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J. Biol. Chem., Vol. 280, Issue 18, 17961-17968, May 6, 2005
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From the
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom and the Department of Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, United Kingdom
Received for publication, December 1, 2004 , and in revised form, February 3, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Although only three tat genes are believed to be important for Tat operation in Escherichia coli, the encoded proteins appear to form at least two types of complex. Tagging of the TatC subunit led to the purification of a TatABC complex with an estimated mass of 
600 kDa according to gel filtration (14), although single particle electron microscopy suggests that detergent may account for much of the mass, and a size of 370 kDa was determined by blue native gel electrophoresis (15, 16). The TatABC subunits have a combined molecular mass of 57 kDa, and this TatABC complex should therefore contain multiple copies of each subunit. Within the complex, TatB and TatC are present in stoichiometric amounts, and they operate even when fused together, indicating that the TatABC complex contains several copies of this heterodimeric unit. The TatA subunit is particularly intriguing; it is homologous to TatB and likewise found in the TatABC complex (14), but the vast majority does not co-purify with the TatBC subunits (14) and instead forms large, homo-oligomeric complexes. In one report, overexpression of TatA alone led to the characterization of a relatively discrete, 460-kDa complex (17). Other work involving coordinated expression of TatABC indicated that TatA forms a wide range of complexes with sizes ranging from <100 to over 500 kDa (16). Estimates of subunit abundance show TatA to be in large molar excess over the other subunits in E. coli (18). Mechanistic studies using in vitro assays show that precursor proteins initially bind to the TatBC subunits (19, 20), strongly suggesting that this heterodimer forms the substrate binding site. In thylakoids, the TatA homolog (Tha4) was only found cross-linked to the other subunits in the presence of an energized membrane and ongoing protein transport (21). This has led to proposals that the binding of substrate triggers the recruitment of the homo-oligomeric Tha4 (or TatA) complex to generate the full translocation system. In this context, it should be noted that the thylakoid and E. coli systems may differ in terms of complex organization. Tha4 was not found associated with the Hcf106/cpTatC complex in blue native gels of solubilized thylakoids (19), whereas part of the TatA pool is tightly associated with TatBC, and this complex is indeed unstable in its absence, with TatB being subjected to proteolysis (22). Either the Hcf106-cpTatC complex differs from its bacterial TatABC counterpart or the Tha4 subunit is lost during analysis.
Another potentially important aspect of the Tat system is its location in bacteria. In a recent report (23), the TatABC subunits were tagged with green fluorescent protein (GFP) derivatives, and the proteins were found to accumulate as punctate regions at the cell poles. This was particularly evident with the TatB-GFP fusion, although some cells also showed a concentration of TatA-GFP toward the poles, whereas a halo was visible in others. TatC-GFP was only detected at low levels, but a polar localization was suggested by the preliminary data. The authors proposed that the core TatABC complex, responsible for binding of substrate, is restricted to these regions, and such a sequestering of the Tat system may have important implications for the mechanism. Here, we have fused GFP and its cyan/yellow derivatives to TatA, -B, and -C. We show that the TatB and TatC fusion proteins are active and able to assemble into TatABC complexes. Although substantial amounts of TatB-GFP are found at the cell poles, we show that both TatA-GFP and TatC-GFP fusions exhibit a predominantly peripheral localization. Because TatC is invariably found in close association with TatB, the data are consistent with Tat-dependent translocation occurring uniformly throughout the plasma membrane.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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tatABCDE has been described before (25). All of the constructs described below were expressed using the arabinose-inducible pBAD24 vector, and arabinose-resistant derivatives were used as described (14, 25). E. coli was anaerobically grown at 37 °C in Luria broth (LB) as described in Ref. 26, supplemented with glycerol (0.5%), trimethylamine N-oxide (0.4%), and ammonium molybdate (1 µM). Medium supplements were used at the following final concentrations: ampicillin, 100 µg/ml; arabinose, 200 µM unless stated otherwise. Generation of GFP/CFP/YFP Fusions—TatA, TatB, and TatC proteins fused to a fluorescent protein were constructed in pBAD-ABCs (14) by creating a unique restriction enzyme site (detailed in Table I) at the 3'-end of the tatA, tatB, or tatC gene, using the QuikChangeTM mutagenesis system (Stratagene) according to the manufacturer's instructions. This system was used to create the same or compatible restriction enzyme sites at either end of the required fluorescent protein, allowing ligation of the fluorescent protein fragment at the 3'-end of the tat gene.
