Uncoupling proteins 2 and 3 interact with ... - Wiley Online Library

Eur. J. Biochem. 267, 2680±2687 (2000) q FEBS 2000

Uncoupling proteins 2 and 3 interact with members of the 14.3.3 family Benoit Pierrat, Moriko Ito, Willy Hinz, Marjo Simonen*, Dirk Erdmann, Michele Chiesi and Jutta Heim Novartis Pharma Inc., Basle, Switzerland

Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms u, b, z were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the g isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 u could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria. Keywords: uncoupling protein; 14.3.3; two-hybrid analysis; mitochondria.

Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP) localized in the inner mitochondrial membrane. Four different UCPs have been identified so far. UCP1 is expressed solely in brown adipose tissue [1], UCP2 is ubiquitous [2], and UCP3 is detected mainly in skeletal muscle in humans [3,4]. A less closely related protein, with major expression in the brain, has recently been identified and named UCP4 [5]. The postulated physiological function of the UCPs is to uncouple respiration from ATP synthesis and thus, to dissipate the energy stored in the proton electrochemical gradient as heat [6,7]. The role of UCP1 in regulating thermogenesis in rodents, where the brown fat plays an important role in hibernation and in cold adaptation, is clearly established. UCP2 and UCP3 could play a more important role than UCP1 in the regulation of thermogenesis and resting metabolic rate in humans, where the brown adipose tissue rapidly disappears after birth. This is particularly true for UCP3, as skeletal muscle is a major thermogenic organ in higher mammals. Many studies have shown that overexpression of UCP2 or UCP3 in host cells induces uncoupling of mitochondria [2,8±10] and increases the thermal power of yeast cells [11]. Recently, the ability of isolated, recombinant UCP2 and 3 to catalyse proton transport across artificial membranes has also been demonstrated [12]. To date, however, direct evidence that endogenous UCP2 and/or UCP3 regulate the level of nonphosphorylating respiration in situ is still Correspondence to J. Heim, Novartis Pharma, Inc., K-125. 13-16, CH-4002 Basle, Switzerland. Fax: 1 41 61 6966323, E-mail: [email protected] Abbreviations: AD, activation domain; UCPs, uncoupling proteins; MATP, mitochondrial anion transport proteins; X-Gal, 5-bromo-4-chloro-3-indoylb-d-galactosidase; MSF, mitochondrial import stimulation factor. *Present address: Brain Research Institute, University of Zurich, Switzerland (Received 6 January 2000, accepted 8 March 2000)

missing. It is justified to assume that mechanisms involved in the regulation of the coupling efficiency of mitochondria must be under strict control at the transcriptional as well as at the post-translational levels. Fine tuning of the UCPs activity could also be sensitive to changes in the concentration of ligands, that sense the level of energy supply to the cell, and of metabolites. In an attempt to identify possible ancillary proteins that might be involved in the regulation of the activity of UCPs, we applied the yeast two-hybrid strategy. Several members of the 14.3.3 protein family have been identified that interact with the C-terminus of UCP2 and 3, but not with UCP1. The relevance of these findings is discussed.

E X P E R I M E N TA L P R O C E D U R E S Strains and media Escherichia coli strain DH5a was used for plasmid manipulations. Cells were grown in L-Broth medium containing ampicillin to select for plasmids. Yeast transformation was carried out as desribed by Hill et al. [13]. Primary positive plasmids from the screening were rescued from yeast by electroporation (2.5 kV, 25 mF and 400 V) of E. coli HB101 strain and selected for the Activation Domain-fused library plasmid using the LEU2 marker to complement the E. coli leuB mutation on M9 minimal medium [14]. The following Saccharomyces cerevisiae strains were used in two-hybrid experiments: L40 [Mata, his3D200, trp1±901, leu2±3,112, ade2, lys2±801am, URA3::(lexAop)8-lacZ, LYS::(lexAop)4HIS3 [15]], and GPY124 [Mata, his3D200, trp1±901, leu2±3, 112, ade2, lys2±801am, URA3::(lexAop)8-lacZ, cyhr]. Strains were grown at 30 8C in YPD (20 g´L21 Difco peptone, 10 g´L21 yeast extract, and 2% glucose) or in synthetic complete medium (SC, 6.7 g´L21 yeast nitrogen base without amino acids, 2% glucose and `dropout' solution that contains all

