The Phosducin-Like Protein PhLP1 Is Essential for G{beta}{gamma} Dimer Formation in Dictyostelium discoideum

Phosducin proteins are known to inhibit G protein-mediated signalingby sequestering Gß ${gamma}$ subunits. However, Dictyosteliumdiscoideum cells lacking the phosducin-like protein PhLP1 displaydefective rather than enhanced G protein signaling. Here weshow that green fluorescent protein (GFP)-tagged Gß(GFP-Gß) and GFP-G ${gamma}$ subunits exhibit drastically reducedsteady-state levels and are absent from the plasma membranein phlp1^– cells. Triton X-114 partitioning suggests thatlipid attachment to GFP-G ${gamma}$ occurs in wild-type cells but notin phlp1^– and gß^– cells. Moreover, Gß ${gamma}$ dimers could not be detected in vitro in coimmunoprecipitationassays with phlp1^– cell lysates. Accordingly, in vivodiffusion measurements using fluorescence correlation spectroscopyshowed that while GFP-G ${gamma}$ proteins are present in a complex inwild-type cells, they are free in phlp1^– and gß^–cells. Collectively, our data strongly suggest the absence ofGß ${gamma}$ dimer formation in Dictyostelium cells lackingPhLP1. We propose that PhLP1 serves as a cochaperone assistingthe assembly of Gß and G ${gamma}$ into a functional Gß ${gamma}$ complex. Thus, phosducin family proteins may fulfill hithertounsuspected biosynthetic functions.

INTRODUCTION

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Introduction

Phosducin family proteins have classically been linked to Gprotein regulation. Both phosducin (Phd) and the related phosducin-likeprotein (PhLP) have been shown to bind Gß ${gamma}$ subunits(18, 32, 53, 57, 64, 69). In so doing, they are thought to functionas a cellular "sink" which sequesters free Gß ${gamma}$ subunitsfollowing their dissociation from receptor-activated G proteins(2, 19, 33, 41, 58). As G protein-coupled receptors only coupleto G ${alpha}$ ß ${gamma}$ trimers, the sequestration of Gß ${gamma}$ attenuatestransmembrane signaling. Thus, phosducin family proteins mayadapt the cell's sensitivity to extracellular signals.

Being interested in factors underlying the adaptation of G protein-mediatedchemotactic signaling in Dictyostelium discoideum, we recentlyidentified three Dictyostelium Phd-like protein genes (3). Totest whether PhLP1, the Dictyostelium protein that is most similarto mammalian Phd and PhLP, is involved in modulating G proteinsignaling, we analyzed phlp1 knockout cells. Surprisingly, Gprotein signaling is completely defective rather than enhancedin phlp1^– cells, which exhibit a phenotype that is remarkablysimilar to that of gß knockout cells. Fluorescenceconfocal microscopy experiments with cells expressing greenfluorescent protein (GFP)-tagged Gß (GFP-Gß)or GFP-G ${gamma}$ fusion proteins indicated that Gß ${gamma}$ complexesare absent from the plasma membrane of phlp1^– cells, providinga possible explanation for the abrogation of signal transduction(3). These findings suggested that Gß and G ${gamma}$ fail tobe assembled into a Gß ${gamma}$ complex in phlp1^– cellsor that the complex is not properly routed to the plasma membranein these cells.

In this paper, we have further investigated the Gß ${gamma}$ defect in phlp1^– cells. We show that steady-state levelsof (GFP-tagged) Gß and G ${gamma}$ subunits are dramaticallyreduced and that these proteins are detected in the cytosolwhen the PhLP1 protein is not present. Triton X-114 partitioningexperiments suggest that G ${gamma}$ is not lipid modified in phlp1 knockoutcells. Prenylation is normally the first step in a sequenceof posttranslational modification events following Gß ${gamma}$ dimerization (22, 70) and is essential for membrane association(45, 60). Moreover, a complementary combination of in vitrobiochemical and in vivo spectroscopic experiments strongly suggeststhat Gß and G ${gamma}$ do not form a complex in the absenceof PhLP1: tagged Gß and G ${gamma}$ subunits are not detectablycoimmunoprecipitated, and cytosolic diffusion measurements usingfluorescence correlation spectroscopy (FCS) reveal that thediffusion of GFP-G ${gamma}$ expressed in phlp1^– cells is similarto that of free GFP-G ${gamma}$ monomers in cells lacking Gß.

Collectively, our data suggest that the phosducin-like proteinPhLP1 serves a role in Gß ${gamma}$ dimer formation. We proposethat PhLP1 may function as a cytosolic cochaperone for Gß ${gamma}$ assembly.

