quantitative imaging of tata-binding protein in living yeast cells

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Quantitative Imaging of TATA-Binding Protein inLiving Yeast Cells

GEORGE H. PATTERSON, STEPHANIE C. SCHROEDER, YU BAI, P. ANTHONY WEIL ANDDAVID W. PISTON*

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, U.S.A.

Received 23 October 1997; accepted 16 January 1998

We describe the quantitative monitoring of TATA-binding protein (TBP) localization and expression in livingSaccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) · TBPfusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM). When GFP · TBPexpression was altered by using various promoters, the levels measured by LSCM correlated well with the levelsdetermined by immunoblot of whole cell extract protein. These results show that GFP · TBP imaging not only offersa method of measurement equivalent to a more conventional technique but also provides real-time quantitation inliving cells and subcellular localization information. Time-lapse confocal imaging of GFP · TBP in mitotic yeast cellsrevealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0·7) between mother anddaughter cells. Based on this and data from a mutant which underexpresses GFP · TBP, we suggest that intracellularlevels of TBP are near rate-limiting for growth and viability. ? 1998 John Wiley & Sons, Ltd.

Yeast 14: 813–825, 1998.

— Green Fluorescent Protein; GFP; confocal microscopy; mitosis

*Correspondence to: D. W. Piston, Department of MolecularPhysiology and Biophysics, 702 Light Hall, Vanderbilt Univer-sity, Nashville, TN 37232, U.S.A. Tel.: (+1) 615 322 7030; fax:

INTRODUCTION

Transcription in eukaryotic cells is catalysed bythree distinct DNA-dependent RNA polymerases(RNAPI, RNAPII and RNAPIII), each of whichtranscribes one class of genes (class I, II or III;Hernandez, 1993; Zawel and Reinberg, 1995).Eukaryotic RNA polymerases cannot specificallyinitiate transcription, and each utilizes a distinctset of general transcription factors (GTFs) thatfacilitate accurate initiation and elongation oftranscription. The TATA-binding protein (TBP),however, is required for transcription by all threeRNA polymerases since TBP is a subunit of SL1,TFIID and TFIIIB (Hernandez, 1993). Conse-quently, TBP is the most central transcriptionfactor in a eukaryotic cell (Hernandez, 1993).Numerous biochemical and genetic studies haveshown the importance of TBP binding to DNA

(+1) 615 322 7236.

CCC 0749–503X/98/090813–13 $17.50? 1998 John Wiley & Sons, Ltd.

and to other proteins, but changes in TBP concen-tration and its subcellular localization in vivo havebeen more difficult to assay.

Changes in expression level and/or subcellulardistribution of TBP within a living yeast cell couldhave significant consequences upon cell physi-ology, presumably by up- or down-regulatingtranscription globally or by modulating expressionof subsets of yeast genes. The consequences ofaltered TBP levels have previously been examinedby transient transfection of TBP-encoding genes inmetazoan cells where it was found that TBPappeared limiting for transcription. For example,overexpression of TBP in Drosophila Schneidercells resulted in increased activity from all sub-classes of RNAPIII promoters (Trivedi et al.,1996) as well as from RNAPII TATA-containingpromoters but not from TATA-less promoters(Colgan and Manley, 1992). Likewise, overexpres-sion of TBP in HeLa and CHO cells was found topotentiate the response of some transactivators butnot others (Sadovsky et al., 1995). Conversely,
overexpression of a TBP-sequestering factor

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known as Dr1 caused a decrease in both RNAPII-and RNAPIII-mediated transcription (Kim et al.,1997). Whether TBP and/or other GTFs are rate-limiting in yeast is less clear. In general, over-expression of TBP in normal cells does notsignificantly affect cell growth rates (Poon et al.,1991), although under certain conditions, over-expression of TBP can increase yeast growth rates(Auble et al., 1994; Bai et al., 1997; Kim et al.,1997). In none of these cases, however, was theeffect of TBP overexpression examined for a largenumber of specific mRNA-encoding genes. More-over, the subcellular localization of overexpressedTBP was not determined. Thus, it is possible thatthe reason TBP overexpression showed such asmall effect on overall cell physiology is the failureof the overexpressed TBP to localize to thenucleus.

Cell fractionation studies performed in mam-malian cells indicate that TBP is normally localizedto the nucleus and is presumably bound to chro-matin during interphase, though Segil et al. (1996)showed that a significant fraction of TBP is cyto-plasmic during mitosis. However even duringmitosis, 210–20% of the TFIID remains associ-ated with the DNA. Similarly, the RNAPI-specificSL1 TBP–TAFI complex localizes with the rRNAgenes throughout the cell cycle (Jordan et al., 1996;Roussel et al., 1996). Presumably, during yeast celldivision, the mother cell transcription machinery isdistributed equally between daughter cells duringthe cell division, although this idea has never beenexplicitly tested. Likewise, both cytoplasmic andchromosome-associated proteins are likely to par-tition equally during telophase and be equallyshared between the dividing cells.

