Localization of GAR transformylase in Escherichia coli and mammalian cells

Lata T. Gooljarsingh*†, Joseph Ramcharan*†, Simon Gilroy‡, and Stephen J. Benkovic*§

*Department of Chemistry, 414 Wartik Laboratory, and ‡Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802

Contributed by Stephen J. Benkovic, April 12, 2001 of the de novo biosynthetic pathway may form a enzymes. Moreover, it has been shown that all of these enzymes multienzyme complex to facilitate flux through the ten bind to the inner surface of the mitochondrial inner membrane, serial steps constituting the pathway. One likely strategy for whereas purified isozymes from other cellular compartments do complex formation is the use of a structural scaffold such as the not possess such binding ability (11). Organized com- cytoskeletal network or subcellular membrane of the cell to me- plexes may possess several kinetic advantages for the cell (12, diate protein–protein interactions. To ascertain whether this strat- 13). One possible advantage is in preventing intermediates from egy pertains to the de novo purine enzymes, the localization escaping into solution where sequestration may occur by other pattern of the third purine enzyme, glycinamide ribonucleotide enzymes for use in different metabolic pathways. A second is the transformylase (GAR Tfase) was monitored in live Escherichia coli proximity of consecutive enzymes of a metabolic pathway that and mammalian cells. Genes encoding human as well as E. coli GAR may serve to increase the metabolic flux through this pathway, Tfase fused with green fluorescent protein (GFP) were introduced especially by channeling the substrate at reduced bulk substrate into their respective cells with regulated expression of proteins concentration. and localization patterns monitored by using confocal fluorescence However, methodological problems are often encountered in microscopy. In both instances images showed proteins to be demonstrating the in vitro existence of such intermediate chan- diffused throughout the cytoplasm. Thus, GAR Tfase is not local- neling within enzyme–enzyme complexes as has been the case ized to an existing cellular architecture, so this device is probably for purine (14). First, many of these interactions not used to concentrate the members of the pathway. However, between enzymes are relatively weak, and isolation of their discrete clusters of the pathway may still exist throughout the complexes from cells is difficult because during the isolation cytoplasm. procedure (gel filtration, affinity chromatography) dilution ef- fects or changes in the ionic strength will tend to dissociate the he de novo purine biosynthetic pathway produces complexes. Different strategies have been used to obtain evi- Tthat are essential for many cellular processes. Purines serve dence for the proximity of active sites of consecutive enzymes as the building blocks for DNA and RNA synthesis, as an energy and to assess the possible kinetic advantages of such an arrange- source for chemical reactions (ATP), in cellular redox reactions ment in several metabolic systems. Datta et al. (15) showed (NADH, NADPH, FAD, etc.), and as signaling molecules in channeling of oxaloacetate (OAA) between mitochondrial various regulatory pathways (cAMP) (1). Because cancer cells malate dehydrogenase (mMDH) and citrate synthase (CS) in a require large amounts of purines to sustain their accelerated polyethylene glycol precipitate of the two enzymes by using growth, the de novo purine biosynthetic pathway has attracted aspartate aminotransferase (AAT) as a trap for the intermediate considerable attention as a target for cancer chemotherapy (2). OAA. Their results indicated that OAA is channeled between Purine biosynthesis is a ten-step enzymatic pathway that involves mMDH and CS, because large amounts of AAT are unable to the conversion of phosphoribosylpyrophosphate to inosine trap OAA in the precipitate of the CS and mMDH. Another monophosphate (IMP; Fig. 1). IMP contains an intact purine approach has been the use of genetic fusions of consecutive nucleus, which serves as the precursor for adenosine and enzymes to approximate the molecular crowding of intracellular guanosine monophosphate formation. The two folate-requiring conditions. For instance, recent experimental work of Lindbladh reactions, glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole ribonucleotide transformylase et al. (16) on connecting yeast mitochondrial CS and MDH via (AICAR Tfase), have attracted particular attention because a short linker sequence into a fusion protein provided channeling some of the most successful anticancer drugs to date have been of OAA between the two active sites. The glycolytic pathway is folate antimetabolites such as methotrexate (3). These two another metabolic pathway that has been intensively studied. enzymes carry out similar chemistry in catalyzing the transfer of Kinetic evidence failed to elucidate the existence of a metabo- BIOCHEMISTRY a formyl group from 10-formyltetrahydrofolate to the amino lon. However, Pagliaro and Taylor (17, 18) showed that the group of the substrates GAR and AICAR to form fGAR and aldolase enzyme localizes in vivo to the cytoskeletal matrix, fAICAR. Investigators have invested their efforts in understand- which may act as a structural scaffold to mediate protein–protein ing the mechanisms of the two transformylases with the express interactions between aldolase and other glycolytic enzymes. We purpose of rationally designing inhibitors that specifically target adopted a similar in vivo approach for studying the locus of the them, and in so doing disrupt purine biosynthesis (4–8). These GAR transformylase enzyme in Escherichia coli and mammalian and other purine enzymes may function in concert as a multien- cells. zyme complex, thus providing another target for chemothera- peutic intervention. Disruption of the complex with concomitant Abbreviations: GAR, glycinamide ribonucleotide; Tfase, transformylase; GFP, green fluo- inhibition of the growth of cancerous cells would introduce an rescent protein; fDDF, N10-formyl-5,8-dideazafolate; AIR, aminoimidazole ribonucleotide; alternative approach to antineoplastic treatment. AICAR, aminoimidazole ribonucleotide. In the last 20 years examples have been found of sequential †L.T.G. and J.R. contributed equally to this work. enzymes, which operate within a metabolic pathway, interacting §To whom reprint requests should be addressed. E-mail: [email protected]. with each other to form highly organized complexes (9, 10). The publication costs of this article were defrayed in part by page charge payment. This Among the mitochondrial enzymes of the Krebs tricarboxylic article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. acid cycle, interactions occur between six of the eight sequential §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.121182998 PNAS ͉ June 5, 2001 ͉ vol. 98 ͉ no. 12 ͉ 6565–6570 Downloaded by guest on September 24, 2021 Fig. 1. The de novo purine biosynthetic pathway. It is a ten-step pathway involving the conversion of phosphoribosylpyrophosphate to inosine monophos- phate. All substrates and intermediates in the pathway are labeled. The third step is catalyzed by the enzyme glycinamide ribonucleotide transformylase (GAR Tfase). This enzyme is monofunctional in E. coli, but exists as part of a trifunctional protein in mammalian cells. The three activities that are condensed are GAR Tfase, GAR synthase, and AIR synthase. The latter enzymes catalyze step 2 and step 5 of purine biosynthesis. Aminoimidazole ribonucleotide transformylase (AICAR Tfase) catalyzes step 9 and exists as a bifunctional, condensed with the tenth activity of the pathway inosine monophosphate (IMP) cyclohydrolase from E. coli to humans. AICAR Tfase uses the same 10-formyltetrahydrofolate as GAR Tfase.