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the essential amino acids and nucleotides) lacking appropriate amino acids. Plasmid construction The potential intermembrane space loops of hUCP2 were linked together as in-frame fusion with the LexA DNA binding domain in the pLexA vector [15]. pLexA-hUCP2A (loop A comprising residues 1±18) was constructed by subcloning annealed oligonucleotides into the EcoRI/BamHI sites of pLexA. Loop B (residues 135±217) was PCR-amplified with primers 5 0 -TACAGGATCCCAGCCCACGGATGTGGTAA-3 0 and 5 0 -AGCTCTGCAGTCCACCTCCGTGGCAAGGGAGGTCATCTGT-3 0 from a human placenta library template and was inserted as a BamHI±PstI fragment into the construct pLexAhUCP2A yielding pLexA-hUCP2AB. Finally, the C-terminal intermembrane domain loop C (residues 290±309) was added to the AB construct by annealing oligonucleotides and subcloning them into the PstI-cleaved bait plasmid containing the AB domains to yield the fusion construct pLexA-hUCP2ABC. pLexA-hUCP2C, containing only the C-terminal residues 290±309, was constructed by subcloning the annealed oligonucleotides covering amino acids 290±309 into PstI digested pLexA. The same strategy was used for the construction of pLexA-hUCP3C (residues 293±312), for hUCP1C (residues 287±306), and for pLexA-hUCP2C-DN (residues 300±309), all the primers being cloned as EcoRI fragments into EcoRI digested pLexA. hUCP2 and hUCP3 loop C Ser/Ala mutants were PCR amplified from the respective pLexA constructs using mutagenic primers (respectively 5 0 -CCCCGGGAATTCTCAGAAGGGAGCCTCTCGGGCAGTGCAGGCAGCCAT-3 0 and 5 0 -CCCCGGGAATTCTCAAAACGGTGCTTCCCGTAACATCTG-3 0 ) in combination with primer 5 0 -CTTCGTCAGCAGAGCTTCACCAT-3 0 and cloned into the EcoRI site of pLexA. All the constructions were confirmed by sequencing using an automated DNA sequencer. Adipocyte library construction Total RNA from human white adipose tissue was isolated from four individuals 35±63 years of age using the guanidium thiocyanate method as described by Chomczyniski and Sacchi [16]. Poly(A)1 RNA was isolated from human total adipocyte RNA using the minikit Oligotex mRNA (Qiagen) and applied as template for cDNA synthesis with an oligo dT primer (5 0 -GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3 0 ). Following the addition of EcoRI adapters (5 0 -AATTCGGCACGAG-3 0 5 0 -CTCGTGCCG-3 0 ), cDNA was processed further with XhoI restriction digestion and introduced into the lambda arms of the HybriZAP-2.1 phage vector (Stratagene). The entire phage library was amplified once and mass excision was performed yielding a total of 6.8 109 colony forming units (c.f.u.; 3.4 108 c.f.u.´mL21). This yielded the plasmid library pAD-GAL4±2.1, containing the adipocyte-derived cDNAs fused with the activation domain of GAL4. Yeast two-hybrid screening A human skeletal muscle cDNA two-hybrid library (Clontech, HL4010AB) was screened using pLexA-hUCP2ABC as a bait. Strain L40 (a gift from S. Hollenberg, Oregon Health Sciences University, Portland, OR, USA) carrying pLexA-hUCP2ABC was transformed with the library and plated on SC±Trp, ±Leu, ±His, ±Ura plates, selecting for histidine-dependent growth.