Unlike mammalian cells, Saccharomycescerevisiae exhibits a closed mitosis. Moreover, thedaughter cell is substantially smaller than themother cell, so that the two cells may containunequal allotments of the mother cell’s machinery.Further, the yeast cell nuclear membrane remainsintact during mitosis (Robinow and Johnson,1987; Wheals, 1987) and may therefore presenta barrier to diffusion and/or redistribution ofnuclear proteins throughout the mitotic cycle.Despite the fact that the daughter cell is smallerthan the mother cell, one could reasonably assumethat a key transcription factor such as TBP isshared equally between the two cells, particularly ifTBP remains bound to the equally distributedDNA. However, this aspect of TBP biology hasheretofore not been examined.

? 1998 John Wiley & Sons, Ltd.

In this study, we used the green fluorescentprotein (GFP) to both quantitatively monitorintracellular TBP levels and investigate its sub-cellular localization and dynamics in living S.cerevisiae cells. Originally isolated and cloned fromthe jellyfish Aequorea victoria (Chalfie et al., 1994),GFP has been used to study protein localizationand gene expression in many heterologous sys-tems. Using the S65T derivative of GFP (Heimet al., 1995) fused to TBP, we have replaced theendogenous TBP-encoding gene with a gene whichencodes a GFP · TBP fusion protein. Althoughthis fusion protein doubles the effective mass ofTBP, it is fully functional and cells containing the‘green’ TBP grow at the same rate as the wild-typeparental yeast cell line. We have validated thequantitative imaging experiments by showing thatmicroscopy results with four different GFP · TBPexpression levels closely correlate with results fromprotein immunoblot analysis. Finally, we havemonitored GFP · TBP localization throughout thecell cycle and have found an asymmetric distribu-tion of this transcription factor between motherand daughter cells. Although the significance ofthis particular finding is currently unknown, wediscuss possible explanations and roles that thisasymmetry may have in yeast cell physiology.

MATERIALS AND METHODS

Plasmids and yeast strainsAll plasmids were propagated in Escherichia coli

XL1Blue in LB containing 100 ìg/ml ampicillin.The S65T derivative of GFP was made with theKunkel method of site-directed mutagenesis(Sambrook et al., 1989) using the TU#65 plasmid(Chalfie et al., 1994) as a template and the muta-genic oligonucleotide, 5*-TGAACACCATAAGTGAAAGTAGTGACA-3* as the annealing primerto give TU#65-S65T. Using the TU#65-S65T as atemplate, PCR amplification products of S65Twere generated using the N-terminal oligonucleo-tide primer 5*-GCGCGTCGACATGAGTAAAGGAGAAGAACT-3* containing a SalI restrictionsite (underlined) and the C-terminal primer 5*-GCGCCTCGAGTTTGTATAGTTCATCCATGC-3*containing an XhoI restriction site (underlined).Since this GFP variant was used for most of thework in this paper, we refer to the S65T variantsimply as GFP hereafter in this report. In the oneexperiment where the wild-type GFP is used, itis denoted as such. The amplification products

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containing GFP encoding sequences were gelpurified and digested with the restriction endo-nucleases, SalI and XhoI, and ligated into a SalI-digested pTFIID plasmid, a yeast expressionplasmid which uses the promoter and regulatorysequences of the TBP-encoding gene to drive lowto moderate levels of inserted ORFs (Poon et al.,1991). SalI cloning of the GFP into pTFIIDgenerated pTFIID-GFP, which expresses lowlevels of GFP. The GFP was also ligated in-frameto the SalI site at the N-terminus of the TBP ORFin pTFIID-WT (Poon et al., 1991) to generatepTFIID-GFP · TBP, which expresses low levels ofthe GFP · TBP fusion protein. Proper insertion ofthe GFP ORF in all expression plasmid con-structs was determined by restriction endonucleasedigestion and DNA sequence analysis.

Three other expression vectors, pPGK, pGALand pTvnPED, were also used to express GFP,TBP and GFP · TBP at various levels. Wepreviously used pPGK-WT and pGAL-WT tooverexpress TBP (Poon et al., 1991). Expressionplasmid pTvnPED expresses the inserted ORFs atsub-wild-type levels, relative to pTFIID becausethe TBP-encoding gene regulatory sequenceswhich drive expression in this plasmid carry atransversion in one of the TBP-encoding genepositive cis elements present in the pTFIID(Schroeder et al., 1994). To generate the variousoverexpression (i.e. pPGK and pGAL) and under-expression (i.e. pTvnPED) constructs, the GFPand GFP · TBP ORFs were removed frompTFIID-GFP and pTFIID-GFP · TBP, respect-ively, by digestion with the restriction endo-nucleases, SalI and BamHI, gel purified, andligated into similarly digested pPGK, pGAL andpTvnPED plasmids.

