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Biochemical and Biophysical Research Communications 270, 570–575 (2000)

doi:10.1006/bbrc.2000.2455, available online at http://www.idealibrary.com on

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nteraction between HIV-1 NEF and Go Proteinsn Transfected COS-7 Cells

rancesca Guzzi,* Elisa Celano,* Giulio Levi,† and Marco Parenti*Department of Experimental and Environmental Medicine and Biotechnology, School of Medicine,niversity of Milano-Bicocca, Via Cadore 48, 20052 Monza, Italy; and †Laboratory of Pathophysiology,

stituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy

eceived March 8, 2000

G-protein-coupled, bombesin receptors [4, 5]. More-owSNtNaSoG[rrr(l

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Nef protein of HIV/SIV lentiviruses affects G-pro-ein-mediated signaling, and physically associates tock, a myristoylated and palmitoylated Src-like ty-osine kinase. To assess whether Nef interacts with-subunits of heterotrimeric G proteins (Ga), carryinghe same lipidation motif as Lck, we transiently ex-ressed Nef and Goa (wild-type or nonpalmitoylated3S mutant), individually or in combination, in trans-

ected COS-7 cells. Recombinant Nef was mostly recov-red in particulate fractions, and a Nef-Green Fluo-escent Protein chimera was localized at the plasma-emma by in vivo fluorescence imaging. Moreover, Nefnd C3S were entirely solubilized by cold Triton X-100,nd excluded from low buoyant density sucrose gradi-nt fractions, containing caveolin-1, whereas wild-ype Goa was partially resistant to Triton extraction,nd colocalized with caveolin-1. After coexpression,ef recruited soluble C3S to membranes, and the tworoteins were coimmunoprecipitated by Goa andef antisera. We conclude that Nef interacts withonpalmitoylated Goa, presumably outside caveolin-ich microdomains of the plasma membrane. © 2000

cademic Press

Key Words: Nef; HIV; AIDS; G protein; palmitoyl-tion; signal transduction; caveolae; caveolin.

The nef gene, highly conserved in all primate lenti-irus (HIV-1, HIV-2, SIV), encodes for a protein ofbout 27 kDa, that is myristoylated on the N-terminallycine (glycine-2) [1]. Recent studies on SIV-infectedacaques monkeys and in severe combined immuno-

eficient (SCID) mice have shown that Nef is essentialor both maintenance of a high virus load in vivo andor AIDS disease progression [2, 3]. However, the mo-ecular mechanism(s) underlying these effects are stillnknown. Recently, it has been shown that Nef expres-ion impairs early events of the signaling cascadesriggered by the activation of tyrosine kinase receptorsor platelet-derived growth factor (PDGF) and of

570006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

ver, a number of cellular proteins directly interactingith Nef have been identified, including p56Lck (Lck), arc-like non-receptor tyrosine kinase that, similarly toef, is myristoylated (myr) on glycine-2, but in addi-

ion is palmitoylated (pal) on cysteine-3, and -5 [6]. The-terminal sequence met-gly(myr)-cys(pal) representscharacteristic motif, shared by other members of therc-like tyrosine kinase family, and by the a subunitsf heterotrimeric Gi, Go, and Gz proteins (Gia, Gza, and

oa) [7]. This motif serves as a signal to target Lck8–10], and Ga [11, 12] polypeptides to detergent-esistant microdomains of the plasma membrane en-iched in cholesterol and glycosphingolipids (glycolipidafts), and containing lipid-modified signaling proteinsG proteins, Src, Ras, etc.), and often caveolins (caveo-ae) [13, 14].