UCP2 and UCP3 interact with 14.3.3 (Eur. J. Biochem. 267) 2681

Colonies that grew in the absence of histidine were assayed for b-galactoside activity using 5-bromo-4-chloro-3-indoylb-d-galactosidase (X-Gal). b-Galactosidase activity was reassayed on filters after retransformation of rescued candidate plasmids from the original positive yeast with pLexA plasmids containing either pLexA-hUCP2ABC or an unrelated bait (human Bax) as negative control in strain L40. Positive clones were sequenced using automated DNA sequencing. BLAST searches were performed with the DNA and putative aminoacid sequences of the positive clones. A second screen was performed with pLexA-hUCP2ABC as a bait in the diploid strain resulting from the mating of strains L40(a) and GPY124(a) expressing the human adipocyte cDNA fusion library. Prior to the mating procedure, strain GPY124a was transformed with 750 mg of the human adipocyte cDNA library using the LiAc method [13]. After transformation, the cells were allowed to grow in SC-Leu for 23 h at 30 8C. Plating efficiency indicated that a total of 2.6 109 transformants had been obtained, including an amplification of about 10-fold during the recovery period. Cells were concentrated and aliquoted at 280 8C, each vial containing about 5.2 108 cells. One vial of these human adipocyte cDNA fusion library transformants was mixed with the same amount of cells from the L40 strain expressing the bait construct and incubated as several drops on YPD plates. After incubation for 6 h at 30 8C, cells were collected and plated on 10 SC±Trp, ±Leu, ±His, ±Ura plates, selecting for histidine-independent growth. Positive clones were assayed as described before.

In vitro transcription/translation of 14.3.3 u and UCP3 The human 14.3.3 u and UCP3 proteins were in vitro transcribed and translated by the TNT(r) T7 Coupled Wheat Germ Extract System (Promega). The 14.3.3 u cDNA was cloned into pcDNA3 (Invitrogen) as an EcoRI±XbaI fragment and UCP3 cDNA in vector pcDNA3.1/Zeo as an EcoRI fragment. Both vectors were linearized by XbaI and HindIII downstream of the cDNA, respectively, and used as templates for transcription. The proteins were radiolabeled with [35S]methionine (Amersham) according to the protocol provided. Immunoprecipitation Radiolabeled 14.3.3 u and UCP3 (5 mL each) were preimmunoprecipitated by overnight incubation at 4 8C with 5 mL of rabbit serum and 100 mL Protein A±Sepharose (1 : 1 v/v suspension in lysis buffer) in a total volume of 500 mL lysis buffer [20 mm Tris/HCl pH 7.5, 1% NP-40, 150 mm NaCl and 50 mm NaF, supplemented with protease inhibitor cocktail tablets (Boehringer, Mannheim) and 1 mm ATP] in the presence or absence of cold UCP3 competitor (10 mg of recombinant UCP3, see below). After removal of the Protein A±Sepharose by centrifugation at 2500 g for 5 min, 100 mL Protein A± Sepharose and 1 mg affinity-purified 14.3.3 u antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added to the supernatant and immunoprecipitation was carried out by overnight incubation at 4 8C. The Protein A±Sepharose associated proteins were centrifuged and washed three times with lysis buffer, boiled in SDS sample buffer and analyzed by 13.5% SDS/PAGE and autoradiography. Recombinant human UCP3 was obtained from E. coli. The ORF was extracted from the plasmid pCR2.1 described below and inserted into the NdeI and EcoRI sites of the vector pET22b(1) (Novagen). Plasmids were transformed into the bacterial strain BL21 (Novagen). Induction was performed by adding 1 mm isopropyl-b-dthiogalactopyranoside at 30 8C when the cells reached a A600

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Fig. 1. Diagrams of UCP2 and UCP2-based constructs. (A) A folding diagram of human UCP2 based on topological and computational studies. Amino-acid numbers indicate the putative junction between transmembrane domains and hydrophilic loops. (B) Schematic diagram of the UCP2-based pLexA fusion constructs. A, B, C, D, and E boxes represent putative interesting segments of UCP2 according to folding diagram shown in A. For the AB and ABC fusions, three additional glycine residues were inserted in between the different domains to introduce flexible spacers. S/A indicates the amino-acid replacement of serine 304 by alanine. CDN represents the N-terminal truncation of the C loop. Numbering refers to the human UCP2 sequence.