GFP plasmids were transformed into theS. cerevisiae strain, YTW22 (Poon et al., 1991),originally derived from the parental strain,YPH252 (MATa ura3-52 trp1-Ä1 his3-Ä200leu2-Ä1 lys2-801amber ade2-101ochre). YTW22 hasa deletion of the chromosomal copy of theTBP-encoding gene and a covering plasmid,pURA-TFIID. The pURA-TFIID plasmid wasreplaced with the pTvnPED, pTFIID, pPGK andpGAL expression vectors using the plasmid shufflemethod. For this experiment, growth of the trans-formed YTW22 cells on 5-fluoroorotic acidselected for the loss of the pURA-TFIID plasmid,leaving only the transformed copy of TBP orGFP · TBP (Boeke et al., 1987). Strains carryingthe TBP expression plasmids pTvnPED-WT,


pTFIID-WT, pPGK-WT and pGAL-WT arereferred to as YTvnPED-TBP, YTFIID-TBP,YPGK-TBP and YGAL-TBP, respectively. Strainscarrying the GFP · TBP expression plasmidspTvnPED-GFP · TBP, pTFIID-GFP · TBP,pPGK-GFP · TBP and pGAL-GFP · TBP arereferred to here as YTvnPED-GFP · TBP,YTFIID-GFP · TBP, YPGK-GFP · TBP andYGAL-GFP · TBP, respectively. YTW22 wasgrown in rich medium and the above-describedstrains were grown in synthetic complete (SC)medium, supplemented with the necessary nutri-ents for selection and the requisite primary carbonsource, depending on the strain. Growth curves foreach strain were generated by monitoring bothculture optical density at 600 nm and cell numberby direct microscopic cell counting while growingin SC media at 30)C.

Expression and purification of protein standardsHis6-tagged GFP protein was expressed in E.

coli and purified as described in Patterson et al.(1997). Recombinant TBP was prepared, asdescribed in Poon et al. (1993) from isopropylthio-â--galactopyranoside (IPTG)-induced E. coliBL21pLysS cells containing a pET11D vectorexpressing TBP. To further purify the TBP, thepeak protein fractions from the heparin-sepharosecolumn were pooled and precipitated by dialysis inSpectro/Por Molecular porous membrane tubing(MWCO 12–14,000) against 200 m-Tris pH 7·9,3·78 -(NH4)2SO4, 1 m-EDTA, and 1 m-â-mercaptoethanol (BME) for 22 h at 4)C. Theprecipitated TBP was pelleted by centrifuga-tion and resuspended in BB500 (20 m-HEPES,10% glycerol, 0·2 m-EDTA, 5 m-MgCl2,10 m-BME, 1 m-benzamidine, 0·1 m-phenylmethylsulfonyl fluoride and 500 m-NaCl)and then applied to a sephacryl S-300 gel fil-tration column (2#100 cm) pre-equilibrated withBB500. The protein concentration of the fractionswas determined by the absorbance at 280 nm using15,000 "1 cm"1 as the extinction coefficientfor TBP. The TBP concentration was verifiedwith known amounts of bovine serum albumin.Purity of the resulting TBP was determined to be>95% by densitometry of a Coomassie brilliantblue-stained 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel (PAGE). GST-TBP wasaffinity purified using glutathione-agarose resin(Sigma, St Louis, MO) from IPTG-inducedE. coli BL21pLysS containing a pGEX

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plasmid (Stratagene, La Jolla, CA) with TBP-encoding cDNA subcloned in-frame with the GST.The cells were lysed in LB200 (20 m-Tris pH 7·9,200 m-NaCl) by sonication and the insolubledebris was removed by centrifugation. The super-natant was incubated with resin prepared accord-ing to the manufacturer’s specifications for 2 h at4)C on a tiltboard. The resin was washed withLB200 and eluted with 20 m reduced glutathionein LB200. Purification efficiency was determined asdescribed above and concentration was determinedby bicinchoninic acid assay (Pierce, Rockford, IL).

Protein blot analysisProtein blots were performed as described by

Bai et al. (1997) with the exception that the trans-fer buffer was 48 m-Tris, 39 m-glycine, and 20%methanol. After protein transfer, the membranewas probed with affinity-purified anti-TBP poly-clonal rabbit IgG (Poon et al., 1991), affinity-purified anti-TAFII30 polyclonal rabbit IgG, oranti-GFP monoclonal antibody (Clontech Labora-tories, Inc., Palo Alto, CA) in TBST (100 m-Tris–Cl pH 7·5, 150 m-NaCl, and 0·1% (v/v)Tween 20) at room temperature for 2 h. Themembrane was incubated at room temperaturefor 1 h with a 1:30,000 dilution of horseradishperoxidase (HRP)-conjugated anti-rabbit IgG forthe TBP and TAFII30 antibodies or a 1:3000dilution of HRP-conjugated anti-mouse IgG inTBST for the GFP antibody and detected byenhanced chemiluminescence (ECL) and a Bio-Rad (Hercules, CA) ECL Phosphorimager screen.The relative amount of TBP and GFP · TBP foreach strain was determined using the mean pixelvalues of the appropriate bands. RecombinantTBP and GST-TBP were used as standards inorder to compare the protein blot data with theimaging data.

Microscopy and analysisLaser scanning confocal microscopy (LSCM)

was performed using a Zeiss LSM410 microscopewith a 40# Plan Neofluar 1·3 NA oil immersionobjective (Carl Zeiss, Thornwood, NY). The488 nm line of an argon-krypton ion laser was usedfor excitation. A Q498LP (Chroma TechnologyCorp., Brattleboro, VT) dichroic mirror and aHQ512/27 emission filter designed specificallyfor GFP was used in the imaging experiments(Niswender et al., 1995).