The structural analogies between Lck and duallycylated Ga led us to verify the possibility that Nefight physically interact with Goa. To test this hypoth-

sis, we first compared by biochemical means, the rel-tive subcellular distribution of Nef, wild-type Goa,nd palmitoylation-defective Goa mutant (C3S), ex-ressed individually or in combination, in transientlyransfected COS-7 cells. The cellular targeting of Nefas further investigated by in vivo fluorescence micro-

copic analysis of the distribution of a fusion proteinetween Nef and the Green Fluorescent Protein (GFP)eporter in transfected CV-1 cells. Finally, the interac-ion between Nef, and coexpressed wild-type Goa, or3S mutant, was assessed by coimmunoprecipitationith antisera directed against either polypeptides.

ATERIALS AND METHODS

Materials. Bru/Lai Nef in the mammalian pSG5 expression vec-or (Stratagene) was a kind gift from Professor V. Erfle (GSF Inst.ol. Virology, Munich, Germany). The cDNAs coding for the wild-

ype Goa, and the C3S Goa mutant, generated as in [15], wereubcloned in the pcDNA3 expression vector (Invitrogen). The GFPDNA was kindly donated by Dr. J. Pines (Wellcome/CRC Institute,

Cambridge, UK). It contains five mutations (F64L, S65T, V163A,IatARDrtsBmAusbcGbf

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Vol. 270, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

167T, S175G) resulting in improved levels of fluorescence at 475 nmnd correct folding at 37°C. The following reagents were obtainedhrough the AIDS Research and Reference Program, Division ofIDS, NIAID, NIH (Rockville, MD): HIV-1 Nef antiserum from Dr.. Swanstrom [16] and HIV-1 Nef monoclonal antibody (EH-1) fromr. J. Hoxie. Other antibodies and their sources were as follows:

abbit anti-Goa polyclonal antiserum (ON1), raised against a syn-hetic peptide corresponding to amino acids 160–169 of the Goaubunit, was a kind gift from Professor G. Milligan (Division ofiochemistry and Molecular Biology, University of Glasgow, UK);ouse monoclonal antibody raised against a1 subunit of Na/K-TPase (gift of Dr. G. Pietrini, CNR Center for Cellular and Molec-lar Pharmacology, Milano, Italy); affinity-purified polyclonal anti-erum against rat NADH cytochrome b5 reductase (kindly donatedy Professor N. Borgese, University of Catanzaro, Italy); anti-aveolin-1 IgG (mAb 2297; Transduction Labs, Lexington, KY); anti-FP antiserum (rabbit polyclonal; MBL, Nagoya, Japan). All otheriochemicals used were of the highest purity available and obtainedrom regular commercial sources.

Generation of Nef-GFP fusion construct. To generate the Nef-FP fusion protein, the cDNA encoding GFP was placed at the 39 end

f the full-length BRUNef. A nucleotide sequence coding for glycine-lycine-glycine-glycine-serine was inserted as a spacer between thewo DNAs. The nef gene was amplified by PCR, using the oligo-ucleotide primer 59-GCGAATTCATGGGTGGCAAGTGGTCA-39,arrying an EcoRI site (underlined) positioned upstream of-terminal Nef sequence (in bold) and a reverse primer 59-CGGATCCACCTCCTCCTGAACCTCCTCCACCGCAGTTCTTG-AGTACTC-39, carrying the BamHI site (underlined) downstreamf C-terminal sequence of Nef after removal of the stop codon. Sim-larly, GFP cDNA was amplified by PCR using the primer 59-GCGGATCCGGAGGTGGCGGATCTAGTAAAGGAGAAGAA-TT-39, carrying the BamHI site (underlined) upstream of GFP-terminal sequence after removal of ATG codon (in bold), and therimer 59-CTCAAGTCTAGA-CTATTTGTATAGTTCATC-39, car-ying the XbaI site (underlined) downstream of C-terminal GFPequence (in bold). Amplified Nef and GFP sequences were subclonednto the EcoRI/BamHI and BamHI/XbaI sites, respectively, of pGEMector (Promega) and the resulting Nef-GFP fusion product clonednto pcDNA3 (EcoRI/XbaI sites). The correctness of construct waserified by DNA sequencing. All DNA manipulations, including liga-ions, bacterial transformations, and plasmid purifications were car-ied out using standard procedures.