of 0.6. Inclusion bodies were purified and the UCP3 was solubilized using lauroylsarcosinate basically according to [17]. After various extensive dialysis steps, the protein was stored in 1% Triton X-100, 300 mm KCl, 1 mm dithiothreitol, and TrisCl, pH 8. The protein contained a His-tag at its N-terminus. Effect of UCP expression in yeast S. cerevisiae Construction of the vector for hUCP3 expression in yeast was described elsewhere [11]. In brief, the open reading frame of hUCP3 cDNA was amplified by RT-PCR from human skeletal muscle poly(A)1 mRNA and cloned into the vector pCR2.1 (Invitrogen). To allow the introduction of the hUCP3 cDNA between the BamHI and the EcoRI site of the yeast expression vector pYeDP60 [18], two corresponding restriction sites at the

flanking ends of the coding sequence were introduced in a second amplification step. AD-containing vectors (pGAD10) expressing various 14.3.3 protein isoforms were directly used as expression vectors for coexpression in yeast with hUCP3. pGAD10 empty vector was used as a control. Yeast W303-B1 [11] transformants were selected on minimal medium lacking Trp and Ura in the presence of glucose. For growth comparison studies, the transformed yeast cultures were grown overnight on a medium containing 0.1% casamino acids, 0.7% yeast nitrogen base, 2% lactic acid, 50 mg´L21 tryptophan and 40 mg´L21 adenine, pH 4.5. The cultures were diluted with 0.9% NaCl solution and 1200 cells were then spotted on the agar plates containing minimal medium and supplemented either with 2% glucose or galactose. The plates were incubated for 48 h at 30 8C before evaluation of growth.

Fig. 2. Immunoblot of cell lysates from S. cerevisiae L40 expressing LexA-hUCP2 fusions. Each lane represents proteins from cells equivalent to A600 of 0.9 probed with a mouse monoclonal anti-LexA antibody. Arrows indicate the corresponding bands of LexA-UCP2 ABC (38 kDa), LexA-UCP2 AB (36 kDa), and LexA-UCP2 A (25 kDa), respectively. Lanes 1 and 10 contain L40 extract. Molecular mass markers are indicated in kDa at the right side of the gels.

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Fig. 3. The hUCP2 yeast two-hybrid screening. Strain L40 expressing the LexA-hUCP2ABC fusion protein was transformed with a GAL4 activation domain fusion skeletal library. From 8.5 106 transformants, only 3 remaining His1/ lacZ1 positives were selected. A parallel screen was initiated with the mating of strain L40 containing the pLexA-hUCP2ABC plasmid and the strain GPY124 previously transformed with the GAL4 AD adipocyte fusion library. From 3.3 107 diploid cells, 31 His1/ lacZ1 positive clones were selected.

R E S U LT S Generation and expression of hUCP2 space loops constructs A bait construct for the yeast two-hybrid system was prepared by linking the full length human UCP2 to the LexA DNAbinding domain. However, as UCP2 contains a number of transmembrane domains, expression of two-hybrid fusion constructs encoding the full-length protein could possibly result in its misfolding or unproductive localization (i.e. the mitochondria). To maximize the probability that functionally relevant domains of UCP2 would be recognized by possible regulatory proteins encoded by the prey libraries, a variety of DNAbinding domain fusion constructs, containing the hydrophilic loops of UCP2 facing the intermembrane space, were also prepared (see Fig. 1). Individual domains such as loops A and C, corresponding to the N-terminus and C-terminus of UCP2, respectively, were named pLexA-hUCP2A and pLexA-hUCP2C. The domain B, which was expected to be fairly soluble even though it comprised the third and forth putative transmembrane loops, was linked to the A and/or C domains to create the AB and ABC fusion constructs, thus giving pLexA-hUCP2AB and pLexA-hUCP2ABC. The different loops in the last fusion constructs were connected via three glycine residues, which would serve as flexible spacers and thus facilitate proper folding of the hybrid constructs. Expression of the fusion constructs pLexA-hUCP2A, pLexAUCP2AB and pLexA-UCP2ABC in strain L40 was analysed by immunoblotting using an antibody against the LexA protein (see Fig. 2). Fusion proteins of the expected masses could be detected. However, while high levels of the individual loop hUCP2A (25 kDa) were regularly expressed, the fusion loops hUCP2AB (36 kDa) and hUCP2ABC (38 kDa) showed a lower and strongly heterogeneous level of expression. Searching for interacting partners of hUCP2 L40 yeast strains, bearing the pLexA-hUCP2ABC fusion bait construct, were used to detect potential interacting proteins by direct transformation with a human skeletal muscle cDNA library (Fig. 3). Approximately 8.5 106 colonies were screened and 45 clones, able to grow on medium lacking histidine (primary hits), were selected and assayed for production of b-galactosidase. Screening was carried out in the presence of 3 mm aminotriazole, which is required to suppress the leakiness of the His3 reporter gene. Three clones that upon re-transformation were shown to specifically activate the reporter gene only when coexpressed with the hUCP2-derived sequences, were further

