dependenton a nonspecific, general activation of endocytosis.Endocytosis was not restricted to the protein moiety of HDLbecause free apoA-I also stimulated the internalization of [ 3 H]-cholesteryl-ether-labelled TG-HDL 2 (Fig. 4a, Chol). The ratio of[ 3 H]cholesteryl-ether to 125 I-labelled protein was also found to bethe same in the original TG-HDL 2 solution and in the materialrecovered from inside the cells after a dissociation step, confirmingthat the whole HDL particle was involved in the endocytosisprocess.HDL endocytosis could occur in hepatocytes through two differentpathways: the first might be dependent on scavenger receptorclass B type I (SR-BI), a widely described HDL receptor 20 thattriggers internalization of the holo-HDL particle, followed byselective transcytosis of lipoprotein cholesterol; the second mightbe independent of SR-BI and involve the uptake and degradation ofthe holo-HDL particle by unknown receptors 21 . To evaluate thepossible involvement of SR-BI in the stimulated HDL endocytosis,we carried out experiments using an IgG against SR-BI that caninhibit the uptake of cholesteryl ester in CHO SR-BI-transfectedcells 4 and in human adrenal NCI-H295R cells (V. Clavey, personalcommunication). This IgG had no effect on cholesteryl ester uptakeFigure 3 Detection of the b-chain of ATP synthase at the cell surface by flow cytometry.HepG2 and CHO cells were analysed by fluorescence-assisted flow cytometry. a, Analysisof HepG2 cells. Unbroken lines represent cells incubated with an antibody against theb-subunit of ATP synthase in the absence (curve 5) or presence of 100 nM apoA-I (curve4) or 250 mgml 21 of F 1 -ATPase (curve 3); broken lines represent cells incubated with anisotypic control mouse IgG2a (curve 1) or an antibody against COX-I (curve 2).b, Histogram showing the mean relative fluorescence of curves 1–5 in a. c, Analysis ofCHO cells, as in described in a (curve 3 not done). d, Histogram showing the mean relativefluorescence of curves 1, 2, 4, 5 in c.Figure 4 Effect of different nucleotides on internalization of TG-HDL 2 by hepatocytes.a–c, Cells were incubated for 10 min at 37 8C with 75 mgml 21 125 I-labelled TG-HDL 2(a, c), [ 3 H]cholesteryl-ester-labelled TG-HDL 2 (a, Chol) or 125 I-labelled LDL (b). In someexperiments, 10 mgml 21 free apoA-I (columns 1 and 7), 100 nM ADP (columns 2 and 8),free apoA-I and ADP at the same concentrations as above (column 3), 100 nM ATP(column 4), 0.2 units ml 21 apyrase (column 5), 100 nM EGF (column 6) or increasingconcentrations of 2MeS-ADP (filled squares, c) and ATP-gS (open squares, c) was addedto the incubation medium. Data are expressed as a percentage above or below the controlvalue (set as 0). d, Cells were pre-incubated for 10 min in serum-free medium with(column 10) or without (column 11) 20 nM EGF, or with 10 mgml 21 free apoA-I (column12) or 100 nM ADP (column 13). The amount of EGFR was measured by flow cytometryusing antibodies against EGFR. An isotypic IgG was also used as a control for the efficacyof the EGFR antibody (column 9). Results are expressed as the mean relative cellfluorescence; receptor internalization is indicated by a drop in fluorescence.NATURE | VOL 421 | 2 JANUARY 2003 | www.nature.com/nature 77


letters to naturein hepatocytes, thus cell-surface SR-BI is not involved in the earlyevents of HDL endocytosis (data not shown).A principal feature of the observed endocytosis is its specificitytowards HDL. Indeed, endocytosis of 125 I-labelled LDL was notstimulated by either apoA-I or ADP (Fig. 4b, columns 7 and 8,respectively). Using flow cytometry, we also measured internalizationof the EGFR, as representative of a tyrosine kinase receptor onHepG2 (Fig. 4d) or IHH cells (data not shown). Whereas EGFstrongly reduced the number of EGFRs at the cell surface (Fig. 4d,column 10) as compared with control cells (Fig. 4d, column 11), freeapoA-I (Fig. 4d, column 12) or ADP (Fig. 4d, column 13) had noeffect on the number of EGFRs on HepG2 cells. Increasing concentrationsof IF 1 protein (a natural inhibitor of the ATP