There is circumstantial evidence for proposing the existence rial media were purchased from Fisher or Difco. Trypsin-EDTA, of a purine complex, such as the clustering of nonsequential FBS, 1ϫ penicillin-streptomycin-fungicide (PSF), and G418 activities onto single polypeptide chains (19), the unstable nature were purchased from GIBCO͞BRL. All protein gel electro- of some of the pathway intermediates (14, 20), and the copuri- phoresis agents and kaleidoscope molecular weight markers fication of the two transformylases (21). In mammals and birds were obtained from Bio-Rad. Polyclonal rabbit anti-GFP anti- the second, third, and fifth activities are all fused within the same bodies were from Molecular Probes, goat anti-rabbit IgG (H ϩ polypeptide chain, whereas in E. coli the activities all exist as L) conjugate with alkaline phosphatase was from Bio-Rad. BCIP discrete proteins. It is possible that the fourth activity nonco- (5-bromo-4-chloro-3-indoyl phosphate) and NBT (nitroblue tet- valently associates with the trifunctional protein. Moreover, the razolium) were from Sigma. All other biochemical reagents were Rosetta Stone Hypothesis (22), which is based on a computa- from either Sigma or Fisher. tional method, postulates that the second, third, and fourth activities in E. coli may associate noncovalently to reconstitute or Purification of GFP, E. coli GAR Tfase, and E. coli GAR Tfase-GFP. The mimic the human trifunctional protein. Substrate channeling plasmids, pLTG82 (E. coli GAR Tfase-GFP) and pMSW2 (E. may occur via multienzyme complexes to protect and rapidly coli GAR Tfase) (25), were transformed into CaCl2 competent process reactive such as the unstable phosphoribo- BL21(DE3) cells, grown in2lofLBat30°C (37°C for GAR sylamine of the first reaction (14), and the carbamate Tfase) until an OD600 of 0.6 was attained. Protein expression was generated when aminoimidazole ribonucleotide (AIR) is con- then induced with 100 ␮M IPTG for 3 h. The proteins were verted into carboxy-AIR (20). purified to 95% homogeneity as determined by SDS͞PAGE, It is highly possible that the purine enzymes therefore use a using the protocol for wild-type GAR Tfase (25). N-terminal structural scaffold or subcellular membrane to mediate protein– His-tagged GFP was expressed from the pRSET vector (gen- protein interactions, as was evidenced for the glycolytic enzyme B erous gift from Klaus Hahn, The Scripps Research Institute, La aldolase (18). To probe for this strategy, green fluorescent Jolla, CA) and purified by using Ni-NTA chromatography. protein (GFP) was used to label the human and E. coli GAR Tfase. The cellular localization patterns of the cells were mon- Construction of the GAR Tfase-GFP Gene Fusion. A cloning vector itored by using confocal fluorescence microscopy. GFP is an was constructed by replacing the multiple cloning site (MCS) of intrinsically fluorescent protein and conveniently does not re- pUC19 with a customized polylinker containing the restriction quire the addition of any exogenous substrates (23, 24). Materials and Methods Table 1. Comparison of unfused GFP and GAR Tfase to Materials. Restriction enzymes were purchased from either New GAR Tfase-GFP England Biolabs or Promega; T4 DNA , Calf Intestinal Alkaline Phosphatase, DNA polymerase I Klenow fragment, In vitro activity In vivo activity and Polynucleotide Kinase were from New England Biolabs. S.A. of GFP Complementation N10-formyl-5,8-dideazafolate (fDDF) was bought from the John Protein GAR Tfase (␭ex; ␭em) of MW12 cells Hynes Laboratory and GAR was synthesized as described (5). GAR Tfase 50 — ϩ G-75 resin was from Sigma, Q-Sepharose resin from Amersham ϩ Pharmacia, and nickel-nitrilotriacetic acid from Qiagen (Chats- GAR Tfase-GFP 46 489 nm; worth, CA). dNTPs were from Boehringer Mannheim. pET22(b) 508 nm was obtained from Novagen and pCI-Neo from CLONTECH. GFP — 489 nm; — 508 nm An Advantage GC DNA PCR Kit was purchased from CLON- TECH. IPTG (isopropyl ␤-D-thiogalactoside) was from Alexis SA, specific activity of E. coli GAR Tfase in ␮mol minϪ1⅐mgϪ1. ␭ex and ␭em, Biochemicals (San Diego, CA). All materials for making bacte- excitation and emission maxima, respectively.

