extfieldsofstudy: Biology Medicine

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Translational Accuracy during Exponential, Postdiauxic, and Stationary Growth Phases in Saccharomyces cerevisiae

ABSTRACT When the yeast Saccharomyces cerevisiae shifts from rapid growth on glucose to slow growth on ethanol, it undergoes profound changes in cellular metabolism, including the destruction of most of the translational machinery. We have examined the effect of this metabolic change, termed the diauxic shift, on the frequency of translational errors. Recoding sites are mRNA sequences that increase the frequency of translational errors, providing a convenient reporter of translational accuracy. We found that the diauxic shift causes no overall change in translational accuracy but does cause a strong reduction in the frequency of one type of programmed error: Ty +1 frameshifting. Genetic data suggest that this effect may be due to changes in the relative amounts of tRNA participating in translation elongation. We discuss possible implications for expression strategies that use recoding.

Translation maintains a balance between speed and accuracy (24). Kinetic proofreading (19,29,44) ensures that correct aminoacyl-tRNAs are selected for each round of translation in the ribosomal A (acceptor) site and that they are in the correct frame, as defined by the previous codon present in the P (peptidyl-tRNA) site. Accurate translation requires a highly discriminating A site. Most tRNAs that enter the A site fail to form three base pairs with the codon displayed there, and so the tRNA rapidly dissociates; only cognate tRNAs are efficiently and quantitatively retained. Hyperaccurate ribosomes actually reject a large proportion of even cognate tRNAs, an event which has been demonstrated in Escherichia coli to increase the translation step time (2,33). It is likely that such a costly step in gene expression is regulated by physiological conditions encountered by the cell. The cell may gain in fitness if translational accuracy varies in response to growth conditions and energy availability.
The yeast Saccharomyces cerevisiae is a eukaryotic microorganism able to ferment carbon sources such as glucose or galactose or to respire on nonfermentable carbon sources such as glycerol or ethanol. Fermentative growth in glucose media produces ethanol as a by-product that is consumed after glucose depletion during a second respiratory growth phase, which begins after a change in cellular metabolism that is called the diauxic shift; after the ethanol is completely consumed, yeast cells stop growing and enter a distinct stationary phase (48). During the postdiauxic phase, many genes undergo large changes in expression (12). After a short lag at the diauxic shift, cells grow much more slowly in the postdiauxic phase; the rate of protein synthesis declines at the shift and recovers only partially (17). Consistent with these changes, genes encoding the translational apparatus, ribosomal pro-teins, and translation factors predominate among the genes most strongly repressed at the diauxic shift (12). Perhaps as importantly, late in the exponential phase, as cells approach the diauxic shift but before the shift in expression begins, the concentration of ribosomes begins to decline, reaching 25% of its initial value at the diauxic shift (22).
Previous studies with bacteria indicated that translational accuracy actually changes with the stage of growth of cells. Several reports showed 3-to 10-fold increases in frameshifting upon entrance into the stationary phase in E. coli (4,16,47) and Salmonella enterica serovar Typhimurium (34). One experiment showed that the effect in E. coli is transient as cells enter the stationary phase (47). We were interested in determining whether such effects occur in a eukaryotic system and whether other translational errors are also affected. We made use of mRNA sequences that program the ribosome to efficiently undergo particular translational errors, such as reading through termination codons (nonsense readthrough) or shifting the reading frame by 1 nucleotide (nt) in the upstream or downstream direction (Ϫ1 or ϩ1 frameshifting). These events, termed programmed alternative decoding or recoding events, occur because the sequence of the mRNA stimulates the ribosome to commit the error. The mechanism underlying these events is quite complex but often involves a translational pause that reduces the rate of normal peptide elongation, allowing a normally inefficient error to occur (reviewed by Farabaugh [14]).
We found an effect in postdiauxic growth opposite that found in bacteria. Programmed ϩ1 frameshifting at a site derived from the yeast retrotransposon Ty1 actually decreases as cells approach the diauxic shift and decreases further as they continue into the postdiauxic and stationary phases. The effect is not general; we found no significant effect of growth stage on two other types of programmed frameshifting or on programmed nonsense readthrough. Since programmed frameshifting is extremely sensitive to changes in tRNA availability, the simplest explanation for this effect is that the ratio of charged tRNAs to the number of elongating ribosomes changes as yeast cells enter postdiauxic growth. We present genetic data supporting this hypothesis.
Media. A synthetic medium was used to select for plasmids carrying nutritional markers introduced during yeast transformation (SD: 0.17% yeast nitrogen base without amino acids and without ammonium sulfate [Difco], 0.5% ammonium sulfate, 2% glucose, appropriate amino acids). SD or a rich glucose medium (YPD: 2% yeast extract [Difco], 1% peptone [Difco], 2% glucose) was used to perform time course and batch growth experiments.
Plasmids. The reporter plasmids used (6,37) consist of a control reporter, the E. coli lacZ gene, encoding ␤-galactosidase, translationally fused to the firefly luciferase gene, luc. Recoding sites are cloned between the genes such that luciferase is expressed only if the ribosome commits a translational error (readthrough or frameshift) at a recoding site. The expression of reporter plasmids previously constructed to study Ty1 ϩ1 frameshifting (pACTy), human immunodeficiency virus type 1 (HIV-1) Ϫ1 frameshifting (pAC1789), and tobacco mosaic virus (TMV) termination readthrough (pACTMV) (37) was compared with that of specific in-frame controls used as a reference to determine the enzymatic activity corresponding to 100% expression of the fusion protein. The control for Ty ϩ1 frameshifting was pACTTy, that for HIV-1 Ϫ1 frameshifting was pAC1790, and that for TMV readthrough was pACTQ (37). Sets of reporter plasmids were constructed for this study based on the ϩ1 frameshift sites of the Ty3 retrotransposon of S. cerevisiae. Ty3 frameshifting occurs at a 21-nt sequence encompassing a GCG-AGU-U recoding site and a downstream 14-nt stimulatory sequence (15,25). The 21-nt region was inserted in place of the recoding signal in pAC1789 to create pACTy3; an in-frame version was constructed by mutating the recoding heptamer from GCG-AGU-U to GCA-GUU to create pACTy3FF. Plasmids were transformed into yeast cells by using a lithium acetate method (18).
Growth stage analysis. To test the effect of growth stage on recoding efficiencies, we cultured yeast transformants in two ways to obtain cells at various stages of the growth cycle. A true single-step growth curve experiment was performed by inoculating yeast cells from an overnight postdiauxic-phase culture in SD minimal medium by diluting 1:1,000 into 60 ml of fresh YPD medium. After 15 to 19 h of incubation at 30°C in shaking cultures, the cells attained early exponential phase (optical density at 600 nm [OD 600 ], 0.5; corresponding to 10 7 cells/ml). Samples were withdrawn after approximately 15,17,19,21,36, and 108 h of incubation of the YPD cultures. The first four samples were in the exponential phase, as judged by their rate of doubling and the fact that over 60% of the cells were budded (10). The last two samples corresponded to the postdiauxic and stationary phases, respectively, as determined by changes in growth rate and cell morphology (48). For reproducibility, samples were obtained at approximately matched optical densities.
The second method of culturing was batch growth. For this method, cells were diluted to various extents as separate 5-ml cultures in fresh YPD or SD medium and grown for a predetermined time of 17 to 20 h to obtain samples of cells in the exponential or postdiauxic phase. To obtain stationary-phase samples, the cultures were incubated for over 60 h. We found that the level of expression observed was determined by the final density of the culture when sampled and was unaffected by the growth stage or density of the inoculum or the length of incubation before sampling (data not shown), indicating that the results reflect differences in growth stage rather than culture history.
Enzymatic activities and recoding efficiencies. Luciferase and ␤-galactosidase activities were assayed by using the same crude extract as described previously (37). For each assay, extracts of three transformants grown under identical conditions were prepared by breakage with acid-washed glass beads (diameter, 0.4 to 0.5 mm) as described previously (37). The concentration of protein in the crude extract was determined by using the Bradford assay (Sigma) (8). The efficiencies of programmed translational errors were determined by calculating the ratio of luciferase activity to ␤-galactosidase activity expressed in the exper-iment and in the control (expression from the in-frame control plasmid). The ratio calculated in the control corresponds to the expression of 100% lacZ-luc fusion protein. This value provides the bench mark for determining recoding efficiencies, which were calculated by dividing the ratio obtained in the experiment by the ratio obtained in the control. The standard errors for recoding efficiencies were generally under 10% throughout the course of this work. Specific activities used for calculations can be obtained as supplemental data at http://www-lmgm.biotoul.fr/grpfayet/stahl/supdata2004.