MATERIALS AND METHODS

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Materials and Methods

Cell culture. All cell lines were grown in 9-cm dishes containing HG5 medium(14.3 g/liter of peptone, 7.15 g/liter of yeast extract, 10g/liter of glucose, 0.49 g/liter of KH₂PO₄, and 1.36 g/literof Na₂HPO₄ · 2H₂O). The Dictyostelium discoideum AX3strain was used as a wild-type control in all experiments. Cellswere grown in HG5 medium that was supplemented with 30 µg/mlG418 (GibcoBRL) for the transfectants expressing tagged Gßor G ${gamma}$ subunits. The gß^– (LW6) and phlp1^–knockout cell lines, as well as cells expressing either GFP-Gßor GFP-G ${gamma}$ , have been described previously (3, 36, 68).

Plasmid construction and transfection. An expression plasmid encoding both GFP-tagged Gßand hemagglutinin (HA)-tagged G ${gamma}$ (HA-G ${gamma}$ ) was constructed as follows.DNA encoding Dictyostelium G ${gamma}$ was isolated as a BglII-SpeI fragmentfrom a pGEM-T G ${gamma}$ -Easy construct (3) and subcloned in pMB74 downstreamof the actin15 promoter. The Dictyostelium expression vectorpMB74 is derived from pMB12Neo (37) by replacement of the 2H3terminator with an actin8 terminator. A double-stranded oligonucleotidecontaining a Dictyostelium translational initiation site (A₅ATG)and a sequence encoding an N-terminal HA epitope (MISYPYDVPDYA)was inserted in frame with the G ${gamma}$ coding sequence by ligatingit as a BamHI-BglII fragment into the BglII site of G ${gamma}$ /pMB74.The resulting expression cassette, with HA-G ${gamma}$ codons flankedby an actin15 promoter and an actin8 terminator, was isolatedby XbaI digestion, Klenow treatment, and ClaI digestion andsubcloned in the SmaI and ClaI sites of a pLB5Neo-based expressionplasmid encoding GFP-Gß (3). The resultant plasmid,pGß ${gamma}$ Express, contains tandem expression cassettesfor GFP-Gß and HA-G ${gamma}$ .

To express a GFP-PhLP1 fusion construct, DNA encoding PhLP1was amplified by PCR using the forward primer 5'-TCTCAGATCTAAAGAATGGAACAAAACATTTTAAATAG-3'and the reverse primer 5'-GGACTAGTATCGTCATTATCATCATCGGAC-3',with the DNA encoding the open reading frame of phlp1 as thetemplate (3). The DNA fragment was subcloned in pGEM-T Easy(Promega) and sequenced. Subsequently, the phlp1 insert wasreleased and ligated into the BglII and SpeI sites of MB74GFP.The MB74GFP plasmid is similar to MB74 but contains the S65TGFP gene behind the SpeI site. The final fusion protein consistsof the open reading frame of PhLP1, two serines, and the completeS65T GFP protein. The plasmids were introduced into Dictyosteliumcells by electroporation following standard procedures, andtransfectants were selected with G418.

Western blot analysis. Cells were lysed either directly by boiling in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) samplebuffer or by filter lysis and detergent solubilization (seebelow). Equal amounts of lysates were loaded; lysates containingGFP-Gß or GFP-G ${gamma}$ were applied to standard 10 or 12%SDS-PAGE gels. Lysates containing the small HA-G ${gamma}$ protein wereapplied to 15% Tricine-SDS-PAGE gels (54). Following electrophoresis,proteins were electroblotted onto polyvinylidene difluoridemembranes (Millipore). For confirming equal loading, blots werestained with Ponceau S. For Western blot analysis, the membraneswere blocked for 2 h in 5% low-fat milk in Tris-buffered salineplus Tween 20 (TBST) (20 mM Tris-HCl [pH 7.4], 137 mM NaCl,0.05% Tween 20). Subsequently, membranes were incubated overnightat 4°C with polyclonal anti-GFP antibody ab6556 (Abcam)diluted 1:5,000 in blocking buffer, washed several times withTBST, incubated for 1 h at room temperature with a peroxidase-coupledsheep anti-rabbit antibody (Roche), washed several times withTBST and once with TBS without Tween 20, and developed withan ECL kit (Roche).

Crude cell fractionation. Cells were harvested from confluent 9-cm dishes and washed twicewith phosphate buffer (pH 6.5). Cell pellets were taken up in150 to 300 µl ice-cold lysis buffer (50 mM Tris-HCl [pH7.5], 5 mM EDTA, 5 mM EGTA, 150 mM NaCl, 1 mM dithiothreitol,containing Complete protease inhibitor cocktail [Roche]) andlysed through a 3-µm Nuclepore filter (Whatman). Lysateswere centrifuged for 5 min in an Eppendorf centrifuge at 4°C,and a sample of the supernatant was carefully removed whiletaking care not to take along any particulate matter. The pelletswere washed and then resuspended in an original volume of lysisbuffer. Equal samples of supernatant (cytosolic) and pellet(particulate) fractions were used for Western blot analysis.