Yeast cells were imaged in Mat-Tek culturedishes (Mat-Tek Corp., Ashland, MA) in SCmedia or on slides coated with 1% agarose contain-ing SC media. When imaging in liquid media, thecells were allowed to settle to the coverslip cover-ing the bottom of the dish before imaging. Steady-state measurements were made on a single opticalsection through the middle of the cells. For theobjective lens and confocal pinhole used here, theoptical sections were 1 ìm thick (Sandison et al.,1995). Time-lapse image sets were acquired inthree dimensions (10 focal planes spaced 1 ìmapart) every 10 min for 12 h.

Two channel (fluorescence and Nomarski DIC)images were analysed using Adobe Photoshop 3.0(Adobe Systems Incorporated, Mountain View,CA), NIH Image 1.61 (National Institutes ofHealth, Bethesda, MD), and Alice 2.4 (HaydenImage Processing Group, Boulder, CO). Meanpixel values from background regions were sub-tracted from regions of interest for normalization.Intracellular GFP concentrations were determinedusing mean pixel values from images of knownconcentrations of purified His6-GFP (S65T) indeep well slides as standards.

RESULTS

GFP · TBP replaces TBP with no effect on cellviability and growth

GFP · TBP experiments were performed in yeaststrains where a plasmid carrying the gene encodingnormal TBP was replaced, by plasmid shuffle,with a plasmid-borne gene encoding a GFP · TBPfusion protein. Although GFP has been success-fully fused to a number of proteins without affect-ing their function (Cubitt et al., 1995; Gerdes andKaether, 1996), we were concerned that taggingsuch a central component of the transcriptionmachinery with such a large probe might interferewith its ability to function properly. We assessedthe ability of GFP · TBP to support yeast cellviability by performing growth curves (Figure 1A),which showed that the YTFIID-GFP · TBP yeaststrain grows at the same rate (doubling time of22·5 h) as the YTFIID-TBP strain. This similarityin the growth rates was also found for two ofthe other yeast strains that we generated (YPGK-TBP and YPGK-GFP · TBP; YGAL-TBP andYGAL-GFP · TBP; data not shown). As discussedfurther below, the two TBP-encoding genepromoter mutant strains (YTvnPED-TBP and

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Figure 1. GFP · TBP replaces TBP with no detriment to yeastcell growth. The plasmid shuffle method was used to replace theTBP-encoding gene with the GFP · TBP-encoding gene. (A)Growth curve comparisons were performed with the yeaststrains YTFIID-TBP and YTFIID-GFP · TBP, which expressTBP and GFP · TBP respectively from the TBP-encoding genepromoter. (B) A protein immunoblot for TBP was performedon WCE from each of the strains. The strain from which theprotein was derived is listed above each lane. The mobilityof molecular weight standards run in an adjacent lane areindicated to the left of the gel.

YTvnPED-GFP · TBP) grew 230% and 280%more slowly than the YTFIID strains, exhibit-ing doubling times of 23·25 h and 24·5 h,respectively (data not shown).

To examine the relative extents of TBPproduction/accumulation in these yeast strainsand, more importantly, prove that the GFP moietywas not simply proteolysed from the GFP · TBPfusion protein, thereby generating ‘TBP’ from the


expressed GFP · TBP, we performed proteinimmunoblot experiments (Figure 1B). Yeast wholecell extracts (WCE) were prepared from equivalentcell numbers of the YTFIID-TBP and YTFIID-GFP · TBP strains, which yielded roughly equalamounts of protein. Equivalent protein amountsof these extracts were fractionated by SDS–PAGE,blotted to a filter, and TBP was detected withanti-TBP IgG. The yeast strains, YTFIID-TBPand YTFIID-GFP · TBP, expressing TBP (lane 1)and GFP · TBP (lane 2), respectively, expressroughly equivalent amounts of the expected sizeproteins (TBP 227 kDa and GFP · TBP254 kDa. The slight difference in signal intensitiesbetween these two forms of TBP is attributable tothe decreased electrotransfer efficiency of the largerGFP · TBP. Importantly, no 27 kDa TBP ispresent in the YTFIID-GFP · TBP extract, indi-cating that the intact fusion protein alone isproviding TBP functionality in these cells. Thedata in Figure 1 indicate that the 27 kDa GFPprotein fused to the N-terminus of TBP doesnot hamper yeast vegetative cell growth andthat GFP · TBP is an adequate replacement forwild-type TBP.

Use of different promoters allows controlledvariation of TBP and GFP · TBP expression levels

We cloned TBP, GFP and GFP · TBP into afamily of expression plasmids which should yield250-fold variation of protein expression levels(Poon et al., 1991; Schroeder et al., 1994). Toassure that we achieved this variation of expressionlevels, protein immunoblot experiments wereperformed. Shown in Figure 2 are protein levelsfor the YTFIID-TBP and YTFIID-GFP · TBPstrains (lanes 1 and 2) in comparison with levelsfrom the YTvnPED yeast strains that underex-press TBP and GFP · TBP (lanes 3 and 4) and theYPGK (lanes 5 and 6) and YGAL (lanes 7 and 8)strains that both overexpress TBP and GFP · TBP.Quantitation of five replicates of this experimentshows that in relation to pTFIID, the pTvnPED,pPGK and pGAL expression plasmids drive 0·6-fold, 19-fold and 28-fold amounts of TBP proteinproduction and 0·5-fold, 13-fold and 14-foldamounts of GFP · TBP. Probing for TAFII30showed that an equal amount of whole cell proteinwas loaded into lanes 1–4 and that lanes 5–8received 1/10 of lanes 1–4, confirming that theobserved protein levels were not a loading artifact(data not shown). The increased degradation of