RESULTS
We have been concerned with the issue of how the translational apparatus maintains an adequate level of accuracy. We were interested in determining how translational accuracy might change as cells progress from the exponential phase through the postdiauxic phase and then into the stationary phase given the drastic changes in the availability of the translational machinery during those phases of growth. Figure 1 shows a typical single-step growth curve for yeast strain Fy1679-18B in rich YPD medium. Cells diluted 1,000fold from a stationary-phase culture rapidly enter a phase of exponential growth that continues for almost 20 h until they reach an OD 600 of 6. For the strain used, an OD 600 of 1.0 is equivalent to 2 ϫ 10 7 cells per ml, as measured by counting with a hemacytometer. Yeast cells grow exponentially by fermentation, doubling every 90 min. After the consumption of all fermentable carbon sources, yeast cells switch to respiration of ethanol, with the switch causing a short lag called the diauxic shift. Cells then continue in a second phase of growth termed the postdiauxic phase, visualized in Fig. 1 as a break in the slope of the growth curve, since yeast cells grow more slowly when respiring. Postdiauxic growth continues for about 30 h, after which the culture density stops increasing and unbudded cells accumulate, signs that the culture has entered the stationary phase. The postdiauxic and stationary phases should not be confused, since they are physiologically quite different (21,48).
Programmed ؉1 frameshifting varies with growth phase. Programmed ϩ1 frameshifting is a very sensitive reporter of the accuracy of A-site selection (5,15,31). At these sites, slow recognition of an in-frame sense codon allows the peptidyl-tRNA bound to the codon immediately upstream to stimulate a ϩ1 shift in frames. This mechanism occurs in the Ty family of yeast retrotransposons (5,15) and in prokaryotic (11) and eukaryotic (1,3,26,27,32,42,43) cellular genes. Members of our laboratory have studied the mechanism of ϩ1 frameshifting in Ty elements for many years and have arrived at a thorough molecular understanding of the process (reviewed by Stahl et al. [39]). At one of the recoding sites derived from Ty1, frameshifting is stimulated by the slow recognition of an AGG codon by the rare tRNA (5). Ty1 ϩ1 frameshifting therefore provides an indirect measure of the rate of AGG decoding.
To test the effect of growth stage on Ty1 ϩ1 frameshifting, we used a plasmid in which the expression of firefly luciferase (luc) depends on frameshifting at the Ty1 frameshift site. The reporter plasmid actually contains two reporter genes separated by a region including the Ty1 recoding site. Translation begins at the initiator of the upstream gene, lacZ. Ribosomes that traverse lacZ encounter the Ty1 recoding site, at which a proportion of them undergo a ϩ1 frameshift event and continue translating into luc. The primary translation product is a fusion of the lacZ product, ␤-galactosidase, to luciferase, the luc product. The frequency of frameshifting is proportional to the ratio of luciferase activity to ␤-galactosidase activity. A construct in which the lacZ and luc genes are in the same reading frame (frame fusion plasmid) and therefore always are translated as a fusion protein provides a bench mark against which to compare expression in the frameshift reporter plasmid. The efficiency of frameshifting is determined by dividing the luciferase/␤-galactosidase ratio expressed from the frameshift reporter plasmid by that expressed from the frame fusion plasmid.
A plasmid (pACTy) carrying the Ty1 frameshift site and a second reporter plasmid (pACTTy), used as the frame fusion control, were transformed into FY1679-18B. For each plasmid, cultures of three transformants that had been grown overnight (OD 600 , 5) in SD medium with glucose as the carbon source were diluted 1:1,000 in fresh YPD medium and grown to the stationary phase. Despite the lack of selection for retention of the plasmid, which requires growth in SD minimal medium lacking leucine, the plasmid, which carries a yeast centromere to stabilize its partitioning, was quantitatively retained during culturing, as judged by plating on minimal medium with and without leucine (data not shown). Samples were periodically withdrawn, and the specific activities of both luciferase and ␤-galactosidase present in crude extracts were determined as described in Materials and Methods. Early during growth, frameshifting efficiency was over 50% (Fig. 2), in agreement with previous results (5,37). As the cells continued into the late exponential phase (OD 600 , 4.4) but before entering the postdiauxic phase, the efficiency of frameshifting began to de-cline to 35%. As the experiment continued, the efficiency continued to decline to 22% during the postdiauxic phase and to 5% in the stationary phase. The decline in efficiency was continuous and appeared to be proportional to culture time.
We tested whether we could use a simpler batch growth experimental system, growing cells overnight in SD-glucose medium, diluting them in fresh YPD medium to various initial concentrations, and then growing them for defined periods to produce simultaneously cultures at various growth stages up to the postdiauxic phase. To achieve the stationary phase, the cultures required more extended incubation. Figure 3 shows the results of such an experiment testing variations in frameshifting at the Ty1 frameshift site with growth phase. The results strongly resemble those obtained in the single-step growth experiment illustrated in Fig. 2. Early in the exponential phase, the efficiency of frameshifting was quite high, and that level declined in cells collected at later stages of growth, with postdiauxic-and stationary-phase cultures showing about 10% frameshifting. Very similar results were obtained when cells were grown in SD-glucose selective minimal medium with the same batch culture approach (data not shown). The absolute values obtained by the two protocols were not identical, although the results obtained with each protocol were reproducible. These results suggest that there may be subtle differences in the state of cells collected at stages of growth that appear to be the same, depending on the history of culture conditions. However, the differences are slight in comparison to the overall effects of declining frameshifting efficiencies as cells approach glucose depletion and beyond. Importantly, these experiments were internally controlled to eliminate the effects of changes in gene expression occurring at levels other than frameshifting. The concept behind the dualreporter system is that it eliminates the effects of various promoter strengths or mRNA stabilities, for example, because the upstream lacZ reporter acts as an internal standard. Doubling the amount of mRNA for the dual reporter would double the expression of each reporter but would leave unchanged the ratio of the two enzymatic activities.
Growth stage does not affect nonsense readthrough or programmed ؊1 frameshifting. Does growth stage affect other recoding events in the same way? The most versatile and bestcharacterized programmed error is Ϫ1 simultaneous slippage frameshifting, found ubiquitously in eukaryotic viruses (reviewed by Brierley [9]) and in cellular genes in prokaryotes, e.g., dnaX (45), and eukaryotes, e.g., Edr (35). Ϫ1 Programmed frameshifting takes place at a slippery heptamer, X XXY YYZ (X and Y can be identical), usually stimulated by a downstream structure, usually a pseudoknot in eukaryotes. Unlike the efficiency of programmed ϩ1 frameshifting, the efficiency of simultaneous slippage Ϫ1 frameshifting is unaffected by the concentrations of the XXY and YYZ decoding aminoacyl-tRNAs, since frameshifting to XXX and YYY occurs after they have been recruited to the ribosome (reviewed by Farabaugh [14]).
We repeated the single-step growth experiment with a lacZluc dual reporter in which the HIV-1 programmed Ϫ1 frameshift site had been inserted between the two reporters so that luciferase expression required the frameshift. The HIV-1 frameshift reporter and the frame fusion control reporter were introduced into FY1679-18B as described above. Triplicate transformants for each reporter were grown to saturation in SD-glucose medium, diluted to an OD 600 of 0.1, and cultured at 30°C. Samples were withdrawn when the cultures had attained average OD 600 values of 0.5, 1.3, 2.7, and 4.4, all in the exponential phase, 7.2 in the postdiauxic phase, and 16 in the stationary phase. As shown in Fig. 2, the level of Ϫ1 frameshifting for the HIV-1 target was 6% in the samples taken in the early exponential phase, and that value remained virtually unchanged in samples taken throughout the course of the experiment. The experiment was repeated with the batch growth protocol. The level of frameshifting was 6 to 10% throughout the course of this experiment (Fig. 3), quite similar to the level obtained in the single-step growth experiment. The data show that the level of programmed Ϫ1 frameshifting is not significantly affected by growth stage.
TMV uses stop codon readthrough to express its polymerase (36). In yeast cells, the peptide release factor inefficiently recognizes the TMV stop codon (in bold type) in CAAUAGCA AUUA, allowing one in five ribosomes to read through, incorporating Tyr, Trp, or Lys at the UAG (7). The efficiency of this event depends first on slow recognition of the UAG codon by peptide termination factor, which allows for noncognate recognition of the UAG by tRNA Tyr , tRNA Trp , or tRNA Lys . Since noncognate tRNAs are generally rejected by the ribosome, the second factor is the efficiency with which the translational accuracy mechanism discriminates against these noncognate tRNAs. Therefore, readthrough efficiency is a measure both of termination efficiency and of missense incorporation of aminoacyl-tRNA at noncognate codons. Using a batch growth approach, we tested whether the efficiency of readthrough was affected by growth stage. We detected no significant change in stop codon readthrough (Fig. 3). Not all programmed ؉1 frameshifts respond to growth stage. The lack of a significant effect on either programmed Ϫ1 frameshifting or programmed nonsense readthrough suggested that the effect on programmed ϩ1 frameshifting must be specific to that type of frameshifting. Rather than the translation system becoming generally hyperaccurate during the postdiauxic and stationary phases, an alteration in some aspect of cell physiology may directly stimulate ϩ1 frameshifting. Recent evidence shows that ϩ1 frameshifting in yeast cells occurs because of the presence of a noncanonical interaction between peptidyl-tRNA and the mRNA in the P site during the shift (41). This interaction can be a weak pyrimidine-pyrimidine wobble interaction, which probably does not involve any base pairing, or a purine-purine wobble clash. The presence of an unconventional pair predisposes the ribosome to shift ϩ1 at the next step in elongation. This fact suggests that the ribosome normally uses the structure of the P-site codon-anticodon complex, especially the wobble pair, to constrain recognition in the A site to the correct reading frame (38). It is conceivable that growth stage has a general effect on the probability of errors at such a site or that its effect is more specific, affecting the probability of frameshifting only at the Ty1 frameshift site.
To examine whether the growth stage effect is general, we tested another programmed ϩ1 frameshift that occurs in a yeast retrotransposon, Ty3, distantly related to Ty1. This frameshift site involves a different heptameric recoding signal. In Ty1, that signal is CUU-AGG-C, presented in codons of the upstream zero frame, while Ty3 uses the sequence GCG-AGU-U. The Ty3 frameshift site stimulates frameshifting quite weakly, with about 2.5% efficiency (15); a downstream mRNA sequence increases that efficiency 7.5-fold (15,25). lacZ-luc dual-reporter plasmids were constructed to carry the Ty3 frameshift site with or without the adjacent stimulator. Frame fusion versions of each were constructed for each plasmid. FY1679-18B yeast cells transformed by these plasmids were grown in SD-glucose medium with the batch growth protocol. Frameshifting at the Ty3 recoding site was constant in the exponential, postdiauxic, and stationary phases. In the presence of the downstream stimulator, about 8% frameshifting occurred in each phase, while in its absence, 2% frameshifting occurred in both the exponential and the postdiauxic phases; expression in the stationary phase was too low to accurately quantify but was no higher than 2%.
Growth stage affects ribosomal pausing at AGG codons. Since the effect of growth stage on Ty1 frameshifting requires only the heptameric sequence CUU-AGG-C, it is likely that the effect can be explained by understanding how this sequence is decoded in the ribosome. Two effects combine to define the efficiency of Ty1 frameshifting: the nature of the codon-anticodon interaction at the P-site codon CUU (41) and the rate of decoding of the A-site codon AGG (5). Slippery stop frameshift sites in which the same CUU P-site codon is immediately followed by a poorly recognized termination codon also are unaffected by growth stage (data not shown). These data imply that the AGG A-site codon rather than the CUU P-site codon may be responsible for the growth stage effect.
The amount of the normally low-abundance AGG cognate tRNA CCU Arg controls Ty1 frameshifting. Overexpressing the tRNA forces in-frame decoding and thus reduces frameshifting  (5). Eliminating tRNA CCU Arg by deleting HSX1, the gene encoding tRNA CCU Arg , forces slow decoding of AGG by the nearly cognate tRNA UCU Arg , normally the AGA decoder (23), stimulating frameshifting. Ty1 frameshifting efficiency decreases as cells leave the exponential phase, suggesting that the availability of the tRNA may increase in the postdiauxic and stationary phases.
In a wild-type strain, the stimulatory pause results from a very slow rate of binding of the low-abundance tRNA CCU Arg . Pausing in a ⌬hsx1 strain results from a different effect. In such a strain, tRNA UCU Arg , which decodes AGG, although highly abundant, is frequently rejected by the ribosome because it is not cognate for AGG (44). The frequent dissociation of tRNA UCU Arg slows AGG recognition sufficiently and strongly stimulates frameshifting. We hypothesized that the growth stage decrease in Ty1 frameshifting results from an increase in the forward rate of binding of tRNA CCU Arg caused by a general increase in tRNA availability. Since pausing in the ⌬hsx1 strain reflects rapid dissociation rather than slow binding, passage into the postdiauxic phase in the ⌬hsx1 strain should not affect frameshifting efficiency, since the rate of dissociation should be unaffected by tRNA availability in solution.
We assayed the growth stage effect on Ty1 frameshifting in strain FY1679-18B ⌬hsx1. The frameshifting efficiency in this strain during the exponential phase was 80%; as expected, it was increased from the level of 55% seen in the congenic parent strain FY1679-18B (Fig. 4). However, as predicted, the frameshifting efficiency in the ⌬hsx1 strain remained essentially unchanged in the postdiauxic and stationary phases, whereas that in the congenic wild-type strain declined to 27 and 9%, respectively (Fig. 4). These results strongly argue that as cells advance through and exit the exponential phase, the normally highly limiting tRNA becomes much more available, causing a change in the way in which the ribosome reads the Ty1 recoding signal in favor of more frequent continued inframe translation. By using specific oligonucleotide probes, we measured the yeast tRNA content in various growth phases and normalized it to that of 5S RNA (data not shown). We were not able to detect any significant change in tRNA content versus rRNA content by using this method.