Triton X-114 partitioning assay. Cells were prepared and lysed as described above. Subsequently,100 µl lysate was diluted with 100 µl lysis buffercontaining 2% Triton X-114, which had been precondensed thrice(4), and rotated for 40 min at 4°C. Insoluble material wasremoved by spinning for 15 min at 4°C in an Eppendorf centrifuge,and supernatants were subjected to phase partitioning by incubationat 30°C for 3 min and spinning for 5 min at room temperature.The upper, water phases were reextracted with an equal volumeof 2% Triton X-114, and the lower, detergent phases were reextractedwith 0.2% Triton X-114. Following partitioning at 30°C,detergent phases were combined, and the volume was adjustedwith lysis buffer to match the volume of the water phase samples.Equal samples of water and detergent phases were used for Westernblot analysis.

Immunoprecipitation. Cells were prepared and lysed as described above. All subsequentprocedures were performed at 4°C. Following lysis, 100 µllysate was diluted with 400 µl lysis buffer containing1.25% Triton X-100 and rotated for 45 min. Insoluble materialwas removed by centrifugation for 15 min in an Eppendorf centrifuge,and the lysate was precleared twice by rotating with 50 µl50% (vol/vol) protein A-Sepharose beads for 1 h and removalof the beads by centrifugation for 10 min. Precleared supernatantswere then rotated for 1 h with 2 µl monoclonal anti-GFPantibody ab1218 (Abcam), and immunocomplexes were collectedby rotation with 25 µl 50% (vol/vol) protein A-Sepharosebeads for 1 h and centrifugation for 1 min. The beads were washedfour times with lysis buffer containing 1% Triton X-100 andonce with lysis buffer without Triton X-100 while transferringthe beads to a fresh tube. Beads were taken up in 2x concentratedSDS-PAGE sample buffer, heated for 3 min at 95°C, and spundown for 5 min in an Eppendorf centrifuge. Supernatants werestored at –20°C prior to Western blot analysis.

Fluorescence correlation spectroscopy. The diffusion measurements of GFP-tagged proteins in cells wereperformed on a ConforCor 2 (Carl Zeiss). The details of thesetup have been described previously (23). In our experiments,GFP was excited with the 488-nm line from an argon-ion laserand focused into the sample with a water immersion C-Apochromat40x lens objective (Zeiss). The excitation intensity was $~$ 11µW. The excitation and emission light was separated bya dichroic beam splitter (HFT 488/633). The fluorescence wasdetected by an avalanche photodiode after being filtered througha band-pass filter of 505 to 550 nm. The pinhole was set at70 µm.

Cells from a confluent dish were transferred to a 96-chamberglass-bottomed microplate (Whatman, Inc.) and washed twice withpotassium phosphate buffer (17 mM, pH 6.5). Measurements wereperformed for cells incubated in buffer at room temperature.Around 100 autocorrelation traces were obtained for each cellline from five measurements made at a randomly chosen spot inthe cytoplasm of around 20 different cells. The expression levelsin cells were too high for FCS measurements, and so the GFPwas photobleached to acceptable fluorescence intensity levelsby exposing the cells to a high-intensity laser beam ( $~$ 1 mW)for 1 s. The measurement duration for all cell lines was 10s.

The data were analyzed using the software FCS Data Processor(61). The autocorrelation traces were fitted with a model describingBrownian motion of a single species in three dimensions (equation1), with an additional offset term to account for artifactscaused by drifts in average fluorescence on time scales of >1s arising from cellular and intracellular movement (5).

(1)

In equation 1, G( ${tau}$ ) is the autocorrelation function, N is theaverage number of molecules in the detection volume, T_dif isthe average diffusion time of the molecules, and sp is the structuralparameter. A global-analysis approach was used to fit up tofive traces simultaneously with the T_dif linked and a fixedvalue of sp. The value of sp, determined from calibration measurementswith a rhodamine 110 solution, was around 5. The translationaldiffusion coefficient D was calculated from the diffusion timeT_dif by using equation 2,

(2)
where ${omega}$ _xy is the equatorial radius of the confocal volume element andis obtained from calibration measurements.

RESULTS

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Results

Expression of GFP-PhLP1 rescues phlp1^– cells. Northern blots revealed that PhLP1 is expressed throughout development(3; unpublished observations). To investigate the cellular localizationof PhLP1 and the potential interaction of PhLP1 with membrane-associatedGß ${gamma}$ complexes, PhLP1 was fused to GFP and expressedin phlp1^– cells. This resulted in a cell line with normaldevelopment and a complete rescue of the aggregation-negativephenotype (unpublished observations). Confocal microscopy revealedthat PhLP1-GFP is localized predominantly in the cytosol, withoutindications for any membrane enrichment (Fig. 1). Stimulationof these cells with 1 µM cyclic AMP in a perfusion chamberdoes not lead to a significant change in the fluorescence intensityof PhLP1-GFP in the cytosol (detection limit of 4% change influorescence intensity [50]) or any detectable translocationof PhLP1-GFP from the cytosol to other cellular compartments(unpublished observations). These observations suggest thatPhLP may function in the cytoplasm and that we have no evidencefor an interaction of PhLP1 with Gß ${gamma}$ complexes at theplasma membrane.