Cell culture and transfection. Monkey kidney COS-7 and CV-1ells were maintained in Dulbecco’s Modified Eagle MediumDMEM) supplemented with 10% newborn calf serum (COS-7), or0% fetal bovine serum (CV-1), 2 mM L-glutamine, 100 U/ml peni-illin and 100 mg/ml streptomycin in an atmosphere of 5% CO2 at7°C. Cells were transfected at approximately 50–70% confluencysing the Lipofectamine reagent (Life Technologies) according to theanufacturers’ instructions and routinely analyzed 48–72 h post-

ransfection.Transfections with Nef-GFP fusion were performed on CV-1 cells

rown on glass coverslips. Protein expression was observed in un-xed cells under a Zeiss Axioplan MC100 fluorescence microscope4 h after transfection.

Western immunoblot analysis. Cellular proteins were resolved byDS–PAGE (12.5% acrylamide) and transferred to nitrocelluloseembranes. Blots were incubated overnight at 4°C in TBST (20 mMris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5%owdered skim milk. After five washes with TBST, membranes werencubated for 3 h at room temperature with the primary antibodiesiluted in milk/TBST and for 1.5 h with horseradish peroxidase-onjugated goat anti-rabbit IgG. Proteins were detected using theuperSignal detection kit (Pierce).

571

l hypotonic buffer (5 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 1 mMGTA, 0.1 mM EDTA, with 0.067 TIU/ml aprotinin (Sigma) and 0.2M phenylmethylsulfonyl fluoride (PMSF; Sigma) as protease in-ibitors. Cell suspensions were incubated on ice for 30 min, freeze/hawed and homogenized with a Teflon/glass homogenizer. Follow-ng a low-speed centrifugation to remove unbroken cells and theuclear pellets, the samples were centrifuged for 30 min at 200,000gt 4°C in a Beckman TL100 centrifuge. The pellets (particulateraction, P) and the acetone-precipitated supernatants (soluble frac-ion, S), were separated by SDS–PAGE (12.5% acrylamide; [17]) andnalyzed by Western blotting [18].To test the differential sensitivity of proteins to Triton X-100

olubilization, cells were lysed at 4°C in Mes-buffered saline [MBS;5 mM 2-(N-morpholino)ethanesulfonic acid (Mes), pH 6.5, 0.15 MaCl] containing 1% (v/v) Triton X-100 and PMSF and aprotinin asbove. Suspensions were passed five times through a 26G needle andncubated on ice for 30 min. After centrifugation at maximal speed in

microfuge for 10 min at 4°C supernatants were collected (Triton-oluble fraction) and pellets resuspended in 10 mM Tris–HCl bufferH 8.0/0.15 M NaCl/1% Triton X-100/60 mM octyl-glucoside/protinin/PMSF, and processed as before (Triton-resistant fraction).roteins from both fractions were acetone-precipitated and subjectedo SDS–PAGE and Western blotting.

Preparation of caveolin-enriched fractions. COS-7 cells werecraped into 2 ml of ice-cold MBS and homogenized with 10 strokesf a loose-fitting Dounce homogenizer. The homogenate was adjustedo 40% sucrose by the addition of 2 ml of 80% sucrose prepared inBS and placed at the bottom of an ultracentrifuge tube. Two 4-ml

olumes of 30 and 5% sucrose in MBS were layered above theomogenate and the gradients centrifuged at 39,000 rpm for 16–20in a SW41 rotor (Beckman Instruments). A light-scattering band

lose to the 5–30% sucrose regions was observed that containedaveolin-1 but excluded most of other cellular proteins. From the topf each gradient, 1 ml gradient fractions were collected to yield aotal of 12 fractions. An equal volume of each gradient fraction waseparated by SDS–PAGE and subjected to immunoblot analysis.