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Figure 2. TBP and GFP · TBP protein levels can be systematicallyvaried in the yeast cell. The plasmid shuffle method was used toreplace the endogenous TBP-encoding gene with TBP- andGFP · TBP-encoding genes expressed from four different pro-moters, the TBP promoter (lanes 1 and 2), TvnPED, a promotermutant (lanes 3 and 4), the PGK promoter and regulatory sequence(lanes 5 and 6), and the GAL10 promoter and regulatory sequence(lanes 7 and 8). Quantitative protein immunoblots were performedon WCE derived from yeast strains expressing TBP and GFP · TBPat these four different expression levels. The strain from which theWCE was derived is listed above each lane. The proteins in lanes5–8 were diluted 1/10 before gel electrophoresis. TAFII30 immuno-blots were performed and quantitated as internal controls (data notshown). The mobility of molecular weight standards run in anadjacent lane are indicated to the left of the gel.

the fusion protein visible in lane 8 may account forthe discrepancy between the TBP and GFP · TBPlevels in the YGAL strain. Most importantly,though, no 27 kDa ‘TBP’ appeared to be generatedfrom GFP · TBP, even when it was overexpressed.

The relative values of TBP production were allexpected from previous studies (Poon et al., 1991)with the exception of the YTvnPED strains, whichhave not been previously analysed for TBP pro-duction levels. Previous work on the cis elementsinvolved in TBP expression (Schroeder et al., 1994)showed that transversion mutagenesis of the TBP-encoding gene PED cis element (nucleotides "147to "128 relative to the transcription start site)dramatically reduced lacZ reporter gene expres-sion levels to <1% of the wild-type promoter level.In contrast, pTvnPED used here produced proteinlevels that were 260% of wild-type levels (258%for expression of TBP and 266% for GFP · TBP).It must be noted, however, that unlike the previouswork, we are demanding that pTvnPED drivesufficient expression of TBP and GFP · TBP tosupport yeast cell growth, and the cells haveresponded to this selection in two ways. First,quantitative Southern blot analyses demonstratedthat the pTvnPED-TBP and pTvnPED-GFP · TBP copy number was amplified (four


copies/cell). Secondly, the cells also utilized post-transcriptional, translational, and/or post-translational mechanisms to increase TBP andGFP · TBP expression to levels sufficient tosupport life.

Quantitative image analysis yields protein valuesthat correlate well with protein blot analysis

We next calibrated our quantitative imagingmethodologies using the family of yeast strainswhich yielded systematic variation of GFP · TBPlevels. A single 1 ìm thick optical section throughthe middle of each ‘green’ yeast strain, YTvnPED-GFP · TBP, YTFIID-GFP · TBP, YPGK-GFP · TBP and YGAL-GFP · TBP, is shown inFigure 3. Quantitative image analysis (>20 cellsanalysed for each strain) revealed 213-foldand 224-fold increase in GFP · TBP levels (overYTFIID) for the YPGK and YGAL strains,respectively, while the YTvnPED strain showeda 20·7-fold reduction in GFP · TBP. TheGFP · TBP was localized exclusively to thenucleus in both the YTvnPED and the YTFIIDstrains. However in both overexpressing strains,only 212-fold wild-type levels of GFP · TBPwere located in the nucleus, while the rest was

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distributed into the cytoplasm except for the vacu-ole (Figure 3). GFP expressed alone (which lacksa nuclear localization signal) was distributeduniformly throughout the yeast cell, excluding thevacuole (data not shown). As shown in Figure 4,the relative expression levels determined by LSCMimaging and protein immunoblotting correlatequite well.

By quantitation of the protein blots, we esti-mated that the average number of TBP moleculespresent in each cell of the YTvnPED-TBP,YTFIID-TBP, YPGK-TBP and YGAL-TBPstrains were 25000, 28500, 2160,000 and2240,000, respectively. To perform a similaranalysis using the LSCM imaging data, we com-pared the total fluorescence intensity in the yeast tothe fluorescence of known amounts of purifiedHis6-tagged GFP (S65T). From these measure-ments, we estimated that 2500, 2700, 210,500and 217,500 GFP · TBP molecules per cell werepresent in the YTvnPED-GFP · TBP, YTFIID-GFP · TBP, YPGK-GFP · TBP and YGAL-GFP · TBP strains. The reason for the discrepancy


between the two measurements is unknown,but several possible causes are discussed below.Regardless, since the data acquired from imagingGFP · TBP expressed at various levels correlatesvery well with the more conventional technologyof protein immunoblotting, we are confident thatthe GFP imaging measurements offer accuraterepresentations of relative protein levels.