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FIG. 1. PhLP1-GFP is localized in the cytosol. An extrachromosomal plasmid expressing a C-terminal fusion of GFP to PhLP1 was expressed in phlp1^– cells, which completely restored the aggregation-negative phenotype. PhLP1-GFP appears to be localized in the cytosol in both weakly and strongly expressing cells, without indications for association to membranes or membrane-associated proteins.

GFP-Gß and GFP-G ${gamma}$ are expressed at reduced levels and do not associate with the plasma membrane in phlp1^– cells. To be able to monitor the fate of Gß and G ${gamma}$ in intactcells, we expressed GFP-Gß or GFP-G ${gamma}$ fusion proteinsin wild-type AX3 cells and in phlp1^– cells (3). Complexesof Gß and G ${gamma}$ tagged with GFP variants have been demonstratedto be fully functional in Dictyostelium amoebae (26, 27). Asa reference, we also employed gß^– cells, whichdo not express Gß and therefore lack normal Gß ${gamma}$ complexes (36, 68).

Western blot analysis revealed that correctly sized proteinsare expressed in all cell lines (Fig. 2A). However, the steady-statelevels of GFP-Gß and GFP-G ${gamma}$ proteins are dramaticallyreduced (at least 20-fold) in phlp1^– cells compared tothe levels in wild-type cells. Such reduced protein levels werealso observed for endogenously expressed Gß by useof an anti-Gß antibody (unpublished observations).Interestingly, the mRNA levels for Gß are not significantlydifferent between control and phlp1^– cells (Fig. 2B),suggesting that the reduced levels of Gß, GFP-Gß,and GFP-G ${gamma}$ proteins are due to increased instability of the proteinsin phlp1^– cells. The cellular level of GFP-G ${gamma}$ in gß^–cells is also significantly lower than in control cells (Fig.2A). This indicates that G ${gamma}$ subunits are less stable in the absenceof Gß. Such a mutual dependency of binding partnersfor stable expression has been previously documented for variousprotein complexes, including Gß ${gamma}$ dimers (24, 51, 55,60, 67).

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FIG. 2. Expression of Gß and G ${gamma}$ in phlp1^– cells. (A) Steady-state protein levels of GFP-Gß and GFP-G ${gamma}$ are reduced in phlp1^– cells. Wild-type AX3 cells (wt) and gß^– and phlp1^– knockout cells were transfected with plasmids encoding GFP-Gß or GFP-G ${gamma}$ . Cell lysates were prepared and analyzed through Western blot analysis with a GFP antibody. The molecular masses of GFP-Gß and GFP-G ${gamma}$ are indicated. The asterisk denotes a cross-reacting AX3 band. (B) Northern blots show normal expression of Gß in phlp1^– cells. Poly(A) mRNA was isolated from wild-type AX3 cells (wt), phlp1^– knockout cells, phlp1^– cells overexpressing PhLP1 (Phlp^OE), and gß^– knockout cells. RNA was size fractionated, transferred, and probed with gß.

Crude cell fractionation of AX3 transfectants revealed thatGFP-Gß and GFP-G ${gamma}$ can be detected both in a cytosolicfraction and in a particulate fraction containing plasma membranes(Fig. 3). Jin and coworkers similarly reported a considerableamount of GFP-Gß (about 30%) to be present in cytosolicfractions of transfected Dictyostelium amoebae and claimed thatthe same holds true for endogenous Gß (27). In strikingcontrast to the situation for wild-type cells, GFP-Gßand GFP-G ${gamma}$ cannot be detected in particulate fractions of phlp1^–transfectants, although they are detected in cytosolic fractions.Again, in gß^– cells, GFP-Gß can combinewith endogenous G ${gamma}$ and exhibits behavior similar to that in wild-typecells, whereas GFP-G ${gamma}$ does not reach the plasma membrane in defaultof a Gß partner. These results are fully in line withour previous fluorescence confocal microscopy studies (3) andexplain why G protein signaling is defective in cells lackingthe PhLP1 protein. Either a Gß ${gamma}$ complex is formed butnot transported to its final destination, or the complex isnot formed at all, with individual Gß and G ${gamma}$ subunitsnot being able to associate with the plasma membrane. In eithercase, this largely results in the demise of the nonfunctionalprotein.

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FIG. 3. GFP-Gß and GFP-G ${gamma}$ are absent from plasma membranes in phlp1^– cells. Cells expressing GFP-Gß or GFP-G ${gamma}$ were subjected to crude fractionation by filter lysis and collection of particulate and cytosolic fractions after centrifugation in a microcentrifuge. Equal samples were used for Western blot analysis with a GFP antibody, as described in the legend for Fig. 2A. wt, wild type.