Coimmunoprecipitation. Transfected COS-7 cells were lysed 30in at 4°C in PBS/Ca21/Mg21 containing 5 mM EDTA, 60 mM octyl-

lucoside, and protease inhibitors (Complete Mini tablets, Boeh-inger-Mannheim). Samples were precleared for 2–3 h at 4°C usingrotein A–Sepharose in PBS/Ca21/Mg21 (20 ml, slurry 1:1) and sub-

ected to overnight immunoprecipitation at 4°C using anti-Nef ornti-Goa ON1 polyclonal antibodies. After incubation for 4–5 h withrotein A–Sepharose (30 ml, slurry 1:1) immunoprecipitates wereashed three times with PBS/Ca21/Mg21 and once with distilledater, and processed as above. Blots were then probed with ON1olyclonal or EH-1 monoclonal anti-Nef antibody.

ESULTS

To explore the possibility that Nef protein may in-eract with Goa, we initially compared the subcellularistribution of both polypeptides after transient ex-ression of each individual gene in COS-7 cells byransient transfection. Figure 1 shows that 48 h afterransfection with pSG5-Nef, a single band migratingn SDS–PAGE with an apparent molecular mass pre-icted for the Nef polypeptide (27 kDa), was detectedy Western blotting of whole cell homogenate with thepecific Nef monoclonal antibody (T), that was absentn the extract from cells transfected with the emptyector (mock). Following ultracentrifugation of post-uclear supernatant of transfected cells at 100,000g,

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Vol. 270, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ef protein was almost entirely recovered in the par-iculate fraction (P) corresponding to cellular mem-ranes, whereas very little was associated to the solu-le cytosolic fraction (S) (Fig. 1).Similarly to Nef polypeptide, most of recombinantild-type Goa, expressed in COS-7 cells, was contained

n the particulate fraction, whereas the less hydropho-ic C3S protein was almost equally distributed be-ween particulate and soluble fractions (Fig. 2A). Co-xpression of Nef did not modify membrane associationf wild-type Goa (data not shown), but significantlyhifted C3S mutant to the particulate fraction (Fig.A), suggesting that Nef insertion into lipid bilayeray recruit some soluble C3S to membranes.Recently, the existence of plasmalemmal microdo-ains, described in the literature as detergent-

nsoluble glycolipid rafts, without, or with caveolinscaveolae), has been demonstrated [12–14]. Thus, thessociation of Nef to such membrane domains wasssessed by solubilization of transfected cells in 1%riton X-100 at 4°C, and subsequent analysis ofetergent-soluble (Ts) and insoluble (Ti) fractions byestern blotting. As shown in Fig. 1 Nef only parti-

ioned in the detergent-soluble phase, thus suggestinghat the polypeptide was not associated to glycolipid-nriched domains. The partitioning of Goa varied, ac-ording to the presence of palmitate. As Nef protein,he C3S mutant was fully soluble in cold Triton,hereas wild-type Goa was partially resistant to solu-ilization (Fig. 2B), thus suggesting that palmitoyl-tion dictates the association of Goa to Triton-insolubleembranes, as previously shown for Lck [8–10] andi1a [11, 12].

FIG. 1. Total expression, subcellular distribution, and Tritonolubility of Nef protein in transfected COS-7 cells. COS-7 cells wereransiently transfected using Lipofectamine reagent with pSG5 vec-or (mock), or pSG5-Nef (T). Fourty-eight hours posttransfection,ells were harvested and lysed in Laemmli sample buffer. Whole cellysates were separated by 12.5% SDS–PAGE and immunoblottedith a monoclonal anti-Nef antibody (EH-1). To determine the sub-

ellular distribution of Nef protein, postnuclear supernatants ofransfected COS-7 cells were fractionated by ultracentrifugation at00,000g into soluble (S) and particulate (P) fractions. Partitioning ofef between fractions was determined by SDS–PAGE and Westernlotting. The solubility of Nef in non-ionic detergents was assayed byncubating transfected COS-7 cells at 4°C in 1% (v/v) Triton X-100,nd subjecting detergent-soluble (Ts) and insoluble (Ti) phases tocetone precipitation, SDS–PAGE and Western blotting. The posi-ions of two apparent molecular mass markers of 23 and 28 kDa arendicated by arrows.