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Expression and Purification of Tat Complexes—Cells expressing these plasmids were induced using 0.1 mM arabinose unless otherwise stated, and membranes were isolated as described (14). The membranes were then solubilized in digitonin, and the Tat complexes were purified by ion exchange chromatography, Streptactin affinity chromatography, and gel filtration (Superose 6HR; Amersham Biosciences), essentially as described in Ref. 14, except that the mono-Q step used in this previous study was found to be unnecessary.
Cell Fractionations—Cells were grown anaerobically on LB with supplements as detailed above in the presence or absence of arabinose (as stated throughout); for studies on tat mutants, the cells were grown aerobically on LB. Periplasm and spheroplasts were prepared by the EDTA/lysozyme/cold osmoshock procedure (14). Spheroplasts were lysed by sonication, and intact cells and cellular debris were removed by centrifugation (5 min at 10,000 x g). Membranes were separated from the cytoplasmic fraction by centrifugation (30 min at 25,0000 x g). Protein concentration was determined using a BCA-linked assay (Pierce). Protein fractions were separated on a 10% nondenaturing polyacrylamide gel and stained for trimethylamine N-oxide reductase (TorA) as described (12).
Quantitation of Fluorescence—Fluorescence was quantified using an SLM-Aminco 8100 series 2 spectrometer set to photon-counting mode (integration time, 1 s; 200-µl sample volume; 3-mm glass cuvette). Detection of fluorescence from the different fluorophores used the following settings: GFP excitation, 475 nm; emission, 515 nm, YFP excitation, 488 nm; emission, 527 nm; CFP excitation, 434 nm; emission, 477 nm. Strains containing parental vectors were used as controls for background fluorescence levels.
Confocal Microscopy, Fluorescence Recovery after Photobleaching (FRAP) Measurements, and Confocal Images—After growth and arabinose induction as appropriate, liquid cultures of E. coli were spotted onto LB agar plates and left for 15–30 min to allow the liquid medium to be absorbed by the agar. Small blocks of agar with the cells adsorbed onto the surface were placed in the well of a custom built temperature-controlled sample holder and covered with a glass coverslip. All measurements were carried out at 37 °C. Fluorescence micrographs and FRAP measurements were performed using a laser-scanning confocal microscope (Nikon PCM2000) equipped with a 100-milliwatt argon laser and a x60 oil immersion objective lens. Excitation was with the 488-nm line from the laser. A 505-nm dichroic mirror was used, and GFP fluorescence was selected with a 515-nm interference filter. For high resolution images, a 20-µm pinhole was used, and images were averages of 10 scans. For FRAP measurements, cells were grown for 3 h prior to measurement in the presence of cephalexin (30 µg/ml), which inhibits cell division and thus produces elongated cells suitable for FRAP (27). One-dimensional FRAP measurements were carried out essentially as previously described (28). A 50-µm pinhole was used. Cells aligned in the Y direction were selected, and the bleach was carried by scanning the confocal laser spot at full power for 2–3 s across the cell in the X-direction. The laser intensity was then decreased by a factor of 32 with neutral density filters, and XY image scans were recorded at 4-s intervals. For both quantitative measurements and FRAP, fluorescence values were extracted from images using Optimas 5.2 image analysis software.
Confocal images shown in Figs. 6 and 7 were taken using a Leica DMRE confocal microscope equipped with the Leica confocal system TCS SP2 with an acousto-optical beam splitter and a 100-milliwatt argon laser. Excitation and emission wavelengths were as follows: CFP excitation, 458 nm; emission, 450–600; GFP excitation, 488; emission, 500–530 nm; YFP excitation, 514 nm; emission, 545–590 nm. Stacks of individual images were taken, and each line was scanned four times and averaged (line average).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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20-fold overexpression of Tat subunits compared with wild-type levels. The vector was expressed in a tatABCDE background in all cases, and a description of the tagged subunits is given in Table I. The experiments described below generally use only one form of tagged TatA/TatB/TatC in each case, but it should be noted that the various tags (GFP/CFP/YFP) behaved identically in comparative tests (data not shown).