Triton X-114 partitioning suggests that GFP-G ${gamma}$ is not prenylated in phlp1^– cells. To further define the stage at which the formation of a functionalGß ${gamma}$ complex goes awry in phlp1^– cells, we assessedwhether G ${gamma}$ proteins are still posttranslationally modified. Duringnormal Gß ${gamma}$ synthesis, a CAAX motif at the C terminusof immature G ${gamma}$ subunits is prenylated, followed by proteolyticremoval of the last three residues (AAX) and carboxyl methylationof the now C-terminal prenyl-cysteine moiety (22, 70). The attachmentof a prenyl group is mandatory for stable membrane associationof Gß ${gamma}$ subunits (45, 60).

Lipid modification of G ${gamma}$ can be demonstrated by metabolic labelingwith radioactive precursors but can also be investigated ina nonradioactive fashion by using the Triton X-114 partitioningassay originally devised for integral membrane proteins (4).In this assay, cell lysates containing Triton X-114 are preparedand then induced to undergo phase separation by transient warmingto 30°C. Proteins carrying a significantly hydrophobic moiety,such as a prenyl group, are drawn from the water phase intothe detergent phase. The partitioning assay has been used notonly for small monomeric G proteins but also for heterotrimericG proteins (28).

As can be seen in Fig. 4, substantial amounts of both GFP-Gßand GFP-G ${gamma}$ expressed in AX3 cells are drawn into the detergentphase after partitioning. This indicates that GFP-G ${gamma}$ is lipidmodified in wild-type cells and that GFP-Gß must forma tight complex with endogenous G ${gamma}$ since it lacks hydrophobicmodifications. When expressed in gß^– cells,GFP-Gß also forms a complex with endogenous G ${gamma}$ proteinsand therefore partitions into the detergent phase. However,GFP-G ${gamma}$ expressed in gß^– cells can only be detectedin the water phase. This indicates that G ${gamma}$ is not (efficiently)modified unless it is present in Gß ${gamma}$ heterodimers.Such a finding corroborates suggestions in the literature thatthe Gß ${gamma}$ complex is the substrate for the prenylationmachinery (22).

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FIG. 4. Triton X-114 partitioning suggests absence of lipid modification on GFP-G ${gamma}$ in phlp1^– cells. Triton X-114-containing extracts were prepared from cells expressing GFP-Gß or GFP-G ${gamma}$ and subjected to phase partitioning at 30°C. Water phases and detergent phases were separated and reextracted, and then equal samples were used for Western blot analysis with a GFP antibody, as described in the legend for Fig. 2A. wt, wild type.

Significantly, GFP-Gß and GFP-G ${gamma}$ expressed in phlp1^–cells could only be detected in the water phase after extractionand partitioning. Thus, G ${gamma}$ remains unmodified in phlp1^–cells and fails to draw Gß into the detergent phase.A lack of lipid attachment in phlp1^– cells might be explainedby defective modification of an otherwise properly formed Gß ${gamma}$ complex or by the failure of Gß and G ${gamma}$ to form a complexin the first place.

Coimmunoprecipitation studies suggest that Gß and G ${gamma}$ do not form a complex in phlp1^– cells. To assess whether there is indeed a lack of Gß ${gamma}$ complexformation in phlp1^– cells, we performed coimmunoprecipitationexperiments. In order to be able to detect both Gßand G ${gamma}$ , we transfected AX3 and phlp1^– cells with a plasmidwhich directs the coexpression of GFP-tagged Gß (GFP-Gß)and hemagglutinin epitope-tagged G ${gamma}$ (HA-G ${gamma}$ ). Cell lysates weresubjected to immunoprecipitation with an anti-GFP antibody,and subsequently aliquots of the precipitate were analyzed byWestern blot experiments with antibodies recognizing GFP (forimmunoprecipitated GFP-Gß) or HA (for coimmunoprecipitatedHA-G ${gamma}$ ).

As can be seen in Fig. 5, GFP-Gß can be immunoprecipitatedfrom both wild-type AX3 and phlp1^– knockout cell lysates.HA-G ${gamma}$ was clearly coprecipitated with GFP-Gß when theseproteins were coexpressed in wild-type cells. In contrast, asimilar band could not be detected in the precipitate of phlp1^–cells coexpressing GFP-Gß and HA-G ${gamma}$ , even though significantamounts of GFP-Gß had been precipitated. Furthermore,we have mixed small amounts of a lysate prepared from wild-typecells expressing GFP-Gß and HA-G ${gamma}$ with large amountsof lysate from phlp1^– cells in a 1:20 ratio and couldeasily observe the presence of the Gß ${gamma}$ dimer (unpublishedobservations). These results suggest that a normal Gß ${gamma}$ complex is not present at significant levels in phlp1^–cells, and we propose that the levels of Gß and G ${gamma}$ in phlp1^– cells are low because they are not in dimersand are hence destabilized, rather than the opposite, that dimersare rare because Gß and G ${gamma}$ levels are too low in theabsence of PhLP1. Unfortunately we have not yet been able toinduce Gß ${gamma}$ dimer formation in vitro by using a mixtureof lysates from GFP-Gß/HA-G ${gamma}$ -expressing phlp1^–cells (providing unassembled GFP-Gß and HA-G ${gamma}$ ) withwild-type cells (providing phosducin).

