A systematic survey of in vivo obligate chaperonin-dependent substrates
2010; Springer Nature; Volume: 29; Issue: 9 Linguagem: Inglês
10.1038/emboj.2010.52
ISSN1460-2075
AutoresKei Fujiwara, Yasushi Ishihama, Kenji Nakahigashi, Tomoyoshi Soga, Hideki Taguchi,
Tópico(s)Enzyme Structure and Function
ResumoArticle1 April 2010free access A systematic survey of in vivo obligate chaperonin-dependent substrates Kei Fujiwara Kei Fujiwara Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, FSB401, Kashiwa, Chiba, Japan Search for more papers by this author Yasushi Ishihama Yasushi Ishihama Institute for Advanced Biosciences, Keio University, Daihoji, Tsuruoka, Yamagata, Japan PRESTO, Japan Science and Technology Agency, Chiyodaku, Tokyo, Japan Search for more papers by this author Kenji Nakahigashi Kenji Nakahigashi Institute for Advanced Biosciences, Keio University, Daihoji, Tsuruoka, Yamagata, Japan Search for more papers by this author Tomoyoshi Soga Tomoyoshi Soga Institute for Advanced Biosciences, Keio University, Daihoji, Tsuruoka, Yamagata, Japan Search for more papers by this author Hideki Taguchi Corresponding Author Hideki Taguchi Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, FSB401, Kashiwa, Chiba, JapanPresent address: Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259-B56 Nagatsuta, Midori-ku, Yokohama 226 8501, Japan Search for more papers by this author Kei Fujiwara Kei Fujiwara Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, FSB401, Kashiwa, Chiba, Japan Search for more papers by this author Yasushi Ishihama Yasushi Ishihama Institute for Advanced Biosciences, Keio University, Daihoji, Tsuruoka, Yamagata, Japan PRESTO, Japan Science and Technology Agency, Chiyodaku, Tokyo, Japan Search for more papers by this author Kenji Nakahigashi Kenji Nakahigashi Institute for Advanced Biosciences, Keio University, Daihoji, Tsuruoka, Yamagata, Japan Search for more papers by this author Tomoyoshi Soga Tomoyoshi Soga Institute for Advanced Biosciences, Keio University, Daihoji, Tsuruoka, Yamagata, Japan Search for more papers by this author Hideki Taguchi Corresponding Author Hideki Taguchi Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, FSB401, Kashiwa, Chiba, JapanPresent address: Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259-B56 Nagatsuta, Midori-ku, Yokohama 226 8501, Japan Search for more papers by this author Author Information Kei Fujiwara1, Yasushi Ishihama2,3, Kenji Nakahigashi2, Tomoyoshi Soga2 and Hideki Taguchi 1 1Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, FSB401, Kashiwa, Chiba, Japan 2Institute for Advanced Biosciences, Keio University, Daihoji, Tsuruoka, Yamagata, Japan 3PRESTO, Japan Science and Technology Agency, Chiyodaku, Tokyo, Japan *Corresponding author. Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259-B56 Nagatsuta, Midori-ku, Yokohama 226 8501, Japan. Tel.: +81 45 924 5785; Fax: +81 45 924 5785; E-mail: [email protected] The EMBO Journal (2010)29:1552-1564https://doi.org/10.1038/emboj.2010.52 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chaperonins are absolutely required for the folding of a subset of proteins in the cell. An earlier proteome-wide analysis of Escherichia coli chaperonin GroEL/GroES (GroE) interactors predicted obligate chaperonin substrates, which were termed Class III substrates. However, the requirement of chaperonins for in vivo folding has not been fully examined. Here, we comprehensively assessed the chaperonin requirement using a conditional GroE expression strain, and concluded that only ∼60% of Class III substrates are bona fide obligate GroE substrates in vivo. The in vivo obligate substrates, combined with the newly identified obligate substrates, were termed Class IV substrates. Class IV substrates are restricted to proteins with molecular weights that could be encapsulated in the chaperonin cavity, are enriched in alanine/glycine residues, and have a strong structural preference for aggregation-prone folds. Notably, ∼70% of the Class IV substrates appear to be metabolic enzymes, supporting a hypothetical role of GroE in enzyme evolution. Introduction A protein must fold into the correct tertiary structure after emerging from the ribosome (Anfinsen, 1973). In the cell, protein folding is assisted by a variety of chaperones, which are also involved in other multiple cellular processes associated with the conformational changes of proteins, such