Fig. 1 shows immunoblots of cells expressing TatAYFPBC, TatABCFPC, and TatABCGFP. The data show the expression of the full fusion proteins of the predicted sizes of 36.6, 45.4, and 55.9 kDa, respectively. In the case of TatC-GFP, the full-length fusion is stable, and there is no evidence for proteolytic clipping. With TatB-CFP, the full-length fusion predominates, although there are indications of smaller polypeptides in the region of 27–45 kDa. Because these are detected by the TatB antibodies but not by GFP antibodies (not shown), these proteins originate from digestion of the C-terminal region of the GFP domain. Free TatB is not generated to any detectable extent. In the case of TatA-YFP, the full fusion protein is again detected by immunoblots, but a series of smaller polypeptides is evident, and, importantly, some mature size TatA is apparent, presumably resulting from proteolytic clipping of the fusion protein. This is significant in terms of determining whether the fusion is active, as explained below. Fluorescence emission spectra were obtained for cells expressing TatA/B/C fusions that contain the various XFP tag, and all were found to exhibit typical emission spectra with the vast majority of the fluorescence localized in the membrane fraction (data not shown).
Evidence That the TatB-XFP and TatC-XFP Fusion Proteins Are Active—We tested whether the TatA/B/C fusion proteins were active, using an assay for the export of a known periplasmic Tat substrate, TorA. Operons encoding TatA-YFP, TatB-CFP and TatC-GFP were induced using 0.1 mM arabinose, after which the cells were fractionated to generate periplasmic, cytoplasmic, and membrane fractions (P, C, and M) as detailed in the legend to Fig. 2. These fractions were analyzed using a native gel assay for TorA activity (12). In the wild-type cells (wt), the periplasm contains substantial amounts of TorA activity, although some activity is also evident in the cytoplasmic fraction; this is commonly observed with this substrate, since export is rarely complete (e.g. Refs. 14 and 15). In the tatABCDE cells (tat), TorA activity is found exclusively in the cytoplasm, as expected. The remaining panels show that TorA is exported in strains expressing TatA-YFP, TatB-CFP, and TatC-GFP. Export is as efficient as in wild-type cells, and this strongly suggests that the TatB-CFP and TatC-GFP fusion proteins are functional because we do not observe mature size TatB or TatC in the blots shown in Fig. 1. However, Fig. 1 showed that mature size TatA is present after expression of TatA-YFP, and this makes it difficult to determine whether the fusion protein is active. The fusion proteins are overexpressed by a factor of 10–20-fold relative to the low levels of TatABC present in wild-type cells, and if only a small proportion of expressed protein is mature size, this may be sufficient to support Tat-dependent export activity.
The XFP Fusion Proteins Are Selectively Assembled into TatABC Complexes, but "Free" TatB-CFP Is Also Present in Unexpectedly High Quantities—To further investigate the nature of the fusion proteins, we tested whether they can be incorporated into TatABC complexes using the purification protocol developed for the isolation of wild-type TatABC complexes (14, 15). Membranes were isolated from cells expressing fusion proteins, after which they were solubilized in digitonin, and the Tat complexes were purified using ion exchange chromatography (Q-Sepharose) followed by Streptactin affinity chromatography that involves recognition of the Strep II tags on TatC or TatC-GFP. This procedure results in effective purification of the TatABC complex (14), and it is invariably found that the bulk of TatB and TatC are found in the Streptactin eluate, whereas the vast majority of TatA is found in the Streptactin wash fraction with only a minor proportion present in the TatABC complex (14, 15).