6566 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.121182998 Gooljarsingh et al. Downloaded by guest on September 24, 2021 BamHI For: 5Ј-GCTGCCGACGAGGGATCCCCCCGTAAT- TAA-3Ј; KpnI Rev: 5Ј-GATAATAGAGCAGGTACCGCGT- TCGCGAA-3Ј. The 1300 bp fragment was ligated into the BamHI͞KpnI sites of pMAK705. The resultant construct was designated pMAK708. The GAR Tfase purN and upstream purM gene sequences were amplified by PCR from genomic JM105 DNA using an NdeI-XbaI forward primer and a XhoI reverse primer which removes the stop codon from the GAR Tfase gene. The primer sequences were as follows. NdeI-XbaI For: 5-GAT- Fig. 2. Image of E. coli GAR Tfase-GFP in BL21(DE3). Cells were grown in LB CAGCGGCATATGTCTAGATTGTCTGCA-3Ј; XhoI Rev: 5Ј- ␮ at 25°C, induced with 50 M IPTG, and imaged with an Olympus BX 60 AATTACGGGGCTCGAGCTCGTCGGCAGCGT-3Ј. The microscope equipped with a Micromax cooled charge-coupled device camera Ϸ1.38 kb fragment was digested with NdeI and XhoI and ligated driven by the METAMORPH software package. Images were captured by using a ͞ U-MWIB excitation cube (Olympus) with a band-pass excitation filter (460– into NdeI XhoI-digested pLTG19. The gene was then subcloned 490 nm) and a long pass barrier filter (Ն515 nm). into pMAK708 by digestion with XbaI and BamHI. The resultant vector, pMAK709, was used for the integration experiment.