Time lapse imaging of dividing yeast cells showsthat TBP retains nuclear localization throughoutthe cell cycle and reveals an asymmetricdistribution of TBP between mothers anddaughters

In addition to the steady-state measurementsdescribed above, we also measured the localizationand distribution of GFP · TBP throughout thecell cycle (Figure 5A). For these experiments, weacquired ten 1 ìm thick optical sections, whichassured that each entire yeast cell would be imagedcompletely at every time point. Imaging each prep-aration of cells for 12 h allowed us to follow cells

Figure 3. Expression of GFP · TBP allows direct observation of TBP inliving yeast cells. Single 1 ìm-thick optical sections of GFP fluorescence(green) from yeast expressing GFP · TBP are shown for each of the fourstrains as indicated (see Figure 2). The fluorescent images are overlaid onNomarski DIC images of the same cells (red). Scale bar is 5 ìm.

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through several cell cycles. In Figure 5A, the tenfluorescence optical sections taken of a group ofliving yeast cells were combined (green) and over-laid upon a single Nomarski DIC image takenthrough the middle of the cells (red). The nucleardynamics and shape changes that we observedappeared to be the same as those reported earlier(Robinow and Johnson, 1987; Wheals, 1987; Joneset al., 1993). During mitosis in the YTFIID andYTvnPED strains, the GFP · TBP remains local-ized to the nucleus throughout the cell cycle. Thisis not the subcellular localization pattern observedin mammalian cells undergoing mitosis. One expla-nation for this data is that S. cerevisiae undergoesa closed mitosis where the nuclear membrane doesnot break down (Robinow and Johnson, 1987;Wheals, 1987) presumably ‘trapping’ TBP in thenucleus.

Analysis of three-dimensional image sets of theYTFIID-GFP · TBP cells revealed an asymmetricdistribution of the GFP · TBP between the motherand daughter cells after nuclear division. Imagequantitation showed that, on average, the daugh-ter cells received 270% of the TBP of the mothercells (Figure 5). Although variations in signal

Figure 4. Quantitative GFP imaging correlates with quantita-tive protein immunoblotting. Quantitative protein immuno-blots were performed on WCE prepared from each of thestrains expressing TBP and GFP · TBP using purified recom-binant TBP and purified recombinant GST · TBP as standards(standards not shown). LSCM and quantitative image analysiswere performed on each of the strains expressing GFP · TBPusing purified recombinant His6-GFP (S65T) as a standard.Each analysis of the TBP and GFP · TBP levels in theYTvnPED, YPGK and YGAL strains is graphically rep-resented relative to levels in the YTFIID strains.


levels between yeast cells were observed in theseanalyses, the temporal series of images in Figure5A shows a typical pattern of GFP · TBP levels ina mother cell and a daughter cell during one cellcycle. Image quantitation is shown in Figure 5B.The GFP · TBP level in the mother cell increasesbefore mitosis and then drops back to a ‘normal’level after budding. The daughter cell clearlyreceives less GFP · TBP than the mother, butGFP · TBP content increases steadily to a ‘normal’level over 21 h. As a control to determine if thisasymmetry is a GFP folding artifact, a time-lapseexperiment was performed on a similar yeast strainexpressing the wild-type GFP fused to TBP. Pre-sumably, if the asymmetry is due to the kinetics ofGFP folding, the more slowly folding (Heim et al.,1995) wild-type GFP would result in an evengreater asymmetry. However, quantitative analysisof these images revealed an average ratio of21:0·65 between mothers and daughters (data notshown). A similar analysis of time-lapse images ofthe YTvnPED-GFP · TBP strain showed no differ-ence between the mother and daughter cellGFP · TBP levels (Figure 5C).

DISCUSSION

Use and limitations of GFP in quantitativeimaging of living cells

After constructing a GFP · TBP fusion, we usedthis intrinsic fluorescent probe to study bothcellular transcription factor content and dynamics.By genetic manipulation of the yeast, S. cerevisiae,we replaced the endogenous TBP-encoding genewith various forms of a GFP · TBP-encoding genethat allowed us to monitor GFP · TBP proteinexpression levels and dynamics without potentialcomplications to interpretation due to the presenceof the wild-type untagged version of TBP. We wereinitially concerned that the introduction of such alarge tag as GFP (227 kDa) on to such a centralcomponent of the transcription apparatus wouldhamper its activity. However, we were able todemonstrate that there were no differences be-tween the growth rates of yeast expressing TBPand of yeast expressing GFP · TBP at normallevels (Figure 1A). Thus, we could conclude that infact GFP does not interfere with TBP function.This evidence coupled with the observation thatGFP · TBP was efficiently and stably produced asan intact fusion protein (Figure 1B) allowed us toreadily monitor TBP in living yeast cells.

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Figure 5. GFP · TBP is asymmetrically distributed between mother cells and daughter cells duringmitosis. (A) A series of time-lapse images of GFP · TBP distribution in the YTFIID-GFP · TBP strainwas acquired by LSCM at 5-min intervals corresponding to the 30–55 min time frame in (B). Scale baris 5 ìm. (B) Quantitative analysis of time-lapse images of YTFIID-GFP · TBP progressing through thecell cycle was performed. These two traces represent the GFP · TBP levels in a typical mother–daughterpair during one cell cycle. (C) Quantitative image analysis was performed on YTFIID-GFP · TBP andYTvnPED-GFP · TBP mother–daughter pairs immediately after completion of nuclear division (corre-sponds to the 25 min time point in panel A). Student’s t tests confirmed a difference in GFP · TBP levelsbetween YTFIID-GFP · TBP mother and daughter cells (*P<0·01) but not between YTvnPED-GFP · TBP mother and daughter cells.