Data for cells expressing TatAYFPBC are shown in Fig. 3. The top two panels show immunoblots of the Streptactin wash and elution fractions (and of the Q-Sepharose eluate loaded onto the column; lane Q), using a mixture of antibodies to TatA and TatB (A) or using antibodies to the Strep II tag on TatC (B). The data show that the bulk of TatA-YFP is present in the wash fractions as expected, but a significant quantity is found in the elution fractions, confirming that the protein does assemble into the TatABC complex. The efficacy of the column is evident from the observation that almost all of the TatB is found in the elution fractions, indicating that most of the TatABC complex had bound to the affinity column. The Strep II blot shows that the vast majority of TatC is present in the elution fractions, as expected from previous data (14, 15). Fig. 3C shows a silver-stained gel of the fractions, in which TatA-YFP is present as a 40-kDa band, and the TatB and TatC proteins, which have similar mobilities of 28 and 30 kDa, respectively, co-migrate in this instance. The data show that the TatAYFPBC is purified to homogeneity (the asterisks indicate a doublet of nonspecific higher molecular mass bands that are present in all of the lanes).
Similar analyses were performed on cells expressing TatAB-CFPC, and membranes were again prepared, solubilized, and subjected to the same purification steps (Fig. 4). Fig. 4A shows a blot using TatA antibodies, in which the subunit is again found in the wash fractions but also in the elution fractions, reflecting partial localization in a TatABC complex. In Fig. 4C, the TatC protein is found almost entirely in the elution fractions, confirming that the TatABC complex binds efficiently to this affinity column. In Fig. 4B, TatB-CFP is found in the same elution fractions, confirming assembly into the TatABC complex, but it is notable that a large proportion of the TatB-CFP is found in the wash fractions. This pool of TatB-CFP is clearly not associated with TatC and is thus exhibiting aberrant characteristics, since overexpression of wild-type TatABC to the same extent yields a TatABC complex that contains essentially all of the TatC, the vast majority of TatB, and a small fraction of the total TatA pool (14, 15).
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In both Figs. 3 and 4, it is notable that the purified complexes contain only the tagged Tat subunit (TatA-YFP or TatB-CFP) with no significant levels of truncated protein evident in either case. Thus, although some truncated protein is present in the immunoblots of TatB-CFP expression (Fig. 1), the full fusion proteins are preferentially incorporated into TatABC complexes. This provides strong evidence that the tagged TatB subunit is indeed functional. It is encouraging that the TatA-YFP is also incorporated into this complex, but it should also be emphasized that TatA also forms large homo-oligomers, and we have no direct means of assessing whether these are correctly formed with the TatA-YFP fusion.
Data for cells expressing TatABCGFP are shown in Fig. 5. In this case, the proximity of the Strep II tag to GFP, rather than the C terminus of TatC, leads to inefficient binding to the Streptactin column, and it did not prove possible to purify the complex to homogeneity. However, the immunoblots of wash and elution fractions clearly show that TatABCGFP is incorporated into the TatABC complex together with TatA and TatB. Again, only a minor part of the TatA pool is present in the elution fractions, whereas a substantial fraction of total TatB and a similar fraction of the available TatCGFP (about half) is found in the elution fractions. The TatABCGFP elutes as a 650-kDa complex during gel filtration chromatography, indicating assembly into a complex of approximately the correct dimensions (not shown).
Confocal Microscopy Confirms That the TatA-XFP Is Primarily Localized in the Cell Periphery, whereas TatB-XFP Is Enriched at the Poles—Fig. 6 shows confocal images of tatABCDE cells expressing TatA, TatB, and TatC fusion proteins after induction with arabinose for 3 h. The TatA-GFP image demonstrates that the fluorescence is visible as a halo in the cells, although some fluorescence is also apparent in the central regions, corresponding to fluorescence from above and below the focal plane. The TatA-GFP is distributed evenly throughout the periphery in most cells, but a minority of cells exhibit punctate regions of fluorescence, sometimes at the poles of the cells. These data resemble those obtained in an earlier study (23) using TatA-GFP.