as stress responses (Hartl and Hayer-Hartl, 2009). In addition to the 'classical' functions, chaperones are proposed to promote protein evolution by buffering the destabilization of proteins caused by harmful genetic mutations (Jenkins et al, 1986; Van Dyk et al, 1989; Rutherford and Lindquist, 1998; Fares et al, 2002; Tokuriki et al, 2009). The absolute requirement of chaperones for cellular functions and for protein evolution might account for the fact that an organism lacking chaperones has not been identified. The bacterial chaperonin GroEL and its co-factor GroES are the only indispensable chaperones for the viability of Escherichia coli (Fayet et al, 1989; Horwich et al, 1993). GroEL consists of two heptameric rings of 57 kDa subunits, and provides binding sites for non-native substrate proteins. GroEL can bind about half of the soluble E. coli proteins in their denatured states in vitro (Viitanen et al, 1992). The co-chaperonin GroES, a dome-shaped heptameric ring of ∼10 kDa subunits, caps GroEL in the presence of adenine nucleotides, forming a central cavity that can accommodate substrate proteins up to ∼60 kDa in size (Xu et al, 1997; Sakikawa et al, 1999; Hartl and Hayer-Hartl, 2002; Fenton and Horwich, 2003). GroEL mutations that affect either the cavity environments or the encapsulation of substrates within the cavity are lethal in E. coli, indicating the in vivo indispensability of the GroEL–GroES cavity (Koike-Takeshita et al, 2006; Tang et al, 2008). An important goal in chaperonin biology is to identify a subset of obligate GroEL/GroES (hereafter, GroE) substrates that absolutely require GroE for folding in cells. Precise identification of the obligate GroE substrates should contribute to the identification of a distinctive role for GroE among chaperones, reveal the structural features of the obligate substrates, and shed light on the role of GroE in protein evolution. (Ewalt et al, 1997; Houry et al, 1999; Kerner et al, 2005). One approach to identifying obligate GroE substrates is a detailed analysis of the phenotypes of GroE-depleted cells. Such analyses identified DapA and FtsE as obligate GroE substrates in the cell lysis and filamentous morphology phenotypes, respectively (McLennan and Masters, 1998; Fujiwara and Taguchi, 2007). Although a detailed phenotypic analysis can precisely identify obligate GroE substrates, this approach is limited, in that the substrates can only be identified one by one, and only in the cells with experimentally tractable phenotypes. Another approach to substrate identification is a proteome-wide analysis. Hundreds of GroEL substrates have been identified using mass spectrometry (MS) (Kerner et al, 2005; Chapman et al, 2006). In particular, Kerner et al (2005) have identified ∼250 substrates that interact with GroE in E. coli, and categorized them into three classes depending on their enrichment in the GroE complex: Class I substrates as spontaneous folders, Class II as partial GroEL-dependent substrates, and Class III as the potential obligate GroE substrates. Notably, ∼84 Class III substrates are estimated to occupy ∼80% of the available GroEL capacity in the cell. This classification was primarily based on the proteomics of the GroE interactors. However, except for DapA, GatY, MetK, ADD, and YajO, which were verified as requiring GroE for folding (Kerner et al, 2005), the in vivo GroE dependency of the Class III substrates has not been tested. In fact, our earlier phenotype analysis revealed that one of the Class III proteins, ParC, was functional even under GroE-depleted conditions (Fujiwara and Taguchi, 2007), raising the possibility that the predicted Class III proteins are not necessarily obligate substrates of GroE. Here, we comprehensively assessed the GroE dependency of the Class III substrates in cells by proteomics, metabolomics, and individual characterization of the GroE requirement (GR), and found that around 40% of the Class III substrates lack GroE dependence. Thus, only ∼60% of the Class III proteins, combined with at least eight substrates not previously categorized as Class III substrates, were GroE dependent in E. coli. As the in vivo GroE obligate substrates were not limited to the Class III substrates assigned previously, we have named the verified in vivo obligate GroE substrates as Class IV substrates. We found that Class IV substrates have a limited range of molecular