hydrolysisactivity of mitochondrial ATP synthase 22 ) strongly inhibited boththe basal (Fig. 5a) and the free apoA-I-stimulated (Fig. 5b) TG-HDL 2 endocytosis, strengthening the idea that ADP generation hasa chief role in basal endocytosis or in endocytosis stimulated byhigh-affinity receptors.The ATP-binding cassette A-1 (ABCA-1) which binds free apoA-I(ref. 23) and is present on HepG2 and IHH cells, might be a partnerin this process. Although no binding parameters for ABCA-1 havebeen measured in this particular cell line 24 , we extrapolated frombinding data in other cell types the binding parameter of free apoA-Ion ABCA-1 (refs 25, 26). It would represent about 4–10% of thetotal free apoA-I binding (25 ng per mg of cell protein) observedunder our conditions on HepG2 cells and IHH, and thus wouldbe almost undetectable. In addition, we observed the stimulation(2–2.5-fold) of cholesterol efflux induced by free apoA-I (a typicalfeature of ABCA-1; ref. 24), but this was not influenced by IF 1 orADP (over the range 1 nM to 10 mM), which allowed us to concludethat our observations did not involve ABCA-1.To estimate the physiological relevance of our data, we carried outin situ internalization experiments using perfused rat liver (ref. 27and Supplementary Table I). Notably, IF 1 protein induced a rapid(45 min) and marked decrease (up to 45%) of TG-HDL 2 internalizationby the liver, indicating that, at least in rodent, the ectopicATP synthase seems to be implicated in hepatic HDL endocytosis.The role that we have proposed for cell-surface ATP synthase inHDL catabolism raises possibilities for the control of cholesterolemia—afundamental issue in cardiovascular disease research. Ourfindings provide a sequence of events whereby the membraneboundF 1 -ATPase elicits a cellular response by generating extracellularADP with the probable involvement of specific downstreamreceptors. With regard to HDL, these mechanisms seem to be part ofa regulation pathway, because the high-affinity binding of HDL tothe b-chain strongly stimulates ADP generation, which in turnpromotes HDL endocytosis. But how the cell directs these proteinstowards the cell surface and how their cell-surface expression (whichseems to be restricted to particular types of cell) is regulated remainunknown and require further investigation.AMethodsReceptor purification using surface plasmon resonanceSurface plasmon resonance measurements and recovery were done at 20 8C using a Biacore3000 (Biacore AB) equipped with a research-grade B1 sensor chip (Biacore AB). ApoA-I(50 fmol mm 22 ) was immobilized on three flow cells using traditional amine-couplingchemistry 28 . The fourth control flow cell lacked immobilized apoA-I. We dilutedsolubilized porcine liver plasma membrane proteins (in 125 mM Tris maleate, 1 mMCaCl 2 , 150 mM NaCl and 8 mM CHAPS, pH 7.4) eightfold in HBS running buffer (10 mMHEPES, 150 mM NaCl, 3 mM EDTA and 0.005% polysorbate, pH 7.4), and injected 100 mgof proteins at a flow rate of 20 mlmin 21 . The APROG microrecovery procedure (BiacoreAB) was used to recover captured proteins in 7 ml of elution buffer (10 mM triethylamineand 6 M urea, pH 11). We resolved the eluates by SDS–PAGE and silver staining. Tomeasure the binding activity of porcine liver plasma membrane proteins, the proteinseluted from apoA-I affinity chromatography were diluted threefold in running buffer, and1.5 mg of proteins was injected into either the apoA-I cell or the control flow cell.Affinity chromatography and sequence analysisApolipoprotein AI, coupled to an Affi-gel 15 support (Bio-Rad), was used to purify apoA-I-binding proteins. Solubilized porcine liver plasma membrane extracts were diluted to afinal concentration of 1 mM in CHAPS buffer and applied to the apoA-I affinity columnfor 1 h at 4 8C. After five column washes with 10 ml 0.1 M sodium acetate buffer, pH 6.5,bound proteins were eluted in 10 mM triethylamine and 6 M urea, pH 11. We resolved theeluates by SDS–PAGE. An amidoblack-stained band of 50K was cut out and digested withendoprotease lysine-C. The