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FIG. 5. GFP-Gß and HA-G ${gamma}$ do not coimmunoprecipitate in extracts of phlp1^– cells. Cell lysates were prepared from nontransfected cells and from cells expressing either GFP-Gß alone or both GFP-Gß and HA-G ${gamma}$ and immunoprecipitated with a monoclonal GFP antibody. Half of the immunoprecipitates were used for Western blot analysis with a polyclonal GFP antibody ( ${alpha}$ -GFP), and the other half were used for Western blot analysis with an antibody recognizing hemagglutinin tags ( ${alpha}$ -HA). wt, wild type.

Similar diffusion of GFP-G ${gamma}$ in phlp1^– and gß^– cells suggests absence of Gß ${gamma}$ complexes in phlp1^– cells. The coimmunoprecipitation experiments described above indicatethat Gß and G ${gamma}$ subunits do not form a complex in cellslacking PhLP1. Further experiments were complicated by the reducedsteady-state levels of GFP-Gß and GFP-G ${gamma}$ in phlp1^–cells. To overcome this problem, and to substantiate our invitro findings in an in vivo environment, we used FCS, whichis a sensitive technique that can be used to monitor the diffusionof proteins expressed at low concentrations in living cells(20, 30, 43). The diffusion characteristics of a protein providevaluable clues about its aggregation state or intracellularinteractions. Thus, the cytoplasmic diffusion coefficients obtainedfrom FCS measurements can be used to discriminate free subunitsfrom Gß ${gamma}$ complexes in different cell lines.

The 7-kDa G ${gamma}$ is much smaller than the 40-kDa Gß and,thus, diffusion characteristics of GFP-G ${gamma}$ would be more stronglyaffected than those of GFP-Gß when Gß ${gamma}$ nolonger forms a complex. Therefore, we chose to analyze the diffusionof GFP-G ${gamma}$ in the cytoplasm of wild-type and mutant cell lines(Fig. 6 and Table 1).

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FIG. 6. GFP-G ${gamma}$ exhibits monomer-type diffusion characteristics in phlp1^– cells. Comparison of the diffusion coefficient distribution of GFP-G ${gamma}$ in wild-type AX3 (black bars), phlp1^– (open bars), and gß^– (gray bars) cells. The diffusion coefficient distribution of GFP-G ${gamma}$ proteins was determined in the cytosol of different cell lines by using FCS. Bars indicate the fraction of the total number of observations in which a specific diffusion coefficient (D) was found.

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TABLE 1. Diffusion of GFP-G ${gamma}$ in wild-type and mutant cell lines

The experimental data fit well to a model describing the diffusionof a single population of fluorescing molecules in three dimensions.Figure 6 shows that the diffusion coefficient distribution forGFP-G ${gamma}$ in phlp1^– cells is shifted to higher values thanthat in wild-type AX3 cells. The slow diffusion of GFP-G ${gamma}$ inwild-type cells indicates that it is predominantly present asa complex in wild-type cells but not in phlp1^– cells.To determine if the diffusion of GFP-G ${gamma}$ in phlp1^– cellswas indeed the diffusion of free GFP-G ${gamma}$ monomers, we measuredthe diffusion of GFP-G ${gamma}$ in gß^– cells. As canbe seen in Fig. 6, the diffusion coefficient distribution forGFP-G ${gamma}$ expressed in gß^– cells strongly overlapswith that obtained for phlp1^– cells, clearly indicatingthat GFP-G ${gamma}$ in phlp1^– cells is free. The average diffusioncoefficient of GFP-G ${gamma}$ molecules in gß^– cellsis very similar to the value obtained for GFP-G ${gamma}$ in phlp1^–cells (14 and 15 µm²/s, respectively). These values areslightly smaller than the value of 20 µm²/s obtained forfreely diffusing GFP monomers in Dictyostelium cells (52). SinceGFP-G ${gamma}$ and GFP are similar in size (35 and 27 kDa, respectively),their diffusion coefficients are also expected to be similar.However, GFP is approximately globular (49) whereas G ${gamma}$ in a Gß ${gamma}$ dimer has a rod-like structure (63), and though it is not knownhow the structure changes when G ${gamma}$ is not associated with Gß,it is likely that it is (partially) unfolded. The differencein the shapes of GFP and GFP-G ${gamma}$ can thus be the reason for thediscrepancy seen between their diffusion coefficients. Takentogether, the FCS data strongly suggest that, in cells thatare devoid of PhLP1, GFP-G ${gamma}$ molecules are present as free monomers.We therefore conclude that Gß ${gamma}$ complexes, if presentat all, form a very small minority in living phlp1^– cells.