sites: NdeI, XhoI, EcoRI, BamHI, SacI, and HindIII. To ensure Cloning the Human Trifunctional GAR Tfase-GFP into a pET-Based that NdeI was unique, an NdeI site 183 bp upstream of the native Vector. The fusion construct was generated by digesting pCC15, pUC19 MCS was removed before insertion of the polylinker. a pET-based plasmid that contains the trifunctional GAR Tfase The polylinker (made by annealing two separate strands: A, gene cloned downstream of the T7 promoter (a generous gift 5Ј-AATTGCATATGGGCCAGTAGCTCGAGGCAAGTA- from Carol Caperelli, University of Cincinnati), with NcoI and CTGGTTCGAATTCAGGGGATCCCAGGAGCTCGGC- XhoI and replacing the gene fragment with a PCR-amplified A-3Ј, and B, 3Ј-CGTATACCCGGTCATCGAGCTCCGTTC- NcoI͞XhoI fragment that removes the stop codon. The resultant ATGACCAAGCTTAAGTCCCCTAGGGTCCTCGAGCCG- mutant plasmid was labeled pCC16. The mutagenesis was car- TTCGA-5Ј) was ligated into EcoRI͞HindIII-digested pUC19. ried out via standard PCR and the primers used were as follows. The new plasmid was designated pLTG19. GFP’s internal NdeI NcoI Trif For: 5Ј-TTGCCGTTGGTGCCATGGAGCGAGAT- site was removed and the PCR-amplified gene was subcloned CAGA-3Ј; XhoI Trif Rev: 5Ј-GTGAGTCTCTATCTCGAGAA- into the EcoRI͞HindIII-digested pLTG19 to give pLTG20. The AAAATAGATG-3Ј. The GFP gene from pLTG82 was flanked E. coli purN gene was then amplified from pMSW2 by using a on either end by XhoI restriction sites that facilitated easy forward primer and a reverse mutagenic primer that converts the subcloning of the gene adjacent to or downstream of the 3Ј end stop codon to an XhoI restriction site. The purN was then cloned of the trifunctional GAR Tfase gene. The proper orientation was into the NdeI and XhoI site of pLTG20 to generate pLTG21. This screened for using HindIII. The final plasmid was designated procedure placed the GFP gene on the 3Ј end of purN. The entire pCC17. fusion construct was subcloned between the NdeI and HindIII sites of the pET22(b) expression vector to give pLTG82. Cloning Trifunctional GAR Tfase-GFP into a Mammalian Expression Vector. The fusion construct was cloned into pCI-Neo (CLON- Integration of GAR Tfase-GFP into E. coli Genome. The method of TECH). This vector carries the human cytomegalovirus (CMV) Hamilton et al. (26) was used, and as such entailed use of a immediate-early enhancer͞promoter region to promote consti- plasmid, pMAK705, possessing a temperature-sensitive origin of tutive expression of cloned DNA inserts in a variety of cell types. replication in conjunction with a bacterial host, JM105, that has The fusion construct was cloned into the pCI-Neo vector by functional recA. This plasmid carries the gene fusion construct, insertion of the GFP sequence first, followed by the trifunctional E. coli GAR Tfase-GFP–uracil phosphoribosyltransferase (upp). gene. The GFP was removed from pLTG82 by digestion with The upp gene is downstream of purN in the native bacterial XhoI, and then inserted into XhoI͞SalI-digested pCI-Neo. SalI genome, and as such drives a recombination event that results in and XhoI have compatible ends, therefore the GFP gene se- integration of the fusion construct into the genome of JM105. quence can be inserted in either orientation. Gene insertion in After the integration event the pMAK plasmid no longer the correct orientation will regenerate a XhoI restriction site at possesses the GFP gene and the plasmid is purged from the cell only the 5Ј end. The correct orientation was screened for using by growing in the absence of chloramphenicol, which is a a double NotI͞XhoI digest. The 3-kb trifunctional gene was then selectable marker for the plasmid. Integration is ascertained via amplified from pCC17 by using a forward mutagenic primer that PCR using primers flanking the GFP gene (data not shown). incorporates an NheI site at the 5Ј end of the gene and a reverse primer that includes the XhoI site at the 3Ј end of the gene. The BIOCHEMISTRY Cloning in the Integration Vector pMAK705. The Upp gene was CLONTECH Advantage PCR kit was used to amplify this large amplified from JM105 genomic DNA using a BamHI forward gene. The Ϸ3-kb pair PCR fragment was digested with NheI and and KpnI reverse primers. The primer sequences were as follows. XhoI and ligated into these sites in the pCI-Neo-GFP vector.