572

nt procedure to isolate detergent-insoluble mem-ranes, based on their low buoyancy on sucrose densityradients, a property conveyed by the high content ofholesterol and glycosphingolipids. Accordingly, weompared the distributions of Nef, wild-type and C3S

oa, with protein markers for subcellular membraneompartments, in fractions generated by ultracentrif-gation of cold Triton extracts of transfected COS-7ells on bottom-loaded sucrose gradients. Based on thenique distribution of caveolin-1 protein, we could as-ume that caveolar membranes floated to fractions 4nd 5, at the boundary between 5 and 30% sucroseayers, whereas the bulk of cellular proteins, includingoluble and non-DIG-associated endogenous polypep-ides, such as NADH cytochrome b5 reductase, aarker for mitochondrial/ER membranes, and a1-

ubunit of Na/K ATPase, a plasma membrane enzyme,tayed within bottom fractions, containing 40% sucroseFig. 3). Nef protein was only recovered in non-caveolar

embrane fractions, thus confirming the Triton solu-ility results. The gradient partitioning of Nef fullyoincided with that of C3S mutant, and of most wild-ype Goa. In fact, wild-type Goa was partially recoveredn the low buoyancy, caveolin-1-containing fractions,hus confirming the association of dually acylated Gao caveolae membranes [11, 12].

To further study the subcellular distribution of Nef,e constructed a chimeric protein by fusion of the

FIG. 2. (A) Effect of Nef coexpression on subcellular distribution of3S mutant in transfected COS-7 cells. COS-7 cells transfected withcDNA3-Goa, or pcDNA3-C3S, individually, or in combination withSG5-Nef, were harvested and postnuclear supernatants fractionatedy ultracentrifugation at 200,000g into soluble (S) and particulate (P)ractions. Partitioning of Goa wild-type and C3S mutant between frac-ions was determined by SDS–PAGE and immunoblotting with poly-lonal anti-Goa antibody (ON1). (B) Partitioning of Goa wild-type and3S mutant into cold Triton-soluble and insoluble extracts of COS-7ells. COS-7 cells transfected with pcDNA3-Goa, or pcDNA3-C3S mu-ant, were incubated at 4°C with 1% Triton X-100 and partitioned intoetergent-soluble (Ts) and insoluble (Ti) phases. The contents of eachoa wild-type, and C3S mutant polypeptides in the two phases wereetermined by SDS–PAGE and Western blotting.

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reen Fluorescent Protein (GFP) reporter to thearboxy-terminus of Nef. Then, the Nef-GFP chimeraas transiently expressed in transfected CV-1 cells, aonkey kidney cell line giving better morphological

esolution than COS-7 cells. As shown in Fig. 4A, GFPuorescence, detected by microscopy on unfixed cells,as stronger around the cell edges and in elongatedxtensions irradiating from cell bodies, thus suggest-ng that the Nef-GFP fusion was concentrated at thelasma membrane. Parallel biochemical analysis inV-1 (and COS-7 cells, data not shown) showed that

he fusion of GFP did not alter subcellular partitioningf Nef polypeptide, since the Nef-GFP chimera, as Neflone (see above), was detected in the particulate, andn the Triton-soluble fractions (Fig. 4B). These resultsuggest that the cellular distribution of the Nef-GFPhimera could presumably be extrapolated to that ofhe wild-type Nef protein.