DISCUSSION

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Discussion

Absence of Gß ${gamma}$ dimers in cells lacking phosducin-like protein PhLP1. Dictyostelium amoebae lacking the phosducin-like protein PhLP1exhibit a phenotype which is strikingly similar to the phenotypeobserved upon gß gene disruption (3). Our presentfindings suggest that the gß^–-like phenotypeof phlp1^– cells is due to defective Gß ${gamma}$ dimerformation. We observed a dramatic reduction in Gßand G ${gamma}$ steady-state protein levels in phlp1 knockout cells, whilemRNA levels were unaltered. This might be explained with thenotion that Gß ${gamma}$ complex formation is required for stableexpression of either partner (24, 51, 55, 60, 67). Our inabilityto detect posttranslational lipid attachment to GFP-G ${gamma}$ in phlp1knockout cells would also be in keeping with a lack of Gß ${gamma}$ dimer formation. It has been suggested that Gß ${gamma}$ assemblyprecedes cytosolic prenylation of the C-terminal CAAX motifof G ${gamma}$ (22). Indeed, when we overexpress GFP-G ${gamma}$ in gß^–cells which cannot form Gß ${gamma}$ complexes, the proteinis unstable and does not seem to be lipid modified in TritonX-114 partitioning assays.

The absence of Gß ${gamma}$ dimer formation is also stronglysuggested by our inability to coimmunoprecipitate GFP-taggedGß and HA-tagged G ${gamma}$ in vitro after coexpression inphlp1^– cells. Furthermore, in vivo FCS experiments revealedthat the diffusion of GFP-G ${gamma}$ in phlp1^– cells is similarto that in gß^– cells, whereas it is significantlyslower in wild-type AX3 cells. The average diffusion coefficientvalues can be explained assuming the presence of free GFP-G ${gamma}$ monomers in phlp1^– and gß^– cells and ofGFP-G ${alpha}$ ß ${gamma}$ complexes in AX3 cells. Therefore, the FCSresults indicate that Gß and G ${gamma}$ are indeed apart incells which lack PhLP1.

Taken together, the above data strongly suggest that withoutthe PhLP1 protein, Gß ${gamma}$ dimer formation is defunct inDictyostelium cells. We emphasize that it cannot be ruled outthat small amounts of Gß ${gamma}$ dimers are still being formedin phlp1^– cells. Maybe such traces of Gß ${gamma}$ canstill provide some modulation of (a subset of) effectors, butin the assays which we reported previously, we failed to detectany Gß ${gamma}$ function (3).

PhLP1: a cochaperone for Gß ${gamma}$ dimer formation? Whereas the G ${gamma}$ subunit is a relatively small and flexible peptidelacking tertiary structure (7, 55), the Gß subunitexhibits complex folding. Gß is a member of the WDrepeat protein family (47, 62), which in turn forms part ofa larger family of ß-propeller proteins (46, 48).Gß proteins contain seven WD repeats which form sevenß sheets making up the blades of a propeller-likestructure. The last blade is made up of one ß stranddonated by the first WD repeat and three ß strandsdonated by the last, C-terminal repeat, forming a noncovalent"Velcro snap" (6) which closes the ring structure of the propeller.Such a repetitive and highly integrated structure may not allowproper Gß folding and/or assembly to occur spontaneously.

The structural difference between Gß and G ${gamma}$ is reflectedin a difference of ease of production. G ${gamma}$ can be produced inany expression system, but Gß is more demanding. Assembly-competentGß cannot be produced in Escherichia coli or wheatgerm extracts (21, 44) and only inefficiently in rabbit reticulocytelysates (11, 12, 44, 55). This indicates the need for an accessoryfactor during Gß synthesis which may have divergedbetween plant and mammalian cells and is in limiting supplyin reticulocyte lysates. The need for one or more cellular factorsis also indicated by the finding that assembly-competent Gßcan only be collected from recombinant baculovirus-infectedSf9 cells during early infection stages, when host cell proteinexpression is not shut down yet (14, 21). Thus, Gßproteins may require molecular chaperones (10, 17) for theirincorporation into functional Gß ${gamma}$ complexes.

A likely candidate as one of these factors is the group II chaperoninCCT (chaperonin containing TCP-1), which is also known as TRiC(34, 66). The barrel-shaped cylinder of CCT, a folding cagemade up of two rings of eight subunits each, may provide a favorableenvironment for, and participate in, the folding and assemblyof various proteins. Intriguingly, an unexpected proportionof yeast proteins interacting with CCT subunits harbor sevenWD repeats (66). Indeed, this set of proteins includes STE4,the yeast Gß subunit. These findings have recentlybeen verified for both STE4 and five other, unrelated WD repeatproteins in yeast (59).

Apart from protein folding, CCT has also been implicated inthe assembly of multimeric proteins. The WD repeat domain ofthe VHL tumor suppressor protein has been shown to bind to CCTprior to VHL assembly with the elongin BC complex (8, 16). Moreover,with the help of five additional cofactors, ${alpha}$ - and ß-tubulinare dimerized following their folding by CCT (15, 66). Thus,CCT, aided by cochaperones, may assist both folding and assemblyof multimeric proteins, including WD repeat proteins such asGß.