Table 2. Intracellular GAR-TFase and GAR Tfase-GFP concentrations in E. coli and fibroblast cells Media (ϩ purine rich) Rate of DDF formation, SA of wt GAR Tfase, Cell (Ϫ purine free) Enzyme assayed ␮mol⅐minϪ1 ␮mol minϪ1⅐mgϪ1 Enzyme, ␮M

JM105 ϩ E. coli GAR Tfase 2.1 ϫ 10Ϫ4 50 1.00 JM105 Ϫ E. coli GAR Tfase 6.3 ϫ 10Ϫ4 50 3.30 JM105-Integrate ϩ E. coli GAR Tfase-GFP 3 ϫ 10Ϫ4 46 0.72 JM105-Integrate Ϫ E. coli GAR Tfase-GFP 7.7 ϫ 10Ϫ4 46 2.00 293 T ϩ Human GAR Tfase 1.1 ϫ 10Ϫ4 2.6 0.18 293 T Stable TGP ϩ Human GAR Tfase-GFP 4.2 ϫ 10Ϫ4 2.6 0.67

All assays were performed in 1 ml. SA, specific activity of wild-type enzyme in ␮mol minϪ1⅐mgϪ1.

Gooljarsingh et al. PNAS ͉ June 5, 2001 ͉ vol. 98 ͉ no. 12 ͉ 6567 Downloaded by guest on September 24, 2021 The standard assay contained 50 mM Tris⅐HCl at pH 7.5, and 350 ␮M(␣, ␤)-GAR and 195 ␮M fDDF. The human trifunctional GAR Tfase was assayed in 0.1 M Hepes at pH 7.5, using 20 ␮M GAR and 50 ␮M fDDF. The reaction was initiated by addition of a small volume of enzyme to a 1-ml final volume aftera3min incubation at 25°C. Specific activity is expressed as ␮mol of product͞min͞mg of enzyme. The concentration of the purified protein was determined by a BCA assay method according to the protocol provided by the manufacturer (Pierce).

Spectral Analysis of GFP. The excitation and emission spectrum of GFP and GAR Tfase-GFP were measured by using a Fluoro- Max-2 fluorimeter (Instruments SA, Edison, NJ).

Western Analysis. Western analysis of the integration strains and the stable transfectants were conducted by using rabbit poly- clonal anti-GFP antibodies (Molecular Probes). The nitrocellu- lose membranes were blocked overnight in Tris-buffered-saline (TBS), 3% BSA. Membranes were incubated for1hatroom temperature, using TBS and primary rabbit anti-GFP antibodies, and washed 3 times for 5 min, using TBST (TBS supplemented with Tween-20). The membrane was then incubated with goat anti-rabbit alkaline phosphatase-linked secondary antibody in TBS-BSA, washed again as before, and then developed with NBT (nitroblue tetrazolium) and BCIP (5-bromo-4-chloro-3- Fig. 3. Image of GAR Tfase-GFP Integration Strains. (A) GFP fluorescence indoyl phosphate). image. (B) Phase contrast image of the JM105 integrate strains. Functional Complementation. GAR Tfase auxotrophic MW12 cells [ara ⌬ (gpt-rpo-lac) thi rbs-221 ilvB2102 ilvH1202 purNЈ-lacZϩ DNA sequencing revealed that there were no mutations of the Yϩ::KanR purT] were used. Electrocompetent cells were trans- trifunctional gene when using the modified PCR procedure. The formed with the appropriate plasmid and the cell strains were resultant plasmid was designated pTGFP. plated on purine-deficient minimal media to confirm enzyme activity in vivo. Transient Transfection of pTGFP into Cos-7 Cells. pTGFP and pGFP were transiently transfected into Cos-7 cells by using the CLON- Microscopy. For the bacterial studies, the microscope system used TECH CLONfectin transfection kit. Protein expression was was an Olympus BX 60 (New Hyde Park, NY) equipped with a determined 48 h after transfections and cells were subsequently MicroMax (Princeton Instruments, Trenton, NJ) cooled charge- imaged. coupled device camera driven by the METAMORPH software package (Version 4.0 from Universal Imaging, Media, PA). The Stable Transfection. 293 T cells were transfected with pTGFP and GFP and phase-contrast images were captured by using a pGFP to generate transformants that stably express the proteins U-MWIB excitation cube unit (Olympus) with a band-pass of interest. The CLONTECH CalPhos mammalian transfection excitation filter (460–490 nm) and a long pass barrier filter kit was used to generate the transfectants. (Ն515 nm). Cos-7 and 293 T fibroblast cells were grown on coverslips in DMEM, 7.7% FBS, and 1ϫ penicillin-streptomy- Quantitation of Enzyme Concentrations in E. coli and Fibroblast Cells. cin-fungicide (PSF), washed twice with PBS, and placed on the Intracellular enzyme concentrations were determined by activity measurements from crude lysates in comparison with the specific activity of purified wild-type protein. The following equation was used to calculate the in vivo concentrations of the protein of interest, P:

͑Rate of DDF formation of crude lysate͒ ϫ ͑103 mg͞g͒ ϫ ͑DF͒ ͓ ͔ ϭ P ͑ ͒ ϫ ͑ ͒ SA of wt Protein MWp ϫ ͑cell volume͒ ϫ ͑number of cells͒

where SA is the specific activity of the protein and DF is the dilution factor, a ratio of the total volume of lysate to the volume used in a 1-ml assay. DF is 6 for JM105 cells, and 2.5 for 293 T fibroblast cells. MWP is the molecular weight of the protein of interest. The volume for E. coli and fibroblast cells are 10Ϫ15 Fig. 4. (A) Western analysis of JM105 integrates. (B) Western analysis of the liters and 4 ϫ 10Ϫ12 liters, respectively. 293 T stably transfected cells. Westerns were performed by using primary rabbit polyclonal anti-GFP antibodies (Molecular Probes) and secondary goat anti-rabbit alkaline phosphatase linked secondary antibody, and developed Enzyme Assay. The purN GAR Tfase and the GAR Tfase-GFP with nitroblue tetrazolium (NBT) and BCIP (5-bromo-4-chloro-3-indoyl phos- proteins were assayed at 25°C with the alternate substrate fDDF phate). Lane 1 and lane 2 are GAR Tfase-GFP from JM105 integrates and lane by monitoring the production of 5,8-dideazafolate (DDF) at 295 3 is a control using purified S65T GFP. (B) Western analysis from 293 T stably Ϫ Ϫ nm (␧ ϭ 18.9 mM 1⅐cm 1) on a Gilford 252 spectrophotometer. transfected cells. M, kaleidoscope molecular weight markers.

6568 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.121182998 Gooljarsingh et al. Downloaded by guest on September 24, 2021 were grown in LB at 25°C and induced with 50 ␮M IPTG. Under these conditions, it was found that the protein was diffuse within the cytoplasm (Fig. 2). In this experiment, very high concentra- tions of protein were being produced within the cell because of the ‘‘all or nothing’’ inducibility of the T7 promoter and hence may have lead to distribution artifacts. To confirm the validity of this result, another experiment was done in which the fusion construct was integrated under control of the native GAR Tfase bacterial genome by replacing the native GAR Tfase gene with the GAR Tfase-GFP chimera. Because the gene is under the control of the native promoter, physiological concentrations of the GAR Tfase are produced inside the cell (Table 2). The cells were grown in minimal purine-deficient media and harvested at ϫ an OD600 nm of 1, washed with 1 PBS, and imaged. The GFP signal was visible above background autofluorescence, and was essentially diffuse throughout the cytoplasm (Fig. 3). This result does not negate the existence of a complex as units of the Fig. 5. Image of Cos-7 cells transiently transfected with pTGFP. Cells were complex may still exist, diffused throughout the cytoplasm, but imaged 48 h after transfection. The cells were imaged by using an axiovert it eliminates the formation of the complex mediated by cytoskel- microscope attached to a LSM 410 confocal microscope (Zeiss), using 488 nm etal or membrane elements in the cell. Western analysis con- (argon laser), 488 dichroic, and 515–560 nm emission filtration. Under these firmed that the intact, full-length fusion protein was produced imaging conditions, the low level of punctate autofluorescence represented within the bacterial and mammalian cells (Fig. 4 A and B). Ͻ 10% of the GFP signal. The nucleus and cytoplasm are labeled N and C, The trifunctional GAR Tfase-GFP protein could not be respectively. purified after its expression in E. coli cells because of the formation of insoluble inclusion bodies. However, green fluo- stage of the microscope for imaging. For the mammalian system, rescence and GAR Tfase activity were detected in crude lysates the cells were imaged by using an axiovert microscope attached of BL21(DE3)pLysS pCC17 cells, suggesting that a properly to an LSM 410 confocal microscope (Zeiss), using 488 nm (argon folded protein could be obtained by directly expressing the laser), 488 dichroic, and 515–560 nm emission filtration. Under fusion in mammalian cells. Cos-7 fibroblast cells were transiently these imaging conditions the low level of punctate autofluores- transfected with the pTGFP vector encoding the trifunctional cence represented Ͻ10% of the GFP signal. GAR Tfase-GFP fusion. Again the signal was diffused through- out the cytoplasm and was excluded from the nucleus (Fig. 5). Results This expression, like the expression of the pLTG82 in E. coli To assess whether the GFP fusion altered the enzyme’s ability to cells, may contain distribution artifacts owing to the production fluoresce, the E. coli GAR Tfase fusion protein was purified and of large quantities of protein within the cell. 293 T fibroblast cells its GAR Tfase activity and GFP spectral properties were com- were therefore stably transfected with the pTGFP construct. In pared with the individual unfused proteins. Table 1 lists the stable transfections, the levels of GFP fusion protein produced comparison of the two proteins. As is evident, the two proteins within the cells are substantially reduced and are similar to were essentially unchanged after the fusion. The fusion protein wild-type levels (Table 2). Once again the signal was found to be had similar in vitro activity to the wild-type GAR Tfase and was diffused throughout the cytoplasm (Fig. 6). active in vivo as is evident from the positive functional comple- mentation test. The spectral characteristics of GFP in the fusion Discussion protein were unchanged. It was then critical to determine The uniform cytoplasmic distribution of the GAR Tfase-GFP whether the fusion protein could be visualized within a live fusion proteins appears to argue against the presence of a bacterial cell via confocal fluorescence microscopy. The cells complex localized by any structural or subcellular architecture. BIOCHEMISTRY