resulting peptides were separated by HPLC on a C18 columnwith a 2–70% gradient of acetonitrile in 0.1% trifluoroacetic acid and sequenced at theInstitut Pasteur.Internalization assaysInternalization assays were done as described 19 . Results are expressed as a percentage of theinternalization measured with 125 I-TG-HDL 2 alone (corresponding to a value of 400 ng ofTG-HDL 2 per mg of cell protein).Flow cytometryThe primary antibodies were from Molecular Probes as follows: clone 7E3-F2 against theb-chain of ATP synthase, 7H10-BD4 against the a-chain of ATP synthase, and 1D6-E1-A8against subunit I of cytochrome oxidase. Antibodies (clone LA1) against EGFR were fromUpstate Biotechnology.To analyse cell-surface EGFR, HepG2 cells were preincubated in medium with orwithout 20 nM EGF for 10 min at 37 8C. For flow cytometry analysis, HepG2 and CHOcells were detached and fixed in 3% paraformaldehyde. We incubated cells at 20 8Cfor1hin PBS, pH 7.4, containing 1% bovine serum albumin (BSA) plus primary monoclonalantibodies. The cells were then washed in PBS plus 1% BSA and incubated at 20 8C for30 min with a goat antibody against mouse IgG conjugated to fluorescein isothiocyanate,before analysis on a Coulter XL 4C flow cytometer (Beckman-Coulter).Immunofluorescence and confocal microscopyGlass coverslips coated with cells were washed with PBS, pH 7.4, fixed for 15 min in 3%paraformaldehyde and saturated for 30 min with 0.2% gelatin (staining buffer). A controlslide was permeabilized for 2 min in 0.2% Triton X-100. Cells were then incubated for 1 hwith the primary antibody diluted to 5 mgml 21 in PBS. Immunostaining was carried outfor 1 h in the dark with antibody against mouse IgG2a conjugated to Alexa 488 diluted to5 mgml 21 in staining buffer.For confocal microscopy, we incubated the cells for 2 h with 100 mgml 21 apoA-I, andthen washed them twice in PBS before fixation. A rabbit polyclonal antiserum againstapoA-I (10 mgml 21 ) was incubated with the primary antibodies as described above.Immunostaining was done with an antibody against mouse IgG2a conjugated to Alexa 488(5 mgml 21 ) and an antibody against rabbit IgG conjugated to rhodamine (5 mgml 21 ). Thecoverslips were examined with a Zeiss Axioskop microscope or with a confocal microscope(LSM510, Zeiss) at a magnification of £ 630.Figure 5 Effect of IF 1 on TG-HDL 2 internalization by hepatocytes. HepG2 cells wereincubated for 10 min at 37 8C in DMEM medium, pH 6.6, with 75 mgml 21 of 125 I-labelledTG-HDL 2 and in the presence of increasing concentrations of IF 1 without (a) or with (b) freeapoA-I (10 mgml 21 ). Data are expressed as a percentage above or below the controlvalue (set as 0).78Measurement of cell-surface ADP and ATPConfluent HepG2 cells in six-well plates were washed in DMEM medium, and thenincubated at 37 8C for 10 min in DMEM, pH 6.6, with 0.1 mCi of [a- 32 P]ATP for the ADPgeneration assay, or with 0.1 mCi 32 P and ADP (100 nM final concentration) for the ATPgeneration assay. Depending on the experiment, IF 1 (100 nM final) or apoA-I (10 mgml 21final) was added to the reaction mixture. We analysed the supernatants by two systems:first, by HPLC coupled to a radioactivity detector on a Whatman Partisphere 5 SAXcolumn as described 29 , with calibration by radiolabelled nucleotides; second, by thin-layerNATURE | VOL 421 | 2 JANUARY 2003 | www.nature.com/nature


chromatography in 2.4% NaCl:NH 4 OH:H 2 O:MeOH (12.5:15:27.5:50 v/v) withquantification of radioactive spots by liquid scintillation.Received 30 July; accepted 7 October 2002; doi:10.1038/nature01250.1. Sviridov, D. & Nestel, P. Dynamics of reverse cholesterol transport: protection against atherosclerosis.Atherosclerosis 161, 245–254 (2002).2. Fidge, N. H. High density lipoprotein receptors, binding proteins, and ligands. J. Lipid Res. 40,187–201 (1999).3. Barbaras, R., Collet, X., Chap, H. & Perret, B. Specific binding of free apolipoprotein A-I to a highaffinitybinding site on HepG2 cells: characterization of two high-density lipoprotein sites.Biochemistry 33, 2335–2340 (1994).4. Martinez, L. O. et al. Characterization of two high-density lipoprotein binding sites on porcinehepatocyte plasma membranes: contribution of scavenger receptor class B type I (SR-BI) to the lowaffinitycomponent. Biochemistry 39, 1076–1082 (2000).5. Boyer, P. D. The ATP synthase: a splendid molecular machine. Annu. Rev. Biochem. 