DISCUSSION
There is ample evidence that the translational machinery is the aspect of yeast cellular physiology most affected by the diauxic shift. The translational machinery makes up a large proportion of cellular mass. rRNA and tRNA make up about 95% of total RNA in yeast cells, and ribosomal protein mRNAs make up almost 20% of the mRNA pool (reviewed by Warner [46]). With 200,000 ribosomes in an average cell, yeast cells must synthesize about 2,000 ribosomes per min to maintain exponential growth, doubling every 100 min (46). At the diauxic shift, the abundance of ribosomal protein mRNAs decreases an average of 4.1-fold (12). As cells approach the diauxic shift, the abundance of rRNA decreases 75%, partly because about 50% of existing cellular ribosomes are destroyed (22). The effect of these changes on yeast translational accuracy was unknown previously. Here we show that as cells shift from exponential to postdiauxic growth, there are effects on certain programmed translational errors, although there does not appear to be a general change in translational accuracy. Ty3 programmed ϩ1 translational frameshifting, simultaneous slippage Ϫ1 frameshifting at the HIV-1 frameshift site, and readthrough of a termination codon derived from TMV each shows little if any effect of the transit into postdiauxic growth. Programmed ϩ1 frameshifts are sensitive indicators of A-site accuracy. These sites appear to have evolved to directly depress the kinetics of cognate, in-frame decoding in the ribosomal A site, a process which indirectly results in an increase in ϩ1 frameshifting (reviewed by Stahl et al. [38]). Similarly, the level of TMV readthrough indicates A-site accuracy as well as the efficiency of translation termination (6). The fact that Ty3 programmed ϩ1 frameshifting and TMV readthrough remain unchanged during the yeast growth phase suggests that A-site translational accuracy does not vary.
Despite the lack of a general effect, ϩ1 frameshifting in the Ty1 system did respond to growth stage. Ty1 frameshifting decreased continuously as cells progressed through single-step growth from exponential to postdiauxic and finally stationary phases. The effect on Ty1 frameshifting occurred in a reporter construct that included only the heptameric recoding site, CUU-AGG-C. Frameshifting at this heptamer occurs during a pause caused by slow recognition of the in-frame codon, AGG, by its cognate tRNA CCU Arg (5). The translational pause occurs because the supply of tRNA CCU Arg is limiting, slowing reading of the AGG codon. The postdiauxic reduction in frameshifting at this site resembled the reduction in frameshifting that results from overexpression of the tRNA. We suspected that tRNA CCU Arg was no longer limiting in translation after the diauxic shift. If this were true, then forcing recognition of the AGG by an abundant isoacceptor, tRNA UCU Arg , would eliminate the effect of the diauxic shift, an event which we observed. These data suggest that Ty1 frameshifting declines as cells enter the postdiauxic phase because of a change in the availability of tRNA for translation, specifically, the availability of the rare tRNA CCU Arg . Measurements of the content of this tRNA during different growth phases showed no variations relative to the cellular amounts of 5S rRNA. This finding clearly contradicts the above hypothesis. It should be noted that this experiment does not distinguish charged tRNA from uncharged tRNA, nor do we know whether the 5S rRNA content reflects the number of ribosomes actively participating in translation. Translation initiation or ribosome biogenesis could decrease after the diauxic shift.
Given these experimental limitations, we favor a hypothesis supported by the three main experimental results reported here: (i) translational accuracy, either at the level of A-site accuracy (measured at the TMV readthrough site) or for Ϫ1 frameshifting (measured at the HIV Ϫ1 frameshift site), does not vary in different growth phases; (ii) Ty3 ϩ1 frameshifting, which is mechanistically related to that of Ty1, is not affected in different growth phases; and (iii) deletion of the scarce tRNA responsible for high levels of Ty1 frameshifting renders Ty1 frameshifting insensitive to the growth phase effect. These data suggest that the availability of tRNA CCU Arg for translation elongation increases relative to the number of translating ribosomes as yeast cells exhaust carbon sources in the media. Why would the availability of tRNA CCU Arg change as cells enter the postdiauxic phase? One possibility is that the massive redirection of cellular metabolism at the diauxic shift (12) reduces the demand for the tRNA. tRNA CCU Arg is among the least abundant of all the yeast tRNAs (20), but it recognizes 21% of the Arg codons in yeast cells, or an average of 4.4 codons per structural gene (28). In contrast, tRNA UCU Arg , which is almost 10 times as abundant (20), decodes only 48% of Arg codons (28). We calculate, based on available data (30,46), that a yeast cell contains approximately 10,000 copies of tRNA CCU Arg , or 1 copy for every 20 ribosomes. The imbalance between tRNA concentration and codon use could explain why the tRNA is so limiting.
Previous experiments showing an effect of cell growth phase on programmed frameshifting or nonsense readthrough suggested that an intrinsic change in ribosomal accuracy may be the cause of the effect. Barak et al. (4) suggested that the reduced availability of GTP in the stationary phase may cause ribosomes to stall at frameshift-inducing sequences. The data presented here clearly imply that although the efficiency of specific programmed events may change, growth phase does not affect translational accuracy in general in S. cerevisiae. The causes of growth phase effects on programmed translational errors likely will prove to be species dependent and to resist generalization. However, the data draw attention to the reason why genes utilize this baroque manner of expression. In general, the event accomplishes a morphogenetic task, provides an autogenous or feedback regulatory goal, or generates an alternative enzymatic activity (reviewed by Farabaugh [14]). However, no rationale is known for the use of programmed errors in several cellular genes, including the ABP140 (3) and EST3 (27) genes in Saccharomyces, the p43 protein gene in Euplotes (1), and the higher eukaryotic genes Edr (35), headcase (40), and others. It is possible that frameshifting in these genes conforms to the previously established types: morphogenesis, regulation, or enzymology. The data presented here suggest the possibility of another general explanation: that the frameshift provides a way to coordinate the expression of a protein product with variations in growth stage or some other general variations in cellular physiology.


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Expression and copy number analysis of TRPS1, EIF3S3 and MYC genes in breast and prostate cancer

The long arm of chromosome 8 is one of the most common regions of amplification in cancers of several organs, especially carcinomas of the breast and prostate. TRPS1, MYC and EIF3S3 genes are located in one of the minimal regions of amplification, 8q23–q24, and have been suggested to be the target genes of the amplification. Here, our goal was to study copy number and expression of the three genes in order to investigate the significance of the genes in breast and prostate cancer. By using fluorescence in situ hybridisation (FISH), we first found that TRPS1 and EIF3S3 were amplified together in about one-third of hormone-refractory prostate carcinomas. Next, we analysed the mRNA expression of the three genes by real-time quantitative RT–PCR and the gene copy number by FISH in six breast and five prostate cancer cell lines. Breast cancer cell line, SK-Br-3, which contained the highest copy number of all three genes, showed overexpression of only EIF3S3. Finally, the expression levels of TRPS1, EIF3S3 and MYC were measured in freshly frozen clinical samples of benign prostate hyperplasia (BPH), as well as untreated and hormone-refractory prostate carcinoma. The TRPS1 and MYC expression levels were similar in all prostate tumour groups, whereas EIF3S3 expression was higher (P=0.029) in prostate carcinomas compared to BPH. The data suggest that the expression of EIF3S3 is increased in prostate cancer, and that one of the mechanisms underlying the overexpression is the amplification of the gene.