Fig. 6. Stable trifunctional GAR Tfase-GFP transfectants of 293 T cells. (A) 293 T cells expressing the trifunctional GAR Tfase-GFP. (B) 293 T fibroblasts expressing GFP. Cells were grown on coverslips, washed twice with 1ϫ PBS, and imaged. The nucleus and cytoplasm are labeled N and C, respectively.

Gooljarsingh et al. PNAS ͉ June 5, 2001 ͉ vol. 98 ͉ no. 12 ͉ 6569 Downloaded by guest on September 24, 2021 Measurement of the in vitro and in vivo activity of the Tfases rule pels one to believe that perhaps some other type of structural out the possibility of inactivation by the fusion. Western analysis scaffold is used. A likely candidate is the fourth purine enzyme, of the fusion proteins by using anti-GFP antibodies also argues formylglycinamidine ribotide (FGAM). This enzyme is quite against the formation of truncation mutants that maintain large—about 130 KDa, much larger than even the human catalytic activity. Moreover, the integration of the fusion protein trifunctional GAR Tfase (106 KDa)—yet it contains only one into the bacterial genome, the equivalent concentrations within known enzymatic activity. It is possible that this enzyme serves the cell of the GAR Tfase-GFP fusion proteins (Table 2), and a dual function in purine biosynthesis and acts as a scaffold their counterpart wild-type enzymes ensure that the observa- tions are not artifactual. Therefore, the purine enzymes do not protein mediating the interactions between the intervening appear to use a subcellular membrane or cytoskeletal network to second GAR Synthase, third GAR Tfase, and fifth AIR Syn- mediate protein–protein interactions, as in the case of the thase enzymes. Alternately, one must entertain the possibility glycolytic enzymes or the mitochondrially bound enzymes of the that a multienzyme complex of the purine biosynthetic enzymes Kreb’s cycle. does not form intracellularly. Of course, one must also remain aware of the possibility that the placement of the GFP within the fusion protein disrupts its We thank Jennifer Kemp for imaging the bacterial cell strains, Dr. Mark interactions with the matrix. Furthermore, units of the complex Warren for his help in constructing the original GFP cloning vector, Dr. may still be present but dispersed throughout the cytoplasm. D. M. Wojchowski for a tutorial in mammalian cell culturing, and Dr. The discovery that the mechanism of complex formation James D. Stevenson for proofreading the manuscript. This work was differs from that established for other metabolic systems com- supported by National Institutes of Health Grant GM 24129

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