Qualitative observations of TBP localization canbe performed in fixed and immunostained cells.However, using GFP genetically fused to TBPopens up the possibility of performing quantitativeinvestigations of TBP in living cells in real time. Apotential problem with quantitation of GFP · TBP(in particular, the S65T derivative) is the tempera-ture dependence of chromophore formation. Tocircumvent this caveat, our imaging experimentswere performed at 25)C, where formation of theGFP (S65T) chromophore was 2100% completewhen expressed in bacteria (Patterson et al., 1997).In the future, we could eliminate this problem byusing a temperature-stable GFP mutant, such asthe EGFP (Cormack et al., 1996; Siemering et al.,1996; Patterson et al., 1997). To address thiscomplication and convince ourselves that our GFP


measurements were indicative of normal intra-cellular TBP action, we compared our LSCMimaging quantitation with measurements of TBPconcentration from protein immunoblotting.

By using protein immunoblotting methods,we previously demonstrated that the expressionplasmids pTFIID-WT, pPGK-WT, andpGAL-WT resulted in expression of 1-fold,210-fold and 230-fold amounts of TBP in yeastcells. Our analysis of GFP · TBP expression fromthis family of expression plasmids by LSCM indi-cated that quantitative imaging of GFP · TBPgives a good relative representation of TBP con-centrations inside cells. However, we do not atpresent understand why the imaging measure-ments of the absolute intracellular TBP levels didnot agree with the biochemical measurements. A

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combination of several factors could be respon-sible for the 210-fold difference between the twosets of measurements. First, it is possible that notall of the GFP is folding into the fluorescent state.Our experiments were performed at a temperaturethat should allow efficient chromophore formation(Patterson et al., 1997), but if for some reasonGFP folds less efficiently in yeast than bacteria,this could affect image quantitation. Secondly, theyeast cell wall could be filtering or scattering someGFP fluorescence resulting in a decrease in theamount of detected fluorescent signal produced inthe cells. Finally, the internal environment of theyeast cell may be suboptimal for GFP fluorescence(Patterson et al., 1997). The intracellular pH of ayeast cell has been estimated to be 26·6–7·0(Garcıa-Arranz et al., 1994). Determination ofGFP (S65T) fluorescence as a function of pHindicates that its brightness is only slightly per-turbed (210–15%) at this pH (Patterson et al.,1997). However, a small loss of signal due to pHcoupled with the other factors could reduce theapparent number of GFP molecules detected, andthus account for the discrepancy between theimaging and biochemical quantitation.

Overexpression of TBP in vivoSince TBP is thought to be limiting for tran-

scription in vivo (Colgan and Manley, 1992), over-expressing it might be expected to increase totalcellular transcription and possibly increase thegrowth rate of the cell. Previous studies whichexamined the effects of overexpressing TBP inyeast found that their growth rate characteristicsdid not change (Poon et al., 1991). However, it wasnot known if the ‘extra’ TBP failed to localize tothe nucleus, which is a trivial explanation for thisresult. The data presented in this report show thatin the YPGK-GFP · TBP and YGAL-GFP · TBPyeast strains, 212-fold wild-type levels ofGFP · TBP is localized to the nucleus (Figure 3)while the rest is cytoplasmic. Whether this 12-foldvalue is a physical limit (i.e. the nucleus is full) orif the extra GFP · TBP simply cannot be trans-ported into or retained in the nucleus (i.e. alltransport machineries, binding sites, transcriptioncomponents, etc. are occupied) is unknown. It isalso possible that the fluorescence observed out-side the nucleus in the YPGK and YGAL strainsderives from degraded GFP · TBP (Figure 2, lanes6 and 8) and is not forced to the cytoplasm by anynuclear localization limit. Regardless, by being


localized to the nucleus, at least 12 times thenormal amount of GFP · TBP has the potentialto participate in transcription, yet this ‘extra’TBP does not result in an increase in yeast cellgrowth. Presumably, another component of thetranscription machinery becomes limiting underthese conditions.

In contrast, the low expression yeast strains,YTvnPED-GFP · TBP and YTvnPED-TBP, hadslower growth rates than normal and expressedGFP · TBP and TBP to 266% and 258% of theirwild-type counterparts, YTFIID-GFP · TBP andYTFIID-TBP, respectively (Figure 4). The twostrains also contained multiple TBP andGFP · TBP gene copies. These data suggest thatthere is, in fact, a lower limit of TBP proteincontent below which yeast cells are non-viable. It isinteresting to note that the TBP steady-state levelsin cells expressing these two forms of TBP from thepTvnPED expression plasmid are the same as thatfound in newly generated daughter cells (60–70%normal). The implications of this lower TBP levelon global gene transcription are currently beinginvestigated.