The picture that emerges from our studies on Dictyostelium phlp1^–cells is that Gß ${gamma}$ complex formation is abolished inthe absence of PhLP1. It is unlikely that PhLP1 deficiency leadsto a loss of function of the CCT machinery, as the CCT machineryis absolutely required for actin and tubulin folding and itsabsence is lethal in yeast (35, 65). Rather, it is temptingto speculate that PhLP1 serves a role as a cofactor cooperatingwith CCT in the formation of native Gß ${gamma}$ heterodimers.Interestingly, mammalian PhLP, but not Phd, has been shown tocoimmunoprecipitate CCT subunits (42).

While the current work was under review, three reports werepublished providing substantial support for the hypothesis thatPhLP is a molecular chaperone for Gß ${gamma}$ assembly (25,39, 40). The structure of the complex between CCT and PhLP revealsPhLP binding at the apical top of CCT, above the folding activity.This leads to the hypothesis that PhLP delivers its bindingpartner (possibly Gß ${gamma}$ ) to the CCT complex for folding(40). Consistent with this hypothesis is the observation thatRNA interference inhibition of the CCT subunit TCP-1 ${alpha}$ leads toa strong reduction of the level of Gß ${gamma}$ subunits (25).Very recently, Lukov et al. (39) have shown that reduced expressionof PhLP by RNA interference results in inhibition of Gß ${gamma}$ expression and G protein signaling, similar to PhLP-null cellsin Dictyostelium (3) and Cryphonectria parasitica (29). Furthermore,Lukov et al. demonstrated that the inhibition of G protein signalingis due to an inability of nascent Gß ${gamma}$ to form dimers,as also demonstrated in the present work. They provide a modelin which PhLP binds to Gß and stabilizes the nascentGß polypeptide until G ${gamma}$ will associate and PhLP willdissociate to catalyze another round of assembly (39). Phosphorylationof PhLP at Ser18 to Ser20 enhances binding to CCT and is essentialfor Gß ${gamma}$ folding (39). However, the role of CCT remainsunclear, since a PhLP mutant with reduced CCT binding (PhLP133-135A)is fully capable in Gß ${gamma}$ folding (39).

The structure of Phd-Gß ${gamma}$ complexes may provide cluesas to what the cochaperoning role of phosducin-type proteinscould possibly be. Phd interacts with blades 6 and 7 of nativeGß ${gamma}$ and can induce conformational changes in that regionwhile transporting Gß ${gamma}$ away from the membrane (13,38). Intriguingly, during Gß biosynthesis, blade 7must be formed by bringing together remote ß strandsfrom the N-terminal and C-terminal WD repeats of Gß(6). Also, assembly-competent Gß has an open structurewith a Stokes radius larger than that of native Gß ${gamma}$ complexes (21, 55, 56). Thus, one might hypothesize that moleculessuch as PhLP facilitate ring closure of the Gß propellerwhile Gß and G ${gamma}$ assemble. The fact that phlp1^–cells grow significantly more slowly than both wild-type andgß^– cells (3) could indicate that the cellularchaperone machinery is to some extent obstructed in the absenceof PhLP1 or that PhLP1 may assist in the folding of other proteinsthat are required for optimal cell growth.

Three families of PhLP proteins have been recognized, with onemember of each family in Dictyostelium (3). The PhLP1 subfamilyincorporates the mammalian PhLP involved in Gß ${gamma}$ folding(39) and the PhLP proteins from Cryphonectria parasitica andDictyostelium that are essential for Gß ${gamma}$ functioningand folding (3, 29). Members of the PhLP2 and PhLP3 familiesbind Gß ${gamma}$ poorly and have no Gß ${gamma}$ -malfunctioningphenotypes. Deletion of PhLP2 is lethal in both Dictyosteliumand yeast (3, 9). Interestingly, in yeast, PhLP2 forms a complexwith CCT and VID27, a protein with a WD40 propeller structuresimilar to that of Gß (1). Deletion of PhLP3 in yeastand Dictyostelium is not lethal (3, 9), but genetic evidencesuggests that the yeast PhLP3 family member (named Plp1p) transfersthe nascent ß-tubulin polypeptides to the CCT foldingapparatus (31). An interesting possibility that the PhLP familiesact as chaperones for the assembly of several proteins arises.

In conclusion, we have found that cells lacking the phosducin-likeprotein PhLP1 are not able to form functional Gß ${gamma}$ dimers,and we propose that this defect is related to an essential cochaperonefunction of PhLP1 during Gß ${gamma}$ assembly.

ACKNOWLEDGMENTS

We thank Peter Devreotes for providing us with the gß^–cell line LW6 and Mark Hink for useful discussions on the FCSdata.

The work by R.E. was financially supported by the Research Councilfor Earth and Life Sciences of the Netherlands Organizationfor Scientific Research.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Phone: 31-50-363 4172. Fax: 31-50-363 4165. E-mail: P.J.M.van.Haastert{at}rug.nl.

These authors contributed equally to this work.

REFERENCES

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