weights and isoelectric points, are aggregation prone, and are structurally distinct. The features of Class IV substrates are consistent with a possible role of GroE in facilitating the evolution of enzymes involved in metabolic reactions and pathways. Results Proteomics of the soluble fraction in GroE-depleted E. coli In GroE-depleted cells, the known obligate GroE substrates either aggregate (e.g. MetK) or are degraded (e.g. DapA, FtsE, and GatY) (McLennan and Masters, 1998; Kerner et al, 2005; Fujiwara and Taguchi, 2007). Thus, we expected that the abundance of other potential GroE obligate substrates would also be reduced in the soluble fraction of GroE-depleted cells. A proteome-wide analysis of the soluble fraction of GroE-depleted cells was therefore conducted to find candidate in vivo obligate GroE substrates. We used a conditional GroE expression strain, MGM100, in which the GroE promoter had been replaced by the arabinose-inducible (and glucose-inhibitable) BAD promoter (Figure 1A; McLennan and Masters, 1998). For proteomics, cells were subjected to a 2-h depletion of GroE in LB medium, during which the level of GroEL was reduced to 50% in MGM100 cells (Figure 1B, top). The tendency for the reduction was also obvious when we compared the emPAI values in MGM100 with those in MG1655 under glucose conditions (Figure 1B, middle). In addition, this reduction was not observed during glucose growth in MG1655 cells (Figure 1B, bottom). Collectively, the drastic reduction of many proteins during glucose growth in MGM100 cells was caused by the GroE depletion. We note that expression levels of a significant number of proteins were increased in the GroE-depleted cells (coloured blue), including methionine biosynthetic enzymes, such as MetE (Horwich et al, 1993; Chapman et al, 2006; Masters et al, 2009), and certain chaperones, such as DnaK and SecB (Supplementary Figure S2), possibly reflecting a stress response induced by GroE depletion. As the levels of chaperones other than GroE were not reduced, the changes we observed can be attributed to the reduced amount of GroE with confidence. The proteome data were used to roughly choose candidate GroE substrates by the following criteria. First, the proteins with soluble abundance that was reduced during depletion to <50% of that found during arabinose growth in MGM100 (as a genetic control) were chosen. The 347 proteins chosen by the first criterion contained many false positives due to a sugar-associated reduction in their levels, and thus were filtered by a second criterion, in which the proteins with expression in MGM100 during glucose growth reduced to <50% of that found during glucose growth in MG1655 were chosen. The cutoff value of 50% was set to minimize false negatives, as the highest solubility of known in vivo obligate GroE substrates was 46%, as found with MetK in both cases. Using the genetic and sugar controls, 252 proteins among the detected 986 proteins met both criteria for rough candidate GroE substrates. The candidates included all of the in vivo tested obligate GroE substrates (MetK, GatY, and DapA), except for FtsE, which was not quantified in the proteomics, confirming the reasonableness of the selected threshold for the growth conditions used here. Then, the percentages of the candidates showing protein reductions in each of the GroEL substrate classes defined by Kerner et al (2005) were calculated. As shown in Figure 1C, 8% of Class I, 32% of Class II, and 56% of Class III substrates were reduced in the GroE-depleted cells. The fraction of class members showing reduced protein amounts increased with the degree of GroEL dependence. It is also noteworthy that about 44% of the Class III substrates (24 out of the 43 quantified proteins) did not meet our criteria for GroE obligate substrates. This again suggests that a significant fraction of Class III members, in addition to ParC, are not obligate substrates in vivo. About 40% of Class III substrates do not require GroE for solubility To assess whether ∼40% of the Class III substrates are not actually obligate GroE substrates, we developed methods, independent of proteomics, to verify their GR for solubility. The methods also aimed to comprehensively cover all of the Class III proteins suggested by Kerner et al (2005), as our proteomics detected only about half of the Class III substrates (43 of 84). We induced the expression of