The TatB-CFP image shows cells expressing TatABCFPC, and the fluorescence is localized in a very different manner. In most cells, a substantial CFP signal is present at the poles of the cells in large punctate areas; these are readily apparent in some cells in this image, but it should be emphasized that other cells are mostly above or below the focal plane, and similar polar accumulations were apparent when the remaining cells were analyzed by altering the plane of focus (not shown). Once again, these data resemble those obtained in a previous study using TatB-GFP (23).
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To understand in greater detail the basis for the polar regions of TatB-XFP fluorescence, we analyzed the distribution of one such fusion under differing levels of expression. We initially attempted to study the protein when expressed at levels equivalent to those of TatB in wild-type cells, but the fluorescence was too low to monitor (not shown). This probably reflects the low abundance of the Tat machinery in wild-type E. coli cells, since the Tat pathway is used for the export of only a minority of periplasmic proteins. To circumvent this problem, we studied the localization of TatB-GFP at different stages of induction. In the absence of arabinose, very little expression occurs using the pBAD24 vector (29), and the zero time image in Fig. 7 shows essentially no signal emanating from TatB-GFP; the low level green is autofluorescence due to the confocal settings used to capture images at a wide range of fluorescence intensities in the remaining images, and an identical signal is obtained using wild-type cells (not shown). The remaining images illustrate the appearance of TatB-GFP after induction with arabinose for 90, 120, 150, and 300 min. After 150 min, many of the cells are highly induced, and they exhibit the type of polar localization shown above in Fig. 6; after 300 min, the cells are fully induced, and virtually all of the cells show this pattern. However, the important images are those taken after 90- and 120-min induction, where the cells contain much lower levels of TatB-GFP fluorescence. Only a few highly induced cells exhibit any form of polar localization, and the vast majority instead contain TatB-GFP that is uniformly distributed around the cell periphery. These data clearly indicate that the polar localization appears only after the synthesis of substantial amounts of protein, and we interpret these data to mean that more natural levels of TatBGFP have no propensity to accumulate at the poles.
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Similar results were obtained with TatB-GFP (data not shown), and Fig. 9 shows a similar type of experiment carried out on cells expressing TatC-GFP, which was shown above to have a relatively uniform distribution in most cells. The fluorescence from these cells is far weaker, but even with these lower resolution images, Fig. 9 shows that after bleaching (region denoted with a thick white bar), the fluorescence rapidly recovers over the next few seconds. This confirms that the TatC-GFP is highly mobile.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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370-kDa TatABC complex (14–16) and separate homo-oligomeric TatA complexes that have been reported to be relatively homogeneous in one report (17) and highly heterogeneous in others (14, 16). However, the mode of interaction between these complexes is unclear. It has been reported (23) that these complexes differ in terms of cellular location, with GFP-tagged TatB found exclusively at the cell poles and tagged TatA distributed more uniformly around the periphery of the cells. This would have important mechanistic implications, and in the present report we have analyzed similar constructs with TatA, TatB, and TatC each linked to GFP, CFP, and YFP. Before discussing the locations of these fusion proteins, it is important to consider whether they are active. With the TatB-XFP and TatC-XFP fusions, the evidence strongly indicates that these constructs are indeed active, because very little proteolytic "clipping" is observed in immunoblots of whole cell extracts, and the full-length fusions are effectively incorporated into purified TatABC complexes. The data for the TatA-XFP fusions, however, are less clear. Immunoblots clearly indicate that a small percentage of the protein is clipped to yield mature size protein, and, given that the fusion protein is overexpressed about 20-fold relative to wild-type TatA levels, this level of mature size protein would almost certainly be able to support Tat-dependent translocation activity. Although we have shown that TatA-XFP can be incorporated into the TatABC complex, which suggests that it may function correctly in this context, the bulk of TatA is found in separate homooligomeric complexes that are believed to be essential for the generation of the active translocation complex (1, 2, 16, 18). We have no means of assessing whether the TatA-XFP fusions function correctly in such complexes. The previous study on Tat-GFP fusions (23) found that expression of TatA, -B, and -C fusions complemented tat mutants lacking the relevant subunit, using growth on SDS as a criterion for Tat-dependent export activity. However, this study did not consider the issue of proteolytic clipping, although smaller polypeptides were apparent. Neither that study nor the present one used export assays that are in any sense quantitative, and this issue therefore awaits resolution in future studies.