Molecular cytogenetic analyses, especially comparative genomic hybridisation (CGH), have indicated that gain of the long arm of chromosome 8 (8q) is one of the most common chromosomal aberrations in many human malignancies, including breast and prostate carcinomas (Forozan et al, 1997). For example, about 70 -90% of metastatic and/or hormone-refractory prostate carcinomas as well as about 50% of untreated breast cancers contain gain of 8q by CGH (Visakorpi et al, 1995;Cher et al, 1996;Tirkkonen et al. 1998;Nupponen et al, 1999). The gain of 8q has also been found to be associated with an aggressive phenotype and poor prognosis in these malignancies Alers et al, 2000). Two minimal regions of amplification, 8q21 and 8q23 -q24, have been identified in prostate cancer by CGH analyses (Cher et al, 1996;Nupponen et al, 1998). Several putative target genes, such as MYC, PSCA, EIF3S3 and TRPS1, have been proposed for the amplification of 8q23 -24 (Jenkins et al, 1997;Reiter et al, 1998;Nupponen et al, 1999;Chang et al, 2000;Rummukainen et al. 2001). However, the significance of each of these genes in tumorigenesis is unclear.
MYC, located at 8q24.1, is a well characterised oncogene, which is involved in early embryogenesis, control of cell growth, cell differentiation and apoptosis (Giaccia and Denko, 2000). Its overexpression or amplification has been found in several cancer types, including breast and prostate cancer (Jenkins et al, 1997;Nupponen et al, 1998;Rummukainen et al, 2001).
We have recently identified two putative target genes for 8q amplification, EIF3S3 and TRPS1 (alias GC79) both located at 8q23 (Nupponen et al, 1999;Chang et al, 2000). Using suppression subtraction hybridisation (SSH), EIF3S3 was found to be overexpressed in breast cancer cell line SK-Br-3, which contains a highlevel amplification at 8q23 -q24 by CGH (Nupponen et al, 1999). Subsequently, it was found that the gene is amplified in about 30 -50% of hormone-refractory, but only in 10 -20% of untreated prostate carcinomas. The amplification of the gene has been found to be associated with high Gleason score . In addition, about 20% of untreated breast cancers contain amplification of EIF3S3 gene (Nupponen et al, 1999). The EIF3S3 gene encodes for a p40 subunit of eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex plays a central role in the translation initiation pathway by binding to 40S ribosomal subunits in the absence of other translational components, and it helps to maintain the 40S and 60S subunits in a dissociated state (Asano et al, 1997). The function of the p40 subunit itself is, however, not known. It has been suggested that also some other translation initiation factors might be involved in the development of malignancies. For example, the overexpression of EIF4E as well as EIF4G1 has been shown to transform normal cells (De Benedetti and Rhoads, 1990;Fukuchi-Shimogori et al, 1997) and increased expression of EIF4E has been found in breast cancer cell lines (Anthony et al, 1996). In addition, a recent cDNA microarray study suggested that upregulation of several genes of translation apparatus is generally involved in metastasis of cancer (Ramaswamy et al, 2003). TRPS1 was recently found to be more expressed in androgendependent than androgen-independent LNCaP prostate cancer cell lines by differential display analysis (Chang et al, 2000). TRPS1 encodes a zinc-finger GATA-type nuclear protein, which has been implicated in apoptosis. Castration leads to increased expression of TRPS1 in rat ventral prostate (Chang et al, 2000(Chang et al, , 2002. Germline mutations in the TRPS1 gene, on the other hand, cause trichorhino-phalangeal syndrome (TRPS) types I and III (Momeni et al, 2000, Lüdecke et al, 2001. In addition, it was reported that xenopus TRPS1 acts as a repressor of the xenopus GATA4 transcription factor (Malik et al, 2001). This suggests that human TRPS1 could be involved in gene regulation of the family of human GATA transcription factors.
In order to evaluate the significance of EIF3S3, MYC, and TRPS1 in breast and prostate cancer, we have analysed the expression and copy number of the three genes in cell lines and tumours using quantitative real-time RT -PCR and fluorescence in situ hybridisation (FISH).

Cell lines and tumour samples
Five prostate cancer cell lines (PC-3, LNCaP, DU145, 22rv1 and NCI-H660) and six breast cancer cell lines (SK-Br-3, ZR75-1, MCF-7, MDA436, EFM19 and T47D) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured under the recommended conditions. Freshly frozen specimens from nine benign prostate hyperplasias (BPH), 35 untreated primary (from 34 prostatectomies and one transurethral resection of the prostate) and 12 locally recurrent hormone-refractory (from transurethral resections of the prostate) prostate carcinomas were obtained from the Tampere University Hospital. The specimens were histologically examined for the presence of more than 60% of cancerous or hyperplastic tissue using haematoxylin and eosinstained slides. The BPH samples were obtained from prostatectomy specimens from cancer patients. However, the specimens were histologically verified not to contain any cancer cells. The TNM distribution of the untreated cases was pT1N0M0, 2; pT2N0M0, 12; pT2N1M0, 2; pT3N0M0, 13; T4NXM0, 1; TXN0M0, 1; TXNXMX, 4. Of the untreated primary carcinomas, 11 were grade I, 16 grade II and seven grade III. Histological grade information was not available for one case. In the hormonerefractory cases, time from the beginning of the therapy to TURP varied from 15 to 60 months. In addition, a tissue microarray (TMA) containing 48 locally recurrent (TURP samples) hormonerefractory formalin-fixed paraffin-embedded prostate carcinomas from the Tampere University Hospital, was constructed according to previously published guidelines (Kononen et al, 1998). The use of clinical tumour material has been approved by the Ethical Committee of the Tampere University Hospital.

RT -PCR
One to three 20 mm frozen sections were cut using a cryotome. The total RNAs were isolated from the sections using Qiagen RNeasy MiniKit (Qiagen Inc., Valencia, CA, USA), and used for the firststrand cDNA synthesis with Superscriptt II reverse transcriptase and oligo d(T) 12 -18 primer according to the manufacturer's protocol (Life Technologies, Gaithersburg, MD, USA). For preparing the standard curve, 5 mg of total RNA from the prostate cancer cell line LNCaP (ATCC, Manassas, VA, USA) was reverse transcribed as described above. After the first-strand cDNA synthesis, serial dilutions (1 : 5) were made to correspond to cDNA transcribed from 500, 100, 20, 4, 0.8 and 0.16 ng of total RNA. Primers for the TRPS1, EIF3S3 and MYC genes were designed with the Primer3 program (available at http://www-genome.wi.mit.edu/cgi-bin/primer3.www.cgi). To avoid amplification of any genomic DNA, the forward and reverse primers for each gene were chosen from different exons. The sizes of the PCR products were designed to be under 400 bp to optimise the RT -PCR measurements. The primer and probe sequences for the genes are given in Table 1.
The PCR reactions were performed with a LightCyclert instrument using the LightCycler -FastStart DNA Master Hybridization Probes Kit (EIF3S3 and MYC) or FastStart DNA Master SYBR Green I Kit (TRPS1) (Roche Diagnostics, Mannheim, Germany). Thermocycling for each reaction was carried out in a final volume of 20 ml containing 2 ml of cDNA sample (or standard), 4 mM MgCl 2 , 0.5 mM of each primer, 0.2 mM of fluorescein and 0.4 mM LC Red640 labelled probes (or SYBR Green I stain in TRPS1 assay), as well as 1 Â ready-to-use reaction mix including Taq DNA polymerase, reaction buffer and dNTP mix. After 10 min of initial denaturation at þ 951C, the cycling conditions of 50 cycles consisted of denaturation at þ 951C for 1 s (EIF3S3 and MYC) or 10 s (TRPS1), annealing at þ 581C for 10 s (EIF3S3 and MYC) or at þ 601C for 7 s (TRPS1) and elongation at þ 721C for 10 s (EIF3S3) or 13 s (MYC) or 7 s (TRPS1). After the PCR reaction and fluorescence measurements, fit point method together with background adjustment was used to determine the cycle in which the log-linear signal was distinguished from the background, and that cycle number was used as the crossing-point value. The software produced the standard curve by measuring the crossing point of each standard and plotting them against the logarithmic values of concentrations ( Figure 1). The expression levels of TRPS1, EIF3S3 and MYC were normalised by the expression level of the housekeeping gene TATA binding protein (TBP), measured as previously described (Linja et al, 2001). The relative expression was illustrated by dividing the EIF3S3, TRPS1 and MYC values with the TBP value, and multiplying by 10. TBP was chosen for the reference gene, because there are no known retro-  pseudogenes for it and its expression is lower than that of many commonly used abundantly expressed reference genes (Bieche et al, 1999). After the PCR, every sample was also run in 1.2% agarose gel electrophoresis to ensure that the right size product was amplified in the reaction. Melting curve analysis was also used to evaluate the quality of the PCR reaction for TRPS1.

Fluorescence in situ hybridisation
Locus-specific PAC probes for human TRPS1 (Chang et al, 2000), EIF3S3 (Nupponen et al, 1999) and MYC (Nupponen et al, 1998) were labelled with digoxigenin-dUTP (Roche Diagnostics), and a pericentromeric probe for chromosome 8 (pJM128) with FITC-dUTP (NEN, Boston, MA, USA), by nick translation. The metaphase preparations from the cancer cell lines were prepared using standard techniques. The dual-colour hybridisation was performed essentially as described previously (Hyytinen et al, 1994). Briefly, the slides were denatured in a 70% formamide/ 2 Â SSC solution (pH 7.0) at 701C for 3 min and dehydrated in an ascending ethanol series. Hybridisation was performed over two nights at 371C. After stringent washes, the slides were stained with antidigoxigenin-rhodamine (Roche Diagnostics) and counterstained with an antifade solution (Vectashied, Vector Laboratories, Burlingame, CA, USA) containing 4,6-diamidino-2-phenylindole (DAPI). The formalin-fixed paraffin-embedded TMAs were pretreated and hybridised as described previously . The FISH signals were scored from nonoverlapping epithelial cells using Olympus BX50 epifluorescence microscope (Tokyo, Japan). Tumours with a tight cluster of signals or at least two-fold higher copy number of the locus-specific probe signals vs centromeric signals or X5 copies of locus specific probe signals were considered to contain a high-level amplification of either gene. Tumours with three to four copies of the gene signals were considered to have a gain of the gene.

Statistical analyses
The associations of the gene copy numbers, tumour types, histological grades and clinical stages with expression levels were calculated with nonparametric Kruskal -Wallis and Mann -Whitney U-tests. Outliers were detected by using extreme studentised deviate method (ESD).

TRPS1, EIF3S3 and MYC gene amplification
Tissue microarrays and FISH were first used to study gene copy number of EIF3S3 and TRPS1 in hormone-refractory prostate carcinomas. High-level amplification (X5 copies) of EIF3S3 and TRPS1 was found in 11out of 40 (28%) and 10 out of 36 (28%) of the cases, respectively. The gain (three to four copies) of EIF3S3 and TRPS1 was found in 19 out of 40 (48%) and 18 out of 36 (50%) of the cases, respectively. In the cases of high-level amplification, the genes were always coamplified. The coamplification was also verified by hybridizing differentially-labelled gene specific probes (biotin labelled EIF3S3 probe and AlexaFluor 594 labelled TRPS1 probe) simultaneously to the TMA. Next, breast and prostate cancer cell lines were analysed for copy numbers of the EIF3S3, MYC and TRPS1 genes. Of the cell lines, the highest copy number of all the genes was found in the breast cancer cell line SK-Br-3, which showed 47 copies of TRPS1 and 21 copies of EIF3S3 and MYC and only one copy of chromosome 8 centromere (Figures 2 and 3). In addition, a high-level amplification (locus/centromere ratio X2) of all three genes was found in MDA436 and PC-3 cancer cell lines. EIF3S3 and MYC were highly amplified also in EFM19 (Figure 3).