The localization of the latter at the plasma mem-rane, where Goa is also found (data not shown),hould then be taken as a further indication that, ateast in principle, the two polypeptides may interact, as

FIG. 3. Subcellular fractionation of Nef-expressing COS-7 cells.OS-7 cells transfected with pSG5-Nef, pcDNA3-Goa, or pcDNA3-3S, were extracted in 1% Triton X-100 at 4°C and subjected toltracentrifugation on sucrose density gradients. One-ml fractionsollected from the top of the gradients were separated by SDS–PAGEnd transferred to nitrocellulose. Fractions 1–4 are the 5% sucroseayer, and fractions 5–8 are the 30% sucrose layer. Fractions 9–12,ontaining 40% sucrose, represent the “loading zone” of theseottom-loaded flotation gradients and contain the bulk of cellularembrane and cytosolic proteins (data not shown). Blots were

robed with antibodies against Nef, caveolin-1 (cav-1), cytochrome b5

eductase (cytR), or a1 subunit of Na/K ATPase to visualize theistribution of transfected Nef, and of endogenously-expressed cav-1,nd protein markers for mitochondrial/ER (cytR) and plasma (Na/KTPase) membranes.

573

bove.To directly address this issue, we performed coim-unoprecipitation experiments. After lysis with octyl

lucoside, extracts of COS-7 cells transfected with Nef,ild-type Goa, or C3S mutant, individually, or in com-ination, were subjected to immunoprecipitation withither Nef, or Goa, polyclonal antisera. Western blot-ing analysis of the immunoprecipitates revealed that

oa (Fig. 5A), and Nef (Fig. 5B) were successfully pre-ipitated by antisera raised against the respectiveartner proteins. Moreover, the results were identical,hether the association with Nef was tested for wild-

ype Goa, or C3S mutant, thus suggesting that thettachment of palmitic acid does not significantly in-uence the interaction.

ISCUSSION

Although the critical role played by Nef protein iniral pathogenesis and AIDS have been unequivocallyemonstrated [3, 4], the underlying biological basiss/are still obscure. To address this issue, a consider-ble effort has been recently devoted to examine theellular consequences of recombinant Nef expression inukaryotic cells. Thus, a number of reports have shownhat Nef modulates intracellular signaling pathways in

FIG. 4. Subcellular targeting of Nef-GFP fusion protein in trans-ected CV-1 cells. (A) Unfixed CV-1 cells expressing Nef-GFP fusionrotein were visualized by fluorescence microscopy. Bar, 20 mm. (B)he subcellular distribution and Triton solubility of Nef-GFP chi-era expressed in CV-1 cells were determined as described in the

egend to Fig. 1 using a monoclonal anti-Nef monoclonal antibody.he same results were obtained with a polyclonal anti-GFP anti-erum (not shown).

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Vol. 270, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

everal cell types. In T cell lines Nef down-regulatesell surface expression of both class I MHC and CD4olecules by accelerating their endocytosis [20–22],

nd prevents antigen receptor-mediated induction ofnterleukin 2 mRNA [23]. In NIH-3T3 fibroblasts con-titutively expressing Nef, the signaling cascades trig-ered by PDGF-induced activation of tyrosine kinaseeceptors, or by stimulation of G protein-coupled recep-ors by bombesin, are substantially attenuated [4, 5].table expression of Nef in a human astrocytic cell line

mpairs the stimulation of DNA synthesis mediated byhe activation of extracellular signal-regulated kinaseERK), that follows stimulation of G protein-coupledeceptors by endothelin-1 [24].

A second strategy to elucidate the pathway(s) byhich Nef functions has consisted in the identificationf the cellular proteins that complex with it. So far, aumber of polypeptides physically interacting withef protein have been described, including Lck [6],-COP [25], a serine kinase [26], and a thioesterase,nzymes [27].Now we report that Nef protein physically interactsith Goa, that shares with Lck an identical myristoy-

ated and palmitoylated N-terminus. Although thisotif may well not be directly involved in the associa-

ion with Nef, as clearly demonstrated for Lck [6], itay act as a signal to target both Lck and Goa to

FIG. 5. Coimmunoprecipitation of Nef and Goa. Detergent ex-racts of COS-7 cells transfected with Nef, Goa wild-type, or C3Sutant, individually or in combination, were immunoprecipitated