characterized. BLAST searches of the sequence databases revealed that the inserts of these plasmids encoded for different fusions of a protein strongly related to the rat 14.3.3 g isoform (GenBank accession No. S55305). In all three plasmids, the fusion proteins contained the N-terminal methionine of 14.3.3 g linked to the GAL4activation domain via a short polypeptide stretch, derived from the primers used for library construction and the 5 0 -noncoding sequences of the natural mRNA. This clone is identical to the recently published human 14.3.3 g isoform [19]. Screening of prey libraries was also performed using the mating strategy (Fig. 3) [20]. This approach, which takes advantage of the ability of two yeast strains of opposite mating type to form diploid cells, was carried out in parallel, as an alternative to the labour intensive, large-scale transformation procedure for expressing two-hybrid proteins in yeast. Optimal mating conditions were obtained when the cells were incubated on YPD plates during the mating process, resulting in an efficiency of 15±20% of diploid formation. The strain L40 expressing the bait pLexA-hUCP2ABC construct was mated with the strain GPY124 (see Experimental procedures), which was previously transformed with the human adipocyte library and amplified eightfold. Mating generated 3.3 107 diploid cells and 35 primary His1 clones were picked and tested for activation of the LacZ reporter gene on filters. Following retransformation with the original bait (pLexA-hUCP2 ABC), 31 colonies could be confirmed. All positive clones contained cDNAs encoding for proteins with strong homologies to different isoforms of the 14.3.3 family: 17 clones corresponded to the u (GenBank X56468 [21]), 12 clones to the b (GenBank X57346 [22]), and one clone to the z isoform of 14.3.3 (GenBank M86400 [23]). The fusion proteins contained additional amino acids between the C-terminal part of the GAL4 activation domain and the potential first methionine of the 14.3.3 protein, that again were derived from the primers used for library construction. Mapping binding sites required for the interaction To investigate the interaction between 14.3.3 proteins and hUCP2 in more detail, the ability of the 14.3.3 family members to interact with the various hUCP2 constructs (i.e. pLexAhUCP2A, pLexA-hUCP2AB and pLexA-UCP2ABC) was studied in the yeast two-hybrid approach. Confirmation of the interaction occurred only in the presence of the pLexA-hUCP2ABC construct (Fig. 4A), whereas no signal was detected when loop AB was expressed. Similarly, no signal was detected when the full-length hUCP2 or loopA constructs were used as bait (not


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Fig. 5. Co-immunoprecipitation of human 14.3.3 u and UCP3. Proteins were in vitro transcribed and translated in the presence of [35S]methionine using a wheat germ lysate system. Coimmunoprecipitation of the radiolabeled proteins was obtained using anti 14.3.3 u antibodies and protein A-sepharose as described in the Materials and methods section. 14.3.3 u was detected as a doublet and varying intensity, as described in the brochure provided with the antibodies. Cold recombinant hUCP3, solubilized from E. coli inclusion bodies, was added as a competitor at a concentration of 20 mg´mL21. Data from three independent experiments gave a 65 ^ 15% (mean ^ SD) decreased level of UCP3 coimmunoprecipitation in the presence of the competitor. Detection and quantification were performed on a PhosphorImager.