individual target proteins from a tac promoter in MGM100 cells after a 2-h depletion of GroE, and measured their total amounts and the proportion in the soluble fraction. The obligate GroE substrates would be expected to become insoluble or be degraded. To validate the strategy, we examined proteins for which the status of GroE dependence had already been verified: Enolase (spontaneously folding in vitro, Class I), GatD (partial GroE-dependent folding in vitro, Class II), MetK, FtsE, DapA (the in vivo obligate GroE substrates, Class III), and ParC (assigned as Class III, but functional in the GroE-depleted cells) (McLennan and Masters, 1998; Kerner et al, 2005; Fujiwara and Taguchi, 2007). Enolase, GatD, and ParC were soluble irrespective of the GroE level, whereas MetF, MetK, FtsE, and DapA aggregated in the GroE-depleted cells (Figure 2A). The disappearance or persistence of the bands under GroE-depleted conditions was almost complete, enabling easy and clear discrimination. Note that DapA was degraded in the earlier report (Kerner et al, 2005), probably reflecting a difference in the expression levels. Except for the difference of whether DapA was degraded or aggregated, the present results are consistent with the earlier data on the GroE dependency, suggesting that the overexpression strategy reflects the in vivo GroE dependency. Figure 2.Solubility of E. coli GroE substrates overexpressed in GroE-normal and -depleted cells. (A) Verification of the method. Solubilities of the proteins, for which the GR had been shown in earlier studies, after overexpression in GroE-normal and -depleted cells. Only the corresponding bands are shown. T, total lysates; S, soluble fractions; GroE-normal, cells cultivated with 0.2% arabinose; GroE-depleted, cells cultivated with 0.2% glucose. I, II, and III in the parentheses indicate the GroE substrate classes. The GroE dependency of ENO, GatD, MetK, and DapA in vitro and in vivo was shown earlier (McLennan and Masters, 1998; Kerner et al, 2005). FtsE in vitro and in vivo, and ParC in vivo were shown in our earlier studies (Fujiwara and Taguchi, 2007). (B) Solubility of substrates essential for the viability of E. coli after overexpression in GroE-normal and -depleted cells. All of the proteins, except for DadA, TrmD, and YbjS, were stained and visualized by Coomassie Brilliant Blue. DadA, TrmD, and YbjS were each fused to an HA tag at the C-terminus and were detected by immunoblotting. GR, GroE requirement. (C) Class III substrates were divided depending on the GroE dependency. Class III substrates with and without GroE dependency were termed Class III+ and Class III–, respectively. Download figure Download PowerPoint Next, we extended the method to all of the essential genes in the three GroEL substrate classes, to test the GR for solubility in cells. The solubility of the essential Class I and Class II proteins (proteins with low expression levels were not measured) was independent of the GroE levels (Figure 2B), confirming that Classes I and II were not dependent on GroE for folding. The GroE-independence of Ppa (Class I), GatD, LpxA, HemL, and FabG (Class II), which were candidates of GroE substrates identified by our proteomics, indicated that not all of the candidates predicted by the proteomics are in vivo obligate GroE substrates. For the Class III substrates, the essential proteins with low expression (DadA, TrmD, and YbjS) were fused with an HA tag sequence at the C-terminus to facilitate western blotting. Seven proteins (MetK, FtsE, and DapA (Figure 2A), Asd, HemB, DadA, and YbjS (Figure 2B)) were found to be aggregated in the GroE-depleted cells. In contrast, six other essential Class III proteins (ParC, FolE, Rsd, SuhB, YcfP, and TrmD) were soluble even after GroE depletion (Figure 2A and B). Taken together, the results showed that the in vivo obligate GroE substrates were enriched in Class III, but not in Class I and II proteins. More importantly, the results also indicated that approximately half of the Class III proteins did not require GroE for solubility, as already suggested by the above proteomics data. Depending on the in vivo GR for solubility, we divided the Kerner's Class III substrates into Class III+ (plus; GroE dependent for solubility in vivo) and Class III− (minus; not GroE dependent for solubility in vivo). Finally, we tested all of the remaining Class III substrates, including the proteins