Expression of TRPS1, EIF3S3 and MYC
The standard curves in the real-time quantitative RT -PCR assay showed wide dynamic range and the linear relationship between cycle number and fluorescent threshold was strong (r 2 B1). In addition to TBP, the expression of b-actin was measured and used alternatively for normalisation of most of the samples (data not shown). The results were similar with both control genes. Due to the potential problems with the b-actin retropseudogenes, TBP was chosen for normalisation of the whole material. Figure 3 illustrates the relative expression of TRPS1, EIF3S3 and MYC in breast and prostate cancer cell lines. In the breast cancer cell line SK-Br-3, which contains the highest copy number of all the three genes investigated, EIF3S3 was the only gene that showed remarkably high-level (three-to 10-fold compared to the other cell lines) overexpression. The relative expression of TRPS1 was highest in ZR75-1, which contains a loss of the gene compared to centromere copy number. Interestingly, in most breast cancer cell lines, the expression of TRPS1 was high compared to the expression levels of EIF3S3 and MYC. The high expression level of TRPS1 in the breast cancer cell lines has also been observed by Northern and Western blot analyses (Chang et al, unpublished). Figure 4 illustrates the relative expression of TRPS1, EIF3S3 and MYC in BPH (n ¼ 9), untreated primary (n ¼ 30 for TRPS1, n ¼ 35 for EIF3S3 and MYC) and locally recurrent hormone-refractory (n ¼ 12 for TRPS1, n ¼ 11 for EIF3S3 and MYC) prostate carcinomas. The expression of EIF3S3 was, on average, three-fold higher (P ¼ 0.029) in carcinomas than in BPH. There was no difference in the level of expression of EIF3S3 in hormone- refractory and untreated prostate carcinomas. MYC and TRPS1 were expressed in equal levels in BPH, untreated and hormonerefractory tumours. There were no significant associations between histological grade or clinical stage (T3 -T4 and/or N þ and/or M þ vs T1-2N0M0) and the expression of any of the three genes in the untreated tumours.

DISCUSSION
Several oncogenes are activated by overexpression of the gene and one mechanism of the overexpression is amplification of the gene (Brodeur and Hogarty, 1998). Gain or amplification of chromosome 8q is one of the most common chromosomal alterations in breast and prostate cancer (Forozan et al, 1997). However, the target gene of the amplification is still unknown. The genes studied here, EIF3S3, TRPS1 and MYC, have been suggested to be putative target genes in 8q23 -q24 (Jenkins et al, 1997;Nupponen et al, 1999;Chang et al, 2000). In order to evaluate the significance of these genes in breast and prostate cancer, we analysed both the gene copy numbers as well as the expressions of the three genes.  (Nupponen et al, 2000). At the bottom, the order and distances in megabases (Mb) of the genes (marked in red) and STSs (marked in black) from the p-telomere (pter) of the chromosome 8 are shown. We have previously shown that EIF3S3 and MYC are coamplified in about one-third of the locally recurrent hormonerefractory prostate carcinomas . Now, it was found that also EIF3S3 and TRPS1 are coamplified in about 30% of the hormone-refractory prostate tumours. The finding that all three genes are commonly coamplified in the hormone-refractory tumours indicates that the size of the amplicon is large. The TRPS1 and EIF3S3 genes are located about 12 and 11 Mb centromeric from MYC, respectively. The large size and the relatively low copy number of the amplicon have previously been implicated also by CGH and FISH studies (Visakorpi et al, 1995;Cher et al, 1996;Nupponen et al, 1998Nupponen et al, , 2000. The majority of cancer cell lines have been established from metastatic lesions of cancer. They typically contain more chromosomal alterations than primary tumours, and the aberrations are more confined. For example, amplicons are often smaller and the copy numbers higher making the cell lines more informative than the primary tumours for mapping the amplicons . Of the breast and prostate cancer cell lines, however, only SK-Br-3 shows a high-level amplification of two (8q21 and 8q23 -24) independent subarm regions by CGH Nupponen et al, 1999). Of the three genes analysed here, TRPS1 showed the highest copy number, about twice as high as for either EIF3S3 or MYC in SK-Br-3. Together with our previous mapping data (Nupponen et al, 2000), the results indicate that SK-Br-3 contains high copy numbers of all the three genes with low copy numbers of the flanking regions ( Figure 1B). Thus, the amplicon from TRPS1 to MYC, covering a chromosomal region of about 12 Mb, contains, at least, three independent subamplicons in SK-Br-3.
We used real-time quantitative RT -PCR approach to measure the expression of TRPS1, EIF3S3 and MYC. In real-time PCR, the quantification of the template is based on detection of the cycle in which the reaction enters the exponential phase, instead of measuring the amount of end product. Thus, none of the reagents is rate limiting in the reaction at the time of measurement of the fluorescence. Several studies have already shown that real-time RT -PCR is a highly quantitative and reliable method (Bieche et al, 1999;Linja et al, 2001;Savinainen et al, 2002). It is also especially useful in analysis of small tumour samples.
We first analysed the expression of EIF3S3, MYC and TRPS1 in the cell lines. In SK-Br-3, which showed the highest copy number of the genes, the expression of only EIF3S3 was remarkably high. The high-level expression of EIF3S3, shown here by quantitative RT -PCR, confirms our earlier Northern blot data (Nupponen et al, 1999). Surprisingly, the expression of TRPS1, whose copy number was highest of the three genes in SK-Br-3, was lower in SK-Br-3 compared to ZR75-1. The expression of TRPS1 was the highest in ZR75-1, which actually contains a relative loss of the gene. The expression of MYC varied among the cell lines. SK-Br-3 containing the highest copy number of the gene did not show clearly increased expression compared to the other cell lines. The data suggest that of the three genes, EIF3S3 is the most likely target gene of amplification in SK-Br-3.
In the clinical prostate cancer specimens, the level of EIF3S3 expression was significantly (P ¼ 0.029) higher in prostate cancer than in BPH. The data are consistent with our previous analyses by semiquantitative mRNA in situ hybridisation, which suggested that EIF3S3 is expressed more in hormone-refractory prostate carcinomas than in BPH (Nupponen et al, 1999). Here, it was found that the expression of EIF3S3 is increased also in untreated prostate cancers. Somewhat surprising, the expression of EIF3S3 was not higher in the hormone-refractory compared to the untreated tumours, despite the fact that hormone-refractory tumours, in general, contain higher frequency of 8q gain (Visakorpi et al, 1995). The data suggest that EIF3S3 is commonly overexpressed in prostate cancer, and also other mechanisms than gene amplification may lead to the overexpression of the gene. There were no The whiskers display the range. Stars depict the outlier values as estimated by extreme studentised deviate method. The expression of EIF3S3 was significantly (P ¼ 0.029) higher in carcinomas than in BPH. There were no significant differences in the expression levels of TRPS1 and MYC between the tumour groups.
significant differences between the expression of either MYC or TRPS1 in BPH, untreated and hormone-refractory carcinomas, suggesting that alterations in the expression of these two genes are not generally involved in the progression of prostate cancer.
In conclusion, the results indicate that overexpression of MYC and TRPS1 are rare in prostate cancer in vivo. In contrast, the expression of EIF3S3 is increased in prostate cancer. One mechanism for the overexpression of EIF3S3 seems to be amplification of the gene as demonstrated by the cell line SK-Br-3. Analyses of the expression levels of EIF3S3 in large clinical materials are now warranted.


extfieldsofstudy: Medicine Biology

text:
Membrane Topology of the DrrB Protein of the Doxorubicin Transporter of Streptomyces peucetius *

Daunorubicin and doxorubicin, two commonly used anticancer agents, are produced by the soil bacterium Streptomyces peucetius . Self-resistance to these antibiotics in S. peucetius is conferred by the drrA B locus that codes for two proteins, DrrA and DrrB. DrrA is an ATP-binding protein. It belongs to the ABC family of transporters and shares sequence and functional similarities with P-glycoprotein of cancer cells. DrrB is an integral membrane protein that might function as a transporter for the efflux of daunorubicin and doxorubicin. Together, DrrA and DrrB are believed to form an ATP-driven pump for the efflux of these drugs. The drrAB locus has been cloned, and the two proteins have been expressed in a functional form in Escherichia coli . A topological analysis of the DrrB protein was performed using gene fusion methodology. Random and site-di-rected fusions of the drrB gene to lacZ , phoA , or gfp reporter genes were created. Based on the fusion data, a topological model of the DrrB protein is proposed in which the protein has eight membrane-spanning domains with both the N terminus and the C terminus in the cytoplasm. Daunorubicin and doxorubicin, two antibiotics produced by the soil bacterium Streptomyces peucetius, are used in chemo-therapy for treatment of various cancers. Self-resistance in S. peucetius to its own antibiotics results from the action of two proteins, DrrA and DrrB. The DrrA protein belongs to slit Fusion lysates fusion the of activity. Indicated amounts of the protein were analyzed on 8.5% ( (cid:1) -galactosidase fusions), 10% (AP fusions), or 12% (GFP fusions) poly-acrylamide-SDS gels. Western blot analysis was then carried out using either the anti-AP (5 (cid:1) 3 3 (cid:1) Inc.), anti- (cid:1) -galactosidase (5 (cid:1) 3 3 (cid:1) Inc.), or anti-GFP (Abscam, Inc.) antiserum. The intensity of the bands was determined by densitometric analysis using GS-710 Calibrated Imaging Densitometer (Bio-Rad). Anti-AP and anti- (cid:1) -galactosidase antibodies were used at a dilution of 1:1500, whereas anti-GFP antibody was used at a dilution of 1:5000. Detection was done with a chemiluminescence kit from Bio-Rad.