Fig. 4. hUCP2 and hUCP3 loop C interact with 14.3.3 isoforms. Either control vectors (pGAD10 and pVP16-Bcl2) or 14.3.3-encoding expression plasmids were cotransformed into yeast reporter strain L40 in combination with (A) UCP2 loop A, AB or ABC in pLexA or (B) hUCP2 or hUCP3 intermembrane space loop C. (C) Control vector (pGAD10) or 14.3.3 u-encoding expression plasmids were cotransformed into yeast reporter strain L40 in combination with pLexA constructs containing hUCP2 C, hUCP1 C, UCP2C C-terminal part (UCP2 00 CDN 00 ), and mutated forms of serine 304 of UCP2 (UCP2CS304/A304) and serine 310 of UCP3CS310/ A310. 3 individual transformants were spotted on synthetic complete medium lacking histidine, leucine and tryptophane. Photograph was taken after 3 days incubation at 30 8C.

shown). Thus, the strategy to dissect a membrane bound protein into individual domains proved to be the successful one. To exclude that the observed interaction could occur only with a particular, non relevant conformation of the hUCP2ABC protein (which contains glycine residues between the different loops of UCP2 to introduce flexibility), experiments with a new bait construct containing only loop C (from Thr290 to Phe309) were performed. Interestingly, expression of the pLexA-hUCP2C construct and of the 14.3.3 isoforms was sufficient to induce a b-galactosidase signal. Similar results were obtained with the

histidine-based growth assay (Fig. 4A). Some variations in terms of the intensity of the signals (either lacZ or histidinebased growth assays) were observed between the different members of the 14.3.3 family, the g subtype being the least effective interacting partner. However, the adipocyte and the skeletal muscle cDNA libraries were of different origins, and different activation domain-containing plasmids were used for the construction of the libraries. The muscle specific 14.3.3 g isoform was in pGAD10 type (Clontech) vectors whereas 14.3.3 u, b and z were in pGAL4±2.1 HybriZap background (Stratagene). In the absence of any quantitative analysis showing comparable levels of expression of the various 14.3.3 isoforms, further experiments are required to conclude which isoform shows the strongest interaction with UCP2. The same yeast two-hybrid approach was used to investigate the specificity of the interaction between the 14.3.3 proteins and the other members of the UCP family. UCP proteins (UCP1, UCP2 and UCP3) share between 55 and 79% aminoacid identity. However, while the UCP3 sequence presents 70% identity with UCP2 over the last 20 amino acids, UCP1 shows only 30% conservation in this part of the protein suggesting that the 14.3.3 interactions with the C-terminus of the UCPs could be isoform specific. Therefore, the ability of constructs containing the C loop of UCP3 (pLexA-hUCP3C, Fig. 4B) and of UCP1 (pLexA-hUCP1C, Fig. 4C) to interact with 14.3.3 members was tested. Co-expression in strain L40 of pLexAhUCP3C with a representative of each subtype of the 14.3.3 family fused to the Gal4 activation domain led to strong b-galactosidase signals and, in addition, supported growth of the strain in the absence of histidine (Fig. 4B). On the other hand, the pLexA-hUCP1C construct did not show any evidence for interaction with any of the 14.3.3 family members in the same yeast-based two-hybrid experiments (Fig. 4C). Similarly to what was previously observed with the pLexA-hUCP2C construct, the muscle-derived 14.3.3 g isoform showed a very weak interaction, whereas the adipocyte-derived isoforms u, b and z were about equally well interactive also with hUCP3C constructs.

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consensus sequence does not exist, amino acids with potential phospho-acceptor sites could be identified within the UCP2 (ser304) and UCP3 (ser310) C-terminal sequences. Mutagenesis experiments were therefore performed to knock out such potential phosphorylation sites. pLexA-hUCP2C (S304/A304) and pLexA-hUCP3C (S310/A310) were constructed and coexpressed in yeast in combination with the 14.3.3 isoforms. Neither mutation affected the levels of the b-galactosidase reporter gene or the histidine-dependent growth of the reporter strain (Fig. 4C) thus excluding phosphorylation of the C-terminus of UCP2 or 3 as necessary for the interaction. 14.3.3 and UCP interact physically in vitro To investigate a direct interaction between 14.3.3 proteins and UCP3, in vitro transcribed and translated radiolabeled proteins were used in coimmunoprecipitation experiments. The results presented in Fig. 5 illustrate that a 14.3.3 u antibody coimmunoprecipitated UCP3 and that addition of cold UCP3, as a competitor of the interaction, significantly decreased the level of radiolabelled UCP3 in the precipitate. This demonstrated that UCP3 and 14.3.3 proteins can, in fact, physically interact with each other. 14.3.3 expression in the yeast S. cerevisiae potentiates UCP3 effects on growth inhibition