66, 717–749(1997).6. Stock, D., Leslie, A. G. & Walker, J. E. Molecular architecture of the rotary motor in ATP synthase.Science 286, 1700–1705 (1999).7. Cabezon, E., Runswick, M. J., Leslie, A. G. & Walker, J. E. The structure of bovine IF(1), the regulatorysubunit of mitochondrial F-ATPase. EMBO J. 20, 6990–6996 (2001).8. Das, B., Mondragon, M. O., Sadeghian, M., Hatcher, V. B. & Norin, A. J. A novel ligand in lymphocytemediatedcytotoxicity: expression of the beta subunit of H þ transporting ATP synthase on thesurface of tumour cell lines. J. Exp. Med. 180, 273–281 (1994).9. Moser, T. L. et al. Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc. NatlAcad. Sci. USA 96, 2811–2816 (1999).10. Chang, S. Y., Park, S. G., Kim, S. & Kang, C. Y. Interaction of the C-terminal domain of p43 and the asubunit of ATP synthase: Its functional implication in endothelial cell proliferation. J. Biol. 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Long-term primary cultures of adult humanhepatocytes. Chem. Biol. Interact. 107, 31–45 (1997).17. Cabezon, E., Arechaga, I., Jonathan, P., Butler, G. & Walker, J. E. Dimerization of bovine F1-ATPase bybinding the inhibitor protein, IF 1 . J. Biol. Chem. 275, 28353–28355 (2000).18. Cabezon, E., Butler, P. J., Runswick, M. J. & Walker, J. E. Modulation of the oligomerization state of thebovine F1-ATPase inhibitor protein, IF1, by pH. J. Biol. Chem. 275, 25460–25464 (2000).19. Guendouzi, K., Collet, X., Perret, B., Chap, H. & Barbaras, R. Remnant high density lipoprotein2particles produced by hepatic lipase display high-affinity binding and increased endocytosis into ahuman hepatoma cell line (HEPG2). Biochemistry 37, 14974–14980 (1998).20. Acton, S. et al. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor.Science 271, 518–520 (1996).21. Silver, D. L., Nan, W., Xiao, X. & Tall, A. R. HDL particle uptake mediated by SR-BI results in selectivesorting of HDL cholesterol from protein and polarized cholesterol secretion. J. Biol. Chem. 276,25287–25293 (2001).22. Walker, J. E. The regulation of catalysis in ATP synthase. Curr. Opin. Struct. Biol. 4, 912–918(1994).23. Wang, N., Silver, D. L., Thiele, C. & Tall, A. R. ATP-binding cassette transporter A1 (ABCA1) functionsas a cholesterol efflux regulatory protein. J. Biol. Chem. 276, 23742–23747 (2001).24. Bortnick, A. E. et al. The correlation of ABC1 mRNA levels with cholesterol efflux from various celllines. J. Biol. Chem. 275, 28634–28640 (2000).25. Burgess, J. W., Kiss, R. S., Zheng, H., Zachariah, S. & Marcel, Y. L. Trypsin-sensitive and lipidcontainingsites of the macrophage extracellular matrix bind apolipoprotein A-I and participate inABCA1-dependent cholesterol efflux. J. Biol. Chem. 277, 31318–31326 (2002).26. Fitzgerald, M. L. et al. Naturally occurring mutations in ABCA1’s largest extracellular loops candisrupt its direct interaction with apolipoprotein A-I. J. Biol. Chem. 277, 33178–33187 (2002).27. Barrans, A. et al. Hepatic lipase induces the formation of pre-b1 high density lipoprotein (HDL) fromtriacylglycerol-rich HDL2. J. Biol. Chem. 269, 11572–11577 (1994).28. Johnsson, B., Lofas, S. & Lindquist, G. Immobilization of proteins to a carboxymethyldextranmodifiedgold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal.Biochem. 198, 268–277 (1991).29. Sultan, C. et al. The novel inositol lipid phosphatidylinositol 3,4-bisphosphate is produced by humanblood platelets upon thrombin stimulation. Biochem. J. 269, 831–834 (1990).Supplementary Information accompanies the paper on Nature’s website(ç http://www.nature.com/nature).Acknowledgements We thank G. Larrieu, for technical help, and P. Maurel INSERMU128, for theprimary human hepatocytes.Competing interests statement The authors declare that they have no competing financialinterests.Correspondence and requests for materials should be addressed to R.B.