Daunorubicin and doxorubicin, two antibiotics produced by the soil bacterium Streptomyces peucetius, are used in chemotherapy for treatment of various cancers. Self-resistance in S. peucetius to its own antibiotics results from the action of two proteins, DrrA and DrrB. The DrrA protein belongs to the ATP-binding cassette (ABC) 1 family of proteins (1), whereas DrrB bears homology to the membrane components of some bacterial ABC transporters (2). Together, DrrA and DrrB are proposed to form an ATP-dependent efflux pump for the export of these antibiotics (3)(4)(5). Based on the nucleotide sequence analysis, it has been proposed that DrrB belongs to a novel subfamily of ABC transporters (2). This subfamily has been termed ABC-2, and it includes four other members, namely NodJ, KpsM, BexB, and CtrC. The membrane components of members of this subfamily (ABC-2) show significant sequence homology to each other but not to other ABC transporters (2). Within this subfamily, DrrB and NodJ are more closely related to each other than to the other three members. Although DrrAB is a drug export system, the other four transporters form the polysaccharide export systems in Gram-negative bacteria (6 -10). DrrA, the ABC component, shows homology to the ATP binding components of these four systems, however, DrrA also shows homology to other members of the ABC family, including the P-glycoproteins (Pgps) from mammalian tumor cells (2,3). Therefore, it has been proposed that DrrAB and the other four systems, mentioned above, comprise a novel subfamily of permeases that utilize an ABC-type ATPase to provide energy for export.
It is well known that the prolonged use of chemotherapeutic drugs, including daunorubicin and doxorubicin, results in multidrug resistance in cancer cells due to the overexpression of Pgp in their cell membranes (11). Pgp, like DrrA, is a member of the ABC family of transporters (1) and is known to bring about efflux of the anticancer drugs in an ATP-dependent manner. It is a large protein, which consists of two similar halves, each half containing one ABC domain and one membrane domain made of six alpha helices. Because DrrAB and Pgp show sequence homology and carry out a similar function, DrrAB in the producer organism and Pgp in the target cells, the bacterial DrrAB system may serve as a good model system to understand the mechanism and evolution of multidrug resistance in cancer cells. Furthermore, elucidation of the structure and function of DrrAB is expected to help in understanding the function of members of the ABC-2 subfamily and elucidate their evolutionary relationships to other ABC transporters.
The drrAB locus has previously been subcloned in an Escherichia coli expression vector, and the proteins have been identified (4). The expression of the two genes was also shown to confer doxorubicin resistance in a sensitive strain of E. coli (4). DrrA, a peripheral membrane protein, contains one consensus ATP-binding domain. It binds ATP in a doxorubicin-dependent manner. DrrB, an integral membrane protein, is believed to form a channel for the export of daunorubicin and doxorubicin (4). Previous studies have shown that the complex of DrrA and DrrB may have a stoichiometry of two subunits of DrrA and two of DrrB (5). Thus the complex of DrrA 2 B 2 would be structurally similar to a molecule of Pgp as well as to most other ABC transporters that are known to consist of four domains, two ATP-binding and two integral membrane domains, assembled from homo-or heterodimers of each type of domain (2,12).
Even though the catalytic and membrane domains in DrrAB are present on separate subunits, there is evidence for strong interaction between them so that their stability and function are dependent on each other (5). It has previously been shown that DrrB protein is not maintained in the cells in the absence of DrrA (5). Moreover, the ATP-binding ability of DrrA is dependent on the presence of DrrB in the membrane (5). Because the two domains, present on separate molecules, are tightly coupled, this property of the DrrAB system offers an advantage in employing this system to understand interaction between the catalytic and the membrane domains, which will also help in elucidating the interaction of domains within Pgp and shed light on its mechanism.
A basic requirement for understanding interaction between DrrA and DrrB is that we first understand how DrrB is oriented in the membrane, which is the topic of the present study. Gene fusions have been successfully used to determine topology of membrane proteins in E. coli (13)(14)(15). In this study, gene fusions of drrB to the promoterless lacZ or phoA reporter genes were created. Because the phoA gene product alkaline phosphatase (AP) is active only in the periplasm and the lacZ gene product ␤-galactosidase is active only in the cytoplasm, active fusions can be localized to the periplasm or cytoplasm of these cells. A third type of fusion with gfp (green fluorescent protein) was also constructed, especially at regions of anomaly where results with lacZ or phoA were either unclear or contradictory. Based on the prediction tools, such as Kyte-Doolittle and Top-pred, DrrB is predicted to contain at least six hydrophobic regions. The information obtained from the complementary gene fusions and the computer-generated hydropathy plots was used in the present work to determine the approximate locations of periplasmic, transmembrane, and cytoplasmic regions, and a model of DrrB in the membrane was developed. We propose that DrrB consists of eight transmembrane domains with both the N and the C termini of the protein in the cytoplasm.

EXPERIMENTAL PROCEDURES
Strains and Plasmids-Strains, plasmids, antisera, and phages used in this study are listed in Table I. DNA Manipulations-The conditions for plasmid isolation, DNA restriction endonuclease analysis, ligation, transformation, and sequencing have been described elsewhere (16).
Isolation of the Random Gene Fusions-CC118 cells containing the indicated plasmids were grown in 50 ml of LB broth containing 10 mM magnesium sulfate, 0.2% maltose, and 25 g/ml chloramphenicol. Phage carrying the transposon (TnlacZ or TnphoA) was added to 2 ml of culture at a multiplicity of one to one. The tube was then slowly shaken for 15 min at 30°C. 20 tubes each containing 0.9 ml of LB broth were inoculated with 0.1 ml of TnlacZ or TnphoA infection from above. After 12 h of aeration at 30°C, 0.2 ml of culture from each tube was spread on plates containing 20 g/ml chloramphenicol and 100 g/ml kanamycin. The plates were incubated at 30°C for 48 h. After incubation, the plasmids were extracted from the pooled cells via alkaline lysis, essentially as described elsewhere (16). The plasmids extracted from the initial infection were transformed into CC118 cells. The transformed cells were then plated on LB agar plates containing 20 g/ml chloramphenicol, 100 g/ml kanamycin, and 40 g/ml 5-bromo-4-chloro-3-indolylphosphate for phoA fusions or 50 g/ml 5-bromo-4chloro-3-indolyl-␤-D-galactopyranoside (X-gal) for lacZ fusions. The plates were incubated overnight at 37°C. Infections that yielded blue colonies on the appropriate media were selected for further analysis. The fusion junctions were determined by nucleotide sequencing of the plasmid DNA.
Construction of the phoA Reporter Plasmid-A phoA reporter vector was constructed by substituting the lacZ gene in pLMB1069 with the phoA gene. pMLB1069 (Table I) was digested with HindIII and CelII to remove the 3.0-kb lacZ fragment. phoA gene was cloned into the remaining 3.25-kb pBR322 fragment as described below. phoA gene was PCR-amplified using pDxP66 (Table I) as the template. The upstream and the downstream primers containing the HindIII and the CelII site, respectively, had the following sequence: upstream, 5Ј-CCCAAGCTTC-CTGACTCTTATACACAA-3Ј; downstream, 5Ј-CCCGCTCAGCTTATT-TCAGCCCCAGAGCGGC-3Ј. The 1.4-kb-amplified PCR product was digested with HindIII and CelII and ligated to the 3.25-kb pBR322 fragment from above to generate 4.7-kb pMLB1070 containing the promoterless phoA gene.
Construction of the gfp Reporter Plasmid-The drrAB genes were PCRamplified using pDx101 as a template and cloned in front of the gfp gene in pGFPuv (a pUC19-based plasmid, Table I) using the XbaI and the KpnI restriction sites. This generated a clone called pGFPuvAB. The upstream and the downstream primers for amplification of drrAB contained the XbaI (5Ј-CTAGTCTAGACATATGAACACGCAGCCG-3Ј) and the KpnI (5Ј-GGGGGTACCAAGTGGGCGTTCTTGTTGC-3Ј) site, respectively. The drrAB-gfp fragment was then amplified using pGFPuvAB as a template and employing the same upstream primer as used above and a downstream primer containing a HindIII site as follows: 5Ј-CCCAAGCT-TCTCAGTTGGAATTCATTA-3Ј. The amplified DNA fragment was digested with AatII and HindIII. The resulting drrB-gfp fragment was inserted in place of the drrB gene in pDx101 (a pSU2718 clone, Table I). The resulting plasmid, pDxGFP, contains wild type drrA and an in-frame fusion of gfp at the 3Ј-end of the drrB gene.
Construction of the Site-directed Gene Fusions-Different lengths of the drrB gene were inserted into the plasmids pMLB1069 or pLMB1070 to create fusions between drrB and lacZ or phoA, respectively. Plasmid pDx101 was used as a template to PCR-amplify drrA along with different portions of drrB. Each amplification employed the same upstream primer but a different downstream primer. Both primers contained a sequence for HindIII restriction site. The upstream primer containing the sequence 5Ј-GCCGCCAAGCTTCCTCTCCCCGCGCGTTGG-3Ј, corresponds to a region upstream of the plac region of the plasmid pSU2718 so that the amplified fragment contains the lac promoter and the operator region. The downstream primers are shown in Table II. The purified PCR products were digested with HindIII and inserted into the HindIII site of the reporter vector. A total of twelve different site-directed lacZ fusions and seven phoA fusions was made. The subsequent fusion proteins are referred to as "L" or "P" (Table III). The Anti-␤Gal Antibody to ␤-galactosidase 5Ј33Ј Inc. Anti-PhoA Antibody to alkaline phosphatase 5Ј33Ј Inc. Anti-GFP Antibody to green fluorescent protein Abscam, Inc. number next to the notation indicates the last residue in DrrB to which the fusion with ␤-galactosidase or AP was made. gfp fusions at different locations in the drrB gene were made by PCR-amplifying different lengths of the drrB gene from pDxGFP using the same upstream primer but a different downstream primer for each amplification. The upstream primer, containing the sequence 5Ј-TG-GCATGACGACGTCCCCCGG-3Ј, corresponds to a region bearing an AatII site at the 5Ј-end of the drrB gene. The downstream primers corresponded to different regions in drrB and each incorporated a KpnI site (Table II). The amplified products were digested with AatII and KpnI. Fragments containing different lengths of the drrB gene were inserted into the same sites in pDxGFP to create in-frame fusions of gfp to different lengths of the drrB gene. This procedure replaced the wild type drrB gene in pDxGFP with a smaller version of drrB in each case. A total of six different GFP fusions (to residues 84, 117, 232, 236, 245, and 283) in DrrB were created. The subsequent fusion proteins are referred to as "G." Measurement of ␤-Galactosidase or AP Activity-The activity of the fusion proteins was determined by the protocols published earlier (17,18). Briefly, CC118 cells containing the fusion plasmid were grown to mid-log phase and induced with 0.25 mM IPTG for 3 h. The cells were harvested and lysed by a single passage through a French pressure cell at 20,000 p.s.i. The lysed cells were centrifuged at 10,000 ϫ g for 15 min to remove unbroken cells. For ␤-galactosidase, 100 g of protein was added to a tube containing 1 ml of Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , and 50 mM ␤-mercaptoethanol). The tubes were placed into a 37°C water bath. The reaction was started by adding 0.1 ml of o-nitrophenyl-␤-D-galactopyranoside (4 mg/ml in Z buffer) to each tube. Reaction was stopped by adding 0.5 ml of 1 M Na 2 CO 3 once sufficient yellow color was developed. The color was analyzed spectrophotometrically at 420 and 550 nm. Alkaline phosphatase activity was determined by adding 100 g of protein to a tube containing 0.9 ml of 1 M Tris-HCl, pH 8.0. The tubes were placed into a 37°C water bath, and the reaction was started by adding 0.1 ml of 0.4% p-nitrophenyl phosphate to each tube. Once sufficient yellow color was developed, the reaction was stopped by adding 0.1 ml of a 1 M K 2 PO 4 . The reaction was analyzed spectrophotometrically at 420 and 550 nm. The ␤-galactosidase or AP activity units were calculated (18).
Fluorescence Microscopic Analysis of Cells Containing the GFP Fusion Proteins-E. coli TG1 cells containing GFP fusion plasmids were grown till mid-log phase and induced with 0.25 mM IPTG for 3 h. Following induction, the cells were stored at 4°C overnight to enhance GFP folding (19). 50 l of cell suspension was washed with buffer A (25 mM Tris, 20% glycerol (w/v), 2 mM EDTA, 1 mM dithiothreitol, pH 7.5) and resuspended in an equal volume of the same buffer. 10 l of the washed cells was positioned on a microscopic glass side and covered with a coverslip. The samples were initially observed under white light, and the same field was then observed using standard UV filters under a fluorescence microscope (AxiomCam). Observations were made using varying exposure times ranging from 1000 to 5000 ms. Both white light and UV-excited images were recorded for each field.
Fluorometric Analysis of the GFP Fusions-E. coli TG1 cells, containing the appropriate GFP fusion plasmid, were grown till mid-log phase and induced with 0.25 mM IPTG for 3 h at 37°C. The cells were further stored at 4°C overnight to enhance GFP folding (19). Cells were then harvested and lysed by a single passage through a French pressure cell at 20,000 p.s.i. The lysate was then centrifuged at 10,000 ϫ g for 15 min to remove unbroken cells. 100 g of lysate was suspended in buffer A and the volume was made up to 3 ml. Fluorometric measurements of triplicate samples were recorded using the Photon Technology International Flurometer at an excitation wavelength of 485 nm. The emission peaks were recorded at 508 nm. Readings were taken at varying slit widths ranging from 1.0 to 2.5 mm.
Western Blot Analysis of the Fusion Proteins-Cell lysates containing fusion proteins were prepared as described above for the measurement of activity. Indicated amounts of the protein were analyzed on 8.5% (␤-galactosidase fusions), 10% (AP fusions), or 12% (GFP fusions) polyacrylamide-SDS gels. Western blot analysis was then carried out using either the anti-AP (5Ј33Ј Inc.), anti-␤-galactosidase (5Ј33Ј Inc.), or anti-GFP (Abscam, Inc.) antiserum. The intensity of the bands was determined by densitometric analysis using GS-710 Calibrated Imaging Densitometer (Bio-Rad). Anti-AP and anti-␤-galactosidase antibodies were used at a dilution of 1:1500, whereas anti-GFP antibody was used at a dilution of 1:5000. Detection was done with a chemiluminescence kit from Bio-Rad.