Fig. 6. Effect of UCP3 and 14.3.3 coexpression on yeast growth. Yeast clones were cotransformed with either empty vectors (pGAD10 and pYeDP60) or 14.3.3 u and UCP3 expressing vectors. Cells (1200 cells) were spotted on agar plates containing either glucose (control) or galactose (to induce expression). After 48 h at 28 8C the plates were analyzed using an image analysis system (scion image 3B for windows). (A) Shows a photograph of the plate after 48 h induction. (B) Represents the mean values ^ SD obtained from nine independent induction experiments.

To better understand the structural requirements for the interaction of the C-terminus of UCP2 and 3 with the 14.3.3 proteins, a limited number of deletion and mutation experiments were performed. A deletion construct was prepared (pLexA-hUCP2C-DN) lacking the first 10 amino acids of the C loop. This N-terminal part of the C loop is highly conserved in all UCPs (including UCP1). Co-expression of pLexA-hUCP2CDN and AD-fused 14.3.3 isoforms in the yeast reporter strain L40 allowed the detection of a measurable, but very weak histidine-dependent growth signal (Fig. 4C). It seems therefore, that this conserved amino-acid sequence, that has been deleted, is necessary but not sufficient for the interaction with 14.3.3 proteins. It has been shown that 14.3.3 proteins bind to proteins containing a R(S)X1,2SPX(P) consensus motif, in which a phosphoserine residue with an arginine at position 2 3 or 2 4 plays a determinant role [24,25]. This motif has been found in a number of signal-transduction proteins such as c-Raf-1 or B-Raf [26], Bcr [27], cdc25 phosphatase [28], tyrosine and tryptophane hydroxylases [29,30]. Even though a perfect

Previous studies have shown that expression of UCP3 in yeast under the control of a galactose-dependent promoter induces a series of phenotypic changes, such as inhibition of growth, uncoupling of cellular respiration or depolarization of the mitochondrial membrane [11]. We found that coexpression of 14.3.3 in the heterologous yeast system enhances, under specific conditions, the activity of UCP3. Cotransformed yeast clones were seeded on agar plates containing either glucose (control) or galactose to induce the expression. As previously reported, expression of hUCP3 strongly inhibited the growth of yeast cells. This effect is normally considered to be a consequence of the uncoupling of mitochondria and thus leads to a limitation of high-energy substrates. The effect on growth was strongly potentiated when both UCP3 and 14.3.3 u were coexpressed (Fig. 6). Similar results were obtained with 14.3.3 b, g or u constructs. This effect was consistently stronger than the small inhibitory activity of control 14.3.3 protein expressed alone as control. Similar high levels of immunodetectable UCP3 were found to be associated with isolated mitochondria when UCP3 was induced alone or in the presence of 14.3.3 proteins (not shown). However, isolation of mitochondria is notably a lengthy procedure so that no information on the kinetics of transport to mitochondria could be obtained.

DISCUSSION The UCPs are considered to be putative regulators of mitochondrial uncoupling and thus, constitute a potential molecular mechanism controlling thermogenesis. It is conceivable that such a critical physiological function of the UCPs needs a tight control by interaction with ligands and, possibly, also with ancillary regulatory proteins. The aim of the present investigation was to identify interacting partners by using a yeast two-hybrid strategy. Constructs derived from hUCP2 were used to search a skeletal muscle and an adipocyte library. Various cDNAs coding for proteins with identities to several isoforms of the 14.3.3 family could be identified (b, u and z from the adipocyte library, g from the muscle library). This