IP) with anti-Nef (A, upper) or anti-Goa (B, upper) polyclonal anti-era and protein A–Sepharose beads. The immunoprecipitates wereubjected to SDS–PAGE and analyzed by Western blotting (WB)ith anti-Goa polyclonal antiserum (A, above), or anti-Nef monoclo-al antibody (B, above). COS-7 cell extracts contained equivalentmounts of wild-type Goa, and C3S (A, below), or Nef (B, below)olypeptides, as determined by Western blot analysis of approxi-ately 1/10 of each cell lysate prior to immunoprecipitation.

574

uctive coupling to partner proteins can occur.Herein we show that a recombinant Nef-GFP chi-era mostly localized at the surface of transfectedOS-7 cells, and this, as well wild-type Nef, wereniquely associated to high-buoyancy, cold Triton-oluble membranes, from where caveolin-1 protein wasxcluded. Based on these observations, we may assumehat Nef protein is not associated to glycolipid- andaveolin-rich rafts of the plasma membrane of COS-7ells. Surprisingly, this is just the opposite of the re-ent findings of Wang et al. [28], who have shown thatef, heterologously expressed in the Jurkat human T

ymphoid cell line, was highly enriched in membraneafts, isolated by the same detergent procedure as ours.t present we can not explain this striking difference

n other terms, than by assuming that the presence ofaveolin-1 within rafts of COS-7 cells, but not in lym-hocytes, may prevent Nef compartmentalization.The distribution of recombinant C3S mutant com-

letely overlapped with that of Nef, suggesting that thewo polypeptides may potentially come into contactith each other. Indeed, direct evidence in support of aef-C3S interaction comes from the observations that

o-expression of Nef shifted some soluble C3S to mem-ranes, and that the two proteins could be co-recipitated by both Nef and Goa antibodies.Differently from Nef and the C3S mutant, wild-type

oa showed a mixed distribution, being partially asso-iated to Triton-resistant, caveolin-enriched mem-ranes. Given the reversibility of palmitoylation reac-ion, the amount of wild-type Goa colocalizing withaveolin would probably reflect the palmitoylated por-ion of wild-type Goa, whereas the unpalmitoylatedild-type Goa, as the C3S mutant, would reside outsidelycolipid domains [11, 12]. This assumption is con-rmed by the finding that wild-type Goa, metabolically

abeled with [3H]palmitate, was immunoprecipitatednly by caveolin-containing fractions from transfectedOS-7 cells [M. Parenti, manuscript in preparation].Based on these findings we speculate that Nef pro-

ein preferably associates with unpalmitoylated Goa,nd the interaction presumably occurs at plasma mem-rane regions distinct from caveolin- and glycolipid-nriched domains.What could be the functional implications of such an

nteraction? Recent evidences show that G protein ac-ivation promotes depalmitoylation of Ga [29]. More-ver, palmitoylation of Ga increases its affinity for bg30], and inhibits GTPase-accelerating activity of RGSroteins [31], thus suggesting that, in its palmitoylatedtate, Ga-subunit is complexed with bg dimer, to formhe inactive ternary complex. Thus, it is possible thatef, by interacting with unpalmitoylated Goa, couldct as a scavenger for Goa monomer, with the conse-uences of preventing its interaction with downstreamffectors, and/or interrupting the reactivation cycle. In

both cases, an impairment of signaling would bea

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nticipated.In conclusion, this study, introducing Goa as a new

utative target for Nef action, adds further strength tohe view that Nef may indeed affect G-protein-ediated signaling in HIV/SIV-infected cells.

CKNOWLEDGMENTS

This work was supported by an AIDS grant (No. 10/A/H) fromstituto Superiore di Sanita to G.L. F.G. is a recipient of a 2-yearellowship from the Italian National AIDS research program fundedy the Istituto Superiore di Sanita.

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