Random Gene Fusions
To determine the topology of the DrrB protein in the membrane, random gene fusions of drrB with the reporter gene lacZ or phoA were generated. TnlacZ or TnphoA was used to create transposon-mediated random gene fusions in the clone pDx101 (Table I), which contains both drrA and drrB genes in cis. The two genes in pDX101 are also translationally coupled, exactly as they occur in the wild type situation. Translational coupling of DrrA and DrrB has previously been shown to enhance translation of DrrB about 2-to 3-fold (5). Furthermore, DrrA has been shown to be required for the stability of DrrB (5). Thus, in the present study, we used plasmid pDx101 to provide optimum conditions for expression of DrrB fusion proteins.
The lacZ gene product, ␤-galactosidase, is active in the cytoplasm, thus ␤-galactosidase fusions were used to identify cytoplasmic domains of DrrB. Complementary gene fusions with phoA result in the active gene product, AP, only if these fusions are located in the periplasmic loops of a membrane protein, thus allowing us to determine periplasmic domains of DrrB. Fusions using TnlacZ or TnphoA were isolated by color screen on the plates. Blue colonies on medium containing X-gal (for lacZ) or 5-bromo-4-chloro-3-indolylphosphate (for phoA) indicated active fusions and were selected for further analysis. The fusion junctions were determined by nucleotide sequence analysis and are shown in Table III.
This method generated three active ␤-galactosidase fusions in DrrB at amino acid positions 37, 107, and 126. Quantitation of the fusion proteins was done by Western blot analysis using anti-␤-galactosidase antiserum, followed by a densitometric analysis of the bands (Fig. 1) (Fig. 1 also includes additional data, which will be described later). A band corresponding to ␤-galactosidase itself or the fusion protein was not detected in the control CC118 cells (Fig. 1), whereas protein bands crossreacting with the anti-␤-galactosidase antiserum were seen in cells containing the fusion plasmids (Fig. 1, L37, L107, and  L126). Because ␤-galactosidase is a large protein of 116 kDa, an increase in the size of ␤-galactosidase, when fused with different length fragments of DrrB (total mass of about 30 kDa), could not be clearly seen on the SDS-PAGE gel shown in Fig. 1. Thus, the bands seen in lanes L37, L107, or L126 in Fig. 1 correspond either to the fusion protein or the degradation prod- uct, ␤-galactosidase. Because the fusion proteins are normally subject to degradation, a sum of intensities of the full-length protein, and the degradation product is routinely used to obtain the synthesis units. This was done for quantitation of all fusion proteins in this study. Synthesis units were then used to calculate normalized activity (Table III).
High level of activity of the three ␤-galactosidase fusions (L37, L107, and L126), as shown in Table III, indicates that these fusions might be located in the cytoplasmic regions of DrrB. An active fusion at position 37 indicates that the N terminus of DrrB might be cytoplasmic. Because random fusions using TnlacZ generated many more active fusions in the soluble protein DrrA (not shown) than in DrrB, site-directed fusions at desirable locations within the drrB gene were subsequently created, as described later.
Using TnPhoA, a total of six active random AP fusions in DrrB were isolated. These are located at amino acid positions 66, 91, 151, 189, 270, and 275 in DrrB (Table III). The fusion proteins were quantitated by Western blot analysis using anti-AP antibody, followed by densitometric analysis of the bands (Fig. 2). The synthesis units were then used to calculate the normalized activity of the fusion proteins. The data in Table III show that all the six fusions indicated above contain significant AP activity, thus suggesting that there are at least four potential periplasmic regions in DrrB. The first periplasmic region might encompass active fusions 66 and 91; second 151; third 189; and fourth 270 and 275.
Based on the data obtained from random gene fusions, some tentative conclusions about the topology of DrrB were made. These include: 1) the N-terminal end of DrrB is cytoplasmic; 2) four regions of DrrB are exposed in the periplasmic space, indicating that there may be more than six transmembrane domains; and 3) the C terminus of DrrB may be periplasmic, which may have resulted in the fourth periplasmic region identified by the active AP fusions at residues 270 and 275. These conclusions were later tested by constructing several site-directed fusions in the putative cytoplasmic or periplasmic regions, as described below.

Site-directed Fusions
␤-Galactosidase-Site-directed ␤-galactosidase fusions were created by using pLMB1069 as the fusion vector. This vector TABLE III ␤-Galactosidase, alkaline phosphatase, or GFP activity of E. coli containing drrAB::lacZ, drrAB::phoA or drrAB::gfp fusion plasmids "L" indicates a fusion with Lac Z, "P" indicates a fusion with PhoA, and "G" indicates a fusion with gfp. The number next to the notation indicates the last residue in DrrB to which the fusion was made. ␤-Galactosidase activity and AP activity were determined in E. coli strain CC118. GFP activity was determined in E. coli TG1 strain. CC118 and TG1 are used as controls. contains a promoterless lacZ gene starting at the 11th codon with a HindIII site preceding it. Gene fragments containing drrA and different lengths of the drrB gene were amplified, and the amplified fragments were subcloned into the HindIII site to create in-frame fusions of drrB with lacZ. A total of 11 site-directed ␤-galactosidase fusions were generated at amino acids 15, 44, 84, 110, 115, 161, 164, 220, 232, 245, or 283 in DrrB. These residues were selected such that fusions to residues 15 and 44 would be in the N-terminal cytoplasmic tail; 84 in the first putative periplasmic loop; and fusions ranging from 110 to 245 in the putative cytoplasmic loops 1, 2, or 3. A ␤-galactosidase fusion to the last residue (283) in DrrB was also constructed. The levels of the fusion proteins were analyzed by Western blotting using anti-␤-galactosidase antibody (Fig. 1). All the ␤-galactosidase fusion proteins could be detected (Fig. 1), however L84 was not seen (data not shown). ␤-Galactosidase is known to exhibit anomalous characteristics, such as jamming of the secretion apparatus, if it is translocated through the membrane (20). Furthermore, if secreted into the periplasm, it aggregates and is degraded by the periplasmic proteases (20,21). ␤-Galactosidase activities of the fusion proteins are shown in Table III. An active ␤-galactosidase fusion at amino acid 37 in DrrB had earlier indicated that the N terminus of DrrB is cytoplasmic (Table III). Site-directed fusions at residues 15 and 44 also showed a high level of ␤-galactosidase activity (Table  III), whereas the fusion at residue 84 was found to be inactive (Table III). Together, these results indicate that roughly the first 50 amino acids of DrrB are cytoplasmic, while residue 84 lies in the first periplasmic loop. It is very likely that, due to its periplasmic location, L84 is degraded, thus it could not be detected by Western blot analysis, as mentioned above. All the remaining fusions showed significant ␤-galactosidase activity (Table III), thus confirming the presence of three cytoplasmic loops in DrrB. Surprisingly, L283, a ␤-galactosidase fusion to the last residue in DrrB, also showed high activity (Table III). These data suggest that the C terminus of DrrB may also be located in the cytoplasm and, in this respect, go contrary to the conclusions made earlier based on the active AP fusions at residues 270 and 275.
Alkaline Phosphatase-A phoA fusion vector, pMLB1070, was constructed as described under "Experimental Proce-  (Table III). An AP fusion at amino acid 84 in DrrB showed high activity (Table III), confirming that residue 84 lies in the first periplasmic loop of DrrB. This is consistent with the low activity obtained with a ␤-galactosidase fusion at the same location (Table III). All the other phoA fusions also showed high AP activity (Table III), indicating that these residues lie in the periplasmic domains in DrrB.
Most of the complementary ␤-galactosidase and AP fusions isolated thus far provided data consistent with each other. A few anomalous fusions were, however, isolated. These consisted of L115 and P117 (and L232, L245, and P236). Both ␤-galactosidase and AP fusions at these locations produced active fusions. These two regions thus needed further analysis. Moreover, high activity of a ␤-galactosidase fusion to the last residue 283 (Table III) indicated the cytoplasmic location for the C terminus of DrrB, which would require further verification. Therefore, at such locations where results obtained with ␤-galactosidase and AP either contradicted each other, or required further verification, a third type of fusion with green fluorescent protein (GFP) was created.