observation strongly suggests that the interactions detected in the two-hybrid assays are not `sequence specific', but rather related to the 14.3.3 function. The g isoform represents the human homologue of the rat 14.3.3 g sequence, which at the time of the experiments had not yet been reported. Our clone is 100% identical to the recently described human 14.3.3 g isoform [19]. Interestingly, Horie et al. [19] have examined the expression profile of this human 14.3.3 g and showed that expression was restricted to skeletal muscle, brain and heart, confirming and extending our results concerning the exclusive finding of the g isoform in skeletal muscle. 14.3.3 proteins are cytoplasmic, acidic dimeric molecules that play a major role in signal transduction pathways [24,31]. They have been identified in many eukaryotic organisms, including plants and fungi. In vertebrate organisms 14.3.3 proteins regulate several aspects of cell physiology including promotion of the activation of tyrosine and tryptophane hydroxylases [30.32], regulation of protein kinases [33±41] or interaction with the cell cycle protein phosphatase Cdc25 [42] and the apoptosis promoting protein BAD with the release of Bcl-XL as a direct consequence [43]. It is interesting to note that 14.3.3 proteins are not found within the mitochondria (personal communication, G. Schatz, Biocenter of the University of Basel, Switzerland), so that it is very unlikely that they can interact and regulate UCP activity, once UCP has been incorporated into the inner mitochondrial membrane. However, interaction with signal-transduction proteins is not the only function proposed for the 14.3.3 isoforms. A cytosolic, ATP-dependent protein factor that stimulates the import of precursor proteins into isolated mitochondria has been shown to be a complex composed of 14.3.3 dimers [44]. This factor, named mitochondrial import stimulation factor (MSF), plays dual functions in mitochondrial import, namely it chaperones the import-incompetent precursor proteins and stimulates their incorporation into the organelles [44±47]. Actually, MSF was shown to facilitate the docking of precursor proteins on an import receptor localized on the mitochondrial outer membrane (i.e. the Tom70±Tom37 complex). Members of the MATP family, such as the ADP/ATP exchanger and the phosphate transporter, preferentially exploit this mechanism to enter the mitochondria. It is therefore tempting to speculate that chaperoning of UCPs by cytosolic 14.3.3 complexes represents a key step for their recognition by the receptor Tom70 and thus for mitochondrial import. We tested this hypothesis by coexpressing the two proteins in a heterologous yeast system. Previous studies have already documented, that UCP3 overexpression in yeast results in mitochondrial uncoupling and inhibition of growth under aerobic conditions [11,47]. In the present study, coexpression of members of the 14.3.3 family was found to potentiate the inhibitory effects of UCP3 on growth (Fig. 6) while, on the other hand, similar amounts of UCP3 were incorporated into yeast mitochondria at steady state. These findings are in line with the hypothesis that 14.3.3 coexpression facilitates the translocation of UCP3 to the mitochondria after induction. This would lead to an earlier onset of uncoupling activity and thus to a more rapid inhibition of growth, while it would have no effect on the activity of UCP3 once inserted into its final membrane location. Surprisingly, the C-terminal part of UCP1 did not interact with the 14.3.3 family members. It is possible that other cytosolic chaperones are involved in mitochondrial targeting of this particular isoform that is expressed exclusively in brown adipose tissue. On the other hand, it is still possible that 14.3.3 proteins can interact with other loops of UCP1. However, interaction with 14.3.3 proteins is not a necessity but only

q FEBS 2000

favors the mitochondrial translocation of MATP members. Recent publications have demonstrated that yeast ectopic expression of the short isoform of UCP3 (UCP3S) that lacks the C-terminal end, also results in uncoupled mitochondria as a consequence of its incorporation [49,50]. Very relevant is the recent finding that UCP3S is incorporated into mitochondria with much reduced kinetics than the long form of UCP3 [51]. 14.3.3 preferentially binds to serine-phosphorylated proteins [25,26,39], but the biochemical significance of this interaction is not clear. Recently, a consensus phosphorylation motif was proposed as a common binding motif for 14.3.3, but although phosphorylation has been shown to be important for the association of a number of proteins with 14.3.3, it is not clear yet whether this particular motif is phosphorylated or not. Our results suggest that potential phosphoacceptor sites present in the last 20 amino acids of UCP2 and UCP3 are not critical for the stringency of the interaction between UCP and 14.3.3.

ACKNOWLEDGEMENTS We greatly acknowledge the expert technical assistance by B. Besenreuther, M. Kamke, S. GruÈninger and I. Pilat. We would also like to thank A. Wanner for the automated DNA sequencing and F. Cumin for advice in the coimmuniprecipitation experiments. Strain GPY124 was constructed by G. Pohlig.

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