Asymmetric distribution of TBP between motherand daughter cells

The asymmetric distribution of GFP · TBPbetween the mother cell and the daughter cell(Figure 5B) is a surprising observation, and severalpossible reasons for this distribution are discussedhere. During mitosis in budding yeast, the nuclearmembrane does not disassemble, but instead thenucleus migrates to the bud site and undergoes adivision much like a dividing cell (Robinow andJohnson, 1987; Wheals, 1987). Since the nucleusappears to divide equally between each mother anddaughter (Jones et al., 1993), a nuclear volumedifference is probably not the underlying reasonfor the observed asymmetric distribution ofGFP · TBP. A second possibility is that the differ-ence in TBP levels between the two nuclei is anartifact produced by GFP · TBP degradation.However, as shown in Figures 1B and 2, only avery small amount of degradation of the fusionprotein can be seen in the TFIID-GFP · TBPstrain WCE, so this is probably not the causefor the asymmetric GFP · TBP distribution. Inaddition, the GFP · TBP in the daughter cellincreases to the mother cell concentration overtime, suggesting that this level is the ‘normal’resting GFP · TBP level. A third possibility is that

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this observation is an artifact due to the kinetics ofthe GFP chromophore formation. This GFP vari-ant (S65T) exhibits a chromophore formation timeconstant of 0·45 h and could result in a ‘fluor-escence lag’ of the GFP · TBP (Heim et al., 1995).However, this possibility seems unlikely since asimilar time-lapse experiment performed onyeast (YWT-wtGFP · TBP) expressing the moreslowly folding wild-type version of GFP fused toTBP gave a similar asymmetry (21:0·65) betweenthe mothers and daughters (data not shown).Another possibility is that the mRNAGFP · TBP isasymmetrically distributed between mother anddaughter cells thus leading to the unequal levels ofGFP · TBP in the divided cells. Molecular geneticanalyses of Ash1p found that this protein wasasymmetrically localized to the daughter cellsduring mitosis (Bobola et al., 1996; Sil andHerskowitz, 1996) and that this asymmetry isdictated by the localization of mRNAASH1 (Longet al., 1997). Therefore, it is possible that themother cell contains more mRNAGFP · TBP whichleads to an asymmetric expression of GFP · TBPprotein; however, summation of the GFP · TBPlevels in the mother–daughter pair after nuclearseparation approximately equals the level ofGFP · TBP observed in the mother cell beforenuclear division (Figure 5B and data not shown).In any case, it remains that the level of GFP · TBPprotein in the daughter cell immediately afternuclear division is less than that in the mother cell.The mechanism for the asymmetric GFP · TBPdistribution is most likely quite complicated, butcould involve partial association of GFP · TBPwith DNA, other transcription factors, or themother cell nuclear matrix during mitosis. Asmentioned earlier, 210–20% of the mammalianTFIID complex (Segil et al., 1996) and all of themammalian SL1 complex (Jordan et al., 1996;Roussel et al., 1996) remain associated with thechromosomes throughout mitosis via mechanismswhich are currently not understood.

In mitotic cells during the M-phase, there is aglobal inhibition of transcription by all threeclasses of DNA-dependent RNA polymerases(Prescott and Bender, 1962). Phosphorylation ofseveral of the GTFs and their components, includ-ing TBP, is thought to play a significant role in thistranscriptional repression (Gottesfeld et al., 1994;White et al., 1995; Leresche et al., 1996; Segil et al.,1996). One of the hypotheses for the mechanismof the mitotic inhibition of transcription (Segilet al., 1996) holds that the redistribution of some


transcription factors may be a mechanism forchanging gene expression either during the cellcycle or during development. The possible effectsupon gene expression that lower GFP · TBP con-centrations could have in the daughter cells isunknown, though it is not unreasonable topostulate that this decreased concentration of TBPcould cause significant changes in specific genetranscription.

Conclusions

A thorough understanding of TBP levels anddynamics inside the living cell will ultimately becrucial to unravelling the complexities of generegulation on a global, genome-wide scale. Thiswas one of the main reasons we attempted toconstruct and utilize the GFP · TBP reporter. Ourdetailed comparisons of quantitative GFP imag-ing with a more accepted biochemical methodvalidates the use of GFP as a quantitative tool forbiological research. Some of the applications ofGFP that we describe here, such as steady-statecomparisons of TBP levels in various strains, couldeasily be made using protein immunoblotting.However, kinetic monitoring of GFP · TBPthroughout the cell cycle is not possible usingbiochemical methods. Notably, it was our kineticobservations of GFP · TBP concentrations andintracellular dynamics that led to the discovery ofasymmetric TBP distributions during yeast celldivision. How or why this distribution occurs ispresently unknown, but the implications fortranscription in a freshly budded cell containing20·7-fold normal TBP levels may lead to a betterunderstanding of the physiological role of TBP.

ACKNOWLEDGEMENTS

We thank Kevin Gerrish and Steven Sanders forassistance with protein immunoblotting. Thesestudies were supported by grants from theBeckman Foundation Young Investigator Pro-gram and the Whitaker Foundation BiomedicalEngineering Research Program to D.W.P., andfrom NIH to P.A.W. (GM52461). During part ofthis work, G.H.P. was an NIH trainee (GM08320).Confocal microscopy was performed at the CellImaging Shared Resource, supported by theVanderbilt Cancer Center (CA68485) andDiabetes Research and Training Center(DK20593).

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quantitative imaging of tata-binding protein in living yeast cells

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