GFP Fusions
GFP has recently been used as a reporter molecule for determining the topology of membrane proteins (22). Based on the principle that the chromophore in GFP is active only in a reducing environment, GFP fusions in the cytoplasm are expected to be fluorescent, whereas those in the periplasm are not (22). To determine if GFP fusions will provide reliable results with DrrAB, a GFP fusion to residue 84 in DrrB (G84) was created. Because both ␤-galactosidase and the AP fusion data (Table III) support the periplasmic location of residue 84 in DrrB, a GFP fusion at this location would be expected to be non-fluorescent. G84, when tested under the fluorescence microscope, was indeed found to be non-fluorescent (Fig. 3), as expected. GFP fusions were then created at regions where contradictory results were obtained with AP and ␤-galactosidase fusions. There were two such regions: near residues 115 and 236 in DrrB. GFP fusions to residues 117, 232, 236, 245, or 283 were created, and fluorescence was determined both by examining under the fluorescence microscope and by UV excitation in a fluorometer. Cells containing G117, G232, or G236 were seen to be fluorescent under the microscope, whereas G245 cells were not fluorescent (Fig. 3). Cells containing G283, a GFP fusion to the last residue in DrrB, were also fluorescent under the microscope (Fig. 3). Fluorescence microscope images of cells containing G84, G117, G245, or G283 are shown in Fig.  3. Relative fluorescence of the cells containing G84, G117, G232, G236, G245, or G283 was also measured in a Photon Technology International fluorometer (excitation: 480 nm, emission: 508 nm), as described under "Experimental Procedures." The fusion proteins were analyzed by Western blot analysis using anti-GFP antibodies (data not shown). The synthesis units were used to calculate normalized activity of the GFP fusion proteins (Table III). The data obtained with the fluorometer (Table III) are consistent with the observations made with the fluorescence microscope (Fig. 3). Cells containing G84 and G245 showed the least fluorescence activity, whereas G117, G232, G236, and G283 that resulted in fluorescence under the microscope also showed higher fluorescence by the fluorometer (Table III).
The data obtained with the GFP fusions, G117, G232, and G236 ( Fig. 3 and Table III), support the data obtained with the ␤-galactosidase fusions, L115 and L232 (Table III). These data, thus, clear up the contradiction between ␤-galactosidase and AP fusions in that region (discussed earlier), indicating that these residues indeed lie in the cytoplasmic loops in DrrB. G245 is the only fusion that gave somewhat ambiguous results. The cells containing G245 were non-fluorescent under the microscope (Fig. 3), whereas the specific activity for G245 was found to be intermediate between G84 and other GFP fusions (Table III).
Interestingly, G283, a GFP fusion to the last residue in DrrB, showed high fluorescence activity ( Fig. 3 and Table III). This supports the ␤-galactosidase data obtained at this location and further strengthens the evidence for the cytoplasmic nature of the C terminus of DrrB. When these data are put together with the active AP fusion data at residue 262 (and other AP fusions supporting the presence of the fourth periplasmic region), we can conclude that there are indeed four periplasmic loops in DrrB and, furthermore, that DrrB contains eight transmembrane helices with both the N and the C termini in the cytoplasm. A model based on the data generated from three different kinds of fusions is proposed in Fig. 4. All the different fusions, generated in this study, are marked on the model. DISCUSSION This study investigates the topology of the membrane component DrrB of the doxorubicin transporter DrrAB of S. peucetius. The DrrB protein occurs in a complex with the peripheral membrane component DrrA. DrrA has been shown to bind ATP and is expected to function as the catalytic subunit in this transporter (4,5). Even though DrrA and DrrB form separate subunits, they exhibit a tight biochemical coupling (5). This coupling results from a specific interaction between the proteins and is essential for their stability and function (5). To understand how DrrA and DrrB interact and regulate the function of each other, it is important to understand how DrrB is localized in the membrane, which was the objective of the present research. In this study, we have made use of the gene fusion methodology employing two commonly used probes, alkaline phosphatase and ␤-galactosidase, to determine topology of the DrrB protein expressed in E. coli. To verify the phoA and lacZ fusion data, we also made use of green fluorescent protein fusions at certain regions. Even though DrrB is originally a Streptomyces protein, our data suggest that the expression of DrrA and DrrB in E. coli results in a functional complex conferring doxorubicin resistance in this host. Moreover, DrrA expressed in E. coli binds ATP in a doxorubicin-dependent manner only if DrrB is also present, suggesting a specific interaction between DrrA and DrrB and between the proteins and doxorubicin (5). Thus, topology of DrrB in E. coli is expected to reflect the topology in S. peucetius. In several other instances, topology of heterologous prokaryotic and eukaryotic membrane proteins has been studied by gene fusion methodology in E. coli cells (23)(24)(25).
The computer-generated hydropathic profile of DrrB shows at least six regions of 18 or more amino acid residues with a hydropathic index greater than 1.5, suggesting the presence of six or more membrane-spanning ␣-helices in DrrB. Fusion data obtained in the present article suggest that DrrB traverses the membrane eight times, and it contains four periplasmic loops and three cytoplasmic loops with both the N terminus and the C terminus in the cytoplasm. A topological model for DrrB based on these results (discussed below) is presented in Fig. 4.
Three active ␤-galactosidase fusions in the N terminus of the protein at residues 15, 37, and 44 indicate that DrrB has a long cytoplasmic N-terminal tail. Even though these three fusions, by themselves, do not conclusively demonstrate the cytoplasmic location of the N-terminal tail, when these data are combined with the results from other fusions, such as P66, P84 or L84, and P99 (Fig. 4), it can be stated with a fair amount of certainty that the N terminus of DrrB is cytoplasmic and that the first periplasmic loop of DrrB lies between residues 66 and 91. An active ␤-galactosidase fusion, L283, at the end of DrrB suggests that the C-terminal of DrrB is also directed inside the cytoplasm. The orientation of the C terminus was further verified by creating a GFP fusion at residue 283. Cells containing G283 were seen to be fluorescent under the microscope, confirming the cytoplasmic location of the C-terminal end of DrrB. Furthermore, the presence of four periplasmic loops in DrrB was indicated by active AP fusions at residues 66, 84, and 91 (loop I); 137, 144, and 151 (loop II); 189 and 197 (loop III); 262, 270, and 275 (loop IV) (Fig. 4), whereas the cytoplasmic loops were shown by active ␤-galactosidase fusions at residues 15, 37, and 44 (N-terminal tail); 107, 110, and 115 (loop I); 161 and 164 (loop II); 220, 232, and 236 (loop III); and 283 (C-terminal tail) (Fig. 4). The current topological model of DrrB in Fig. 4 is based on the weight of the majority of the data. The results of two different types of fusions in this study were generally consistent with each other. Some anomalous fusions were isolated; however, a third type of fusion with GFP at these locations helped resolve this contradiction, as described under "Results." Some ambiguous data have also been reported in other studies (24).
Most members of the ABC superfamily are predicted to contain either six or twelve transmembrane ␣ helices, which in several instances has been shown to be the case experimentally (26,27). Several exceptions to this rule, however, do exist in literature. For example, Tap1, a eukaryotic ABC transporter associated with antigen presentation, has been shown to contain eight transmembrane domains so that the complex of Tap1 and Tap2 contains more than 12 ␣ helices (23). Some other eight transmembrane domain-containing proteins include NixA, the nickel transporter in Helicobacter pylori (28), and HlyB, the hemolysin transporter, of E. coli (29). ProW, which forms the membrane component of the ProU locus, has been shown to contain seven transmembrane ␣ helices instead of the predicted six helices (30).
To date, crystal structure information for three ABC transporters is available. These include E. coli MsbA (31), E. coli BtuCD (32), and V. cholerae MsbA (33). These structures have provided valuable insights into the topology of membrane components of the ABC transporters as well as suggested possible modes of their interaction with the nucleotide binding components. However, crystal structure or other topological information for members of the ABC-2 family is so far not available. The topology of the DrrB protein reported in this study constitutes first such report. Previous studies from this laboratory have clearly shown that DrrA and DrrB form a complex and biochemically regulate the stability and function of each other (4,5). A better understanding of the topology of DrrB will help us elucidate the domains involved in direct interaction between DrrA and DrrB. This study suggests that DrrB contains a long N-terminal cytoplasmic tail, three cytoplasmic loops, and a short cytoplasmic C-terminal tail, which may form the potential sites of interaction with DrrA. Future studies will analyze the roles of these domains. FIG. 4. A model for the topology of DrrB in the membrane. This model is based on data obtained from three different kinds of reporter gene fusions (shown in the model) constructed in DrrB. According to this model, the DrrB protein contains eight transmembrane ␣ helices. Both the N terminus and the C terminus of DrrB are directed in the cytoplasm. The protein has a long N-terminal cytoplasmic tail of about 50 amino acids. The suggested placement follows the positive-inside rule (26) and includes an equal number of intramembrane positive and negative charges. Acidic (squares) and basic (diamonds) residues are indicated.