Regarding the localization of Tat-XFP constructs, our data on TatA-XFP constructs are similar to those of the previous study (23), and the data all point to a rather uniform distribution of TatA-XFP throughout the plasma membrane, with a minor proportion of protein located in punctate areas (in the region of 24%, according to the data shown in Fig. 1). Our data with TatB-XFP constructs also resemble those obtained in Ref. 23, and we likewise find that the protein is found at the poles of the cells. However, quantitation of the images shows that a significant proportion of this fusion protein (about half) is present in the periphery.
Our data with TatC-XFP differ substantially from the findings described in Ref. 23. TatC-GFP was expressed at low levels in this study and could not be analyzed using the expression system used for TatA and TatB fusions. Some evidence pointed to a polar localization for TatC-GFP, although it was emphasized that the data were less clear than those obtained with the other fusions. Here, we have clearly shown that the distribution of TatC-CFP and TatC-GFP is very different from that of TatB-XFP constructs. Although some polar localization of TatC-XFP is observed, the bulk of protein is found in the form of a definite halo around the cells, indicative of a relatively uniform distribution throughout the plasma membrane. These confocal data demonstrate that the polar TatB-XFP fusions are not complexed with TatC in the stoichiometric ratio found for wild-type TatBC (14, 15). It is also significant that a similar localization of TatB-GFP is observed when the cells are induced for shorter time periods, suggesting that the polar localization may stem from either longer periods of synthesis or the increased levels of protein or both. Our FRAP data indicate that there is no rapid exchange of TatA-GFP or TatC-GFP between the punctate areas and the remainder of the plasma membrane. This is consistent with the idea that the protein in the punctate areas is not physiologically active.
Biochemical studies provide additional data on this issue. Affinity chromatography by virtue of the Strep II tag on TatC results in the partial or complete purification of complexes that contain both TatA and TatB-XFP, clearly demonstrating that TatB-XFP and TatC form a complex. This tagged TatABC complex is of the correct size, and, as detailed above, it is almost certainly active. The logical interpretation is that the halo of TatC-XFP is correctly complexed with TatB (and TatA). In turn, this strongly implies that the polar areas of TatB-XFP are not complexed with TatC, and they must, therefore, be inactive because there is clear evidence that TatB and TatC function together (14, 15). We cannot rule out the possibility that these areas represent stored forms of TatB that may be physiologically relevant. However, the confocal data do demonstrate that both of the primary types of Tat complex (TatA homo-oligomer and TatABC core) are uniformly distributed around the cell periphery. The one caveat is that we do not understand the significance of the smaller punctate areas that are often observed with the TatA and TatC fusion proteins.
The biochemical data are consistent with the imaging work. The Streptactin affinity column is highly effective at binding the Strep II-tagged TatC, which is accordingly found almost exclusively in the elution fractions. In previous studies, we have shown that the majority of TatB elutes in the same fraction as TatC, in keeping with the tight binding of this subunit to TatC within the TatABC complex (14, 15). However, our data with TatABCFPC-expressing cells show that a much higher proportion of TatB-CFP does not co-purify with TatC and instead elutes in the wash fractions of the Streptactin column. Overall, it appears that TatB-XFP fusions are expressed at abnormally high levels relative to TatC, since similar studies on wild-type TatB and TatC do not indicate the presence of comparable quantities of unbound TatB (14). The TatB-XFP that is not complexed to TatC is, in our opinion, likely to correspond to the polar TatB-XFP that does not co-localize with TatC in the imaging studies.
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FOOTNOTES |
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¶ Supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust.
|| To whom correspondence should be addressed. Tel.: 44-2476-523557; Fax: 44-2476-523568; E-mail: Crobinson{at}bio.warwick.ac.uk.
1 The abbreviations used are: Tat, twin arginine translocation; GFP, green fluorescent protein; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; XFP, green/cyan/yellow fluorescent protein; FRAP, fluorescence recovery after photobleaching; TorA, trimethylamine N-oxide reductase; Strep, Streptactin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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