not detected in our proteomics survey, except for the plasmid origin protein Ypt1, for an in vivo GR for solubility. A total of 59 Class III proteins were tested by an overexpression-Coomassie staining method (Supplementary Figure S3) or the HA tag fusion expression-immunoblotting method (Supplementary Figure S4A). In some cases, the proteins that were expressed in the GroE-normal cells had entirely disappeared from the GroE-depleted cells. As the disappearance after GroE depletion of obligate substrates, such as DapA, GatY, and FtsE, had previously been reported, we regarded the seven Class III proteins that disappeared in the depleted cells as Class III+ substrates. As the overexpression of some proteins resulted in aggregate formation even in the GroE-normal cells, we reduced their expression to that which 'leaked' from the tac promoter in the absence of inducer (Supplementary Figure S4B and C). After confirmation of the method using the several substrates validated above (Supplementary Figure S4B), 11 HA tag-fused Class III proteins (Supplementary Figure S4C) were tested. When all of the solubility assays for the Class III substrates were combined (Figure 2A and B; Supplementary Figures S3 and S4), Class III was divided into 49 Class III+ (7 in Figure 2; 20 in Supplementary Figure S3; 22 in Supplementary Figure S4) and 34 Class III− substrates (Figure 2C). When measured by proteomics data, we found that all of the Class III+ substrates (except for AraA and Nfo) were specifically reduced in the GroE-depleted cells (Supplementary Figure S5). As the proteomics method did not involve overexpression, we conclude that the aggregation found in the GroE-depleted cells in which overexpression was used was not merely the consequence of the overexpression of the individual candidate substrates. Regarding the Class III− proteins, ∼80% of the Class III− members quantified in the proteomics were not reduced. However, the amounts of the remaining proteins, such as SuhB, TrmA, and YcfP, were specifically reduced, prompting us to test whether the Class III− members are physiologically functional when synthesized in the absence of GroE. Activities of Class III− proteins in GroE-depleted cells In contrast to the Class III+ proteins, the Class III− proteins not only retained their solubility in the absence of GroE, but the amounts of 2/3 of them remained unchanged, as assessed by proteomic measurements. As the ultimate test of GroE dependence is whether it is required for functionality, we tested whether the Class III− enzymes that retained their solubility were also functional. Our first test used metabolomics, using a capillary electrophoresis (CE)-MS system (Ishii et al, 2007). We quantified the concentrations of 187 metabolites, and found that 9 metabolites were significantly increased and 20 were reduced when the GroE was depleted (Supplementary Table S1). Dysfunction of an enzyme should cause an accumulation of its precursor and/or a reduction in the product of the catalysed reaction (Supplementary Figure S6A). Indeed, 5-aminolevulinate and N-acetylornithine, the precursors of the reactions catalysed by the Class III+ proteins HemB and ArgE, respectively, accumulated in GroE-depleted cells (Supplementary Figure S6B; Supplementary Table S1). Likewise, S-adenosylmethionine and N-acetylglucosamine, the products of the Class III+ enzymes MetK and NagZ, respectively, were decreased. Thus, the metabolomics data support the dysfunction of several Class III+ substrates (Supplementary Figure S6C; Supplementary Table S1). For Class III−, we found that the intracellular thymidine concentration was not decreased in the GroE-depleted cells (Figure 3A), implying that FolE, one of the essential Class III− proteins, is functional in the cells, as FolE-defective cells only grow in thymidine-supplemented rich medium (El Yacoubi et al, 2006). Figure 3.Functions of essential Class III− proteins in GroE-depleted cells. (A) The concentration of thymidine in GroE-normal and -depleted cells. Thymidine is known to be reduced in FolE-depleted cells. (B) Inositol monophosphatase activity of SuhB in cell lysates of the SuhB-overexpressing GroE-normal and -depleted cells. Relative activities were determined in the cell lysates under each GroE condition, in the presence or absence of 1 mM IPTG. (C) Time course of tRNA(Leu) methylase activity of TrmD in GroE-normal (+GroE) and -depleted (−GroE) cell lysates. + IPTG: TrmD was induced by 1 mM IPTG; -IPTG: cells without IPTG treatment. Download figure Download PowerPoint Second, the enzymatic activities of the Class III− proteins were directly assayed in the E. coli lysates. The activities of two essential Class III− proteins, an inositol monophosphatase, SuhB, and a tRNA methylase, TrmD, were measured in the lysates after the overexpression of Class III− proteins. The enzymes were active in both the GroE-depleted and -normal cells (Figure 3B and C), indicating that the enzymes are both soluble and functional in the GroE-depleted cells. Among the Class III− substrates, only four proteins (ParC, FolE, SuhB, and TrmD) are essential. Although the functionality of the remaining Class III− proteins was not tested, we could show that at least all of the essential Class III− proteins were physiologically functional even in the GroE-depleted cells, further supporting the validity of the Class III+ and III− grouping. Identification of other in vivo obligate GroE substrates that were not previously assigned as Class III substrates After the complete survey of the Class III substrates, we searched for other novel GroE obligate substrates besides the identified Class III proteins. The metabolomics data showed that the level of O-phosphoserine, the product of a Class II substrate, SerC, was reduced in the GroE-depleted cells, suggesting that SerC was not active in the cells (Supplementary Figure S6C; Supplementary Table S1). In addition, the proteomics data also suggested that SerC was reduced in the supernatant of the GroE-depleted cells (Figure 4A). We then tested the solubility of SerC by the overexpression method, and found that it was aggregated in the GroE-depleted cells (Figure 4B), strongly suggesting that the in vivo obligate GroE substrates are not confined to the identified Class III substrates. Figure 4.Obligate GroE substrates that were not derived from Class III substrates. (A) Expression levels of Class II substrates and proteins that were not identified as GroEL interactors, but were significantly reduced in GroE-depleted cells. Expression levels were determined by emPAI (Ishihama et al, 2005). ND, not detected. (B) Solubility of the proteins referred to in (A) in GroE-normal and -depleted cells. The number in parentheses indicates the GroE substrate class of the protein. 'O' indicates that the protein had not appeared among the GroEL interactors. GR, GroE requirement. (C) Homologs of Class III and II proteins. Percents indicate the amino-acid sequence identities between the indicated proteins pairs. Class IV (red), III− (blue), and all E. coli cytosolic proteins (green). (D) Solubility of the homologs of Class III+ substrates in GroE-normal and -depleted cells. (E) Class IV substrates defined as the in vivo obligate GroE substrates. In total, 49 Class III+ substrates, combined with eight newly identified obligate substrates, were classified as Class IV substrates. Download figure Download PowerPoint Other putative GroE substrates were also identified. First, using the proteomics data, we verified the GR for a dozen drastically reduced proteins in the GroE-depleted cells. A severe cutoff value, ∼12% of solubility, was introduced to choose the candidates, as the solubility of SerC was 12%. These included three Class II proteins (KdsA, PyrC, and NuoC) and six proteins that had not appeared among the GroEL interactors (GuaC, ThiL, SdaB, PyrD, NemA, and GdhA). The solubility assays of these overexpressed candidate proteins revealed that all of the Class II candidates and two of the other six candidates (PyrD and GdhA) behaved as in vivo GroE obligate substrates (Figure 4B; Supplementary Figure S7). We also identified the homologs of Class III+ substrates on a database, and evaluated the GR for their solubility in GroE-depleted cells. Several Class III+ proteins share homology (Figure 4C). These proteins include two pairs, DapA and NanA (28% identity), and FrdA and SdhA (32% identity). YjhH (a homolog of DapA and NanA), NadB (a homolog of FrdA and SdhA), and TatD (a homolog of YcfH and a Class II protein YjjV) were examined by the solubility assay. Among these proteins, TatD and YjhH required GroE for solubility, whereas NadB did not (Figure 4D). Taken together, we found eight new in vivo GroE obligate substrates not previously id
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