(e-mail: Ronald.Barbaras@toulouse.inserm.fr).letters to nature..............................................................Chloroplast to nucleuscommunication triggeredby accumulation ofMg-protoporphyrinIXÅsa Strand*†, Tadao Asami‡, Jose Alonso*, Joseph R. Ecker*& Joanne Chory*†* Plant Biology Laboratory; and † the Howard Hughes Medical Institute; andThe Salk Institute, La Jolla, California 92037, USA‡ Plant Functions Laboratory, Riken, Wako, Saitama 351-0198, Japan.............................................................................................................................................................................Plant cells coordinately regulate the expression of nuclear andplastid genes that encode components of the photosyntheticapparatus. Nuclear genes that regulate chloroplast developmentand chloroplast gene expression provide part of this coordinatecontrol. There is evidence that information also flows in theopposite direction, from chloroplasts to the nucleus 1,2 . Until now,at least three different signalling pathways have been identifiedthat originate in the plastid and control nuclear geneexpression 3,4 but the molecular nature of these signals hasremained unknown. Here we show that the tetrapyrrole intermediateMg-protoporphyrin (Mg-ProtoIX) acts as a signallingmolecule in one of the signalling pathways between the chloroplastand nucleus. Accumulation of Mg-ProtoIX is both necessaryand sufficient to regulate the expression of many nuclear genesencoding chloroplastic proteins associated with photosynthesis.Communication between plastids and the nucleus is necessaryfor the initiation of chloroplast development in the light, and alsofor the ability of the plant cell to respond correctly to fluctuations inthe environment. Evidence that nuclear genes are regulated bysignals originating in the plastid came from studies with mutantsof carotenoid biosynthesis. These mutants bleach when exposed tohigh irradiances of light, and show decreased expression of nuclearphotosynthetic genes 5 . This photobleaching response was laterreplicated by treating wild-type plants with norflurazon, a noncompetitiveinhibitor of carotenoid biosynthesis 6 . Norflurazontreatedplants suffer from photooxidation of the thylakoid membranesand the resulting inhibition of chloroplast development andfunction leads to decreased transcription of nuclear encodedphotosyntheticgenes 7,8 . Using the effect of norflurazon on nucleargene expression, we have previously undertaken a genetic approachto identify components of the plastid to nucleus signalling pathway(s)9 . Five non-allelic loci were identified as genome uncoupledmutants (gun1–5); these mutants express nuclear-encoded photosyntheticgenes in the absence of proper chloroplast development.Three of these loci encode enzymes in the tetrapyrrole pathway;GUN2 encodes haem oxygenase (allelic to HY1), GUN3 phytochromobilinsynthase (allelic to HY2), and GUN5 the H-subunit of Mgchelatase(Fig. 2) 3 . Here we use the gun1, gun2 and gun5 mutants, aswell as new mutants, to unravel the source of the plastid signalregulating nuclear-encoded genes.To further characterize the extent of nuclear genes whoseexpression is regulated by the plastid signal, we used the AffymetrixArabidopsis oligoarray, containing about 8,200 genes, and showedthat, in addition to the previously described photosynthetic genes(LHCB and RBCS), 322 genes change their expression more thanthreefold (182 repressed and 140 induced), in wild-type seedlingsgrown on 5 mM norflurazon (Supplementary Information). Geneticanalysis of the different gun mutants suggests there are two separategun pathways, defined by mutations in GUN1 and GUN2-5 (ref. 3),so we compared the expression profiles of three gun mutants, gun1,gun2 and gun5. Of the 322 genes whose expression levels wereNATURE | VOL 421 | 2 JANUARY 2003 | www.nature.com/nature 79

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