Analysis of the RNA degradosome complex in Vibrio angustum S14 Melissa A. Erce, Jason K. K. Low and Marc R. Wilkins Systems Biology Laboratory, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia Introduction Post-transcriptional control of gene expression is an important regulatory mechanism, as the length of time that a transcript is available for translation limits the expression of its protein product. Messenger RNA (mRNA) half-lives can differ by as much as two orders of magnitude within a single cell. In Escherichia coli, where the average message has a half-life of about 5 min, individual mRNA half-lives can be as short as several seconds or as long as 1 h [1]. Through the reg- ulation of mRNA stability, patterns of protein synthe- sis in the cell can be modulated in response to changes in growth conditions [2]. In addition, selective mRNA decay results in the differential expression of gene products in some polycistronic mRNAs [3]. The multi- Keywords degradosome; microdomains; RhlB; RNase E; two-dimensional Blue Native-SDS ⁄ PAGE Correspondence M. Wilkins, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia Fax: +61 2 9385 1483 Tel: +61 2 9385 3633 E-mail: m.wilkins@unsw.edu.au (Received 28 June 2010, revised 28 September 2010, accepted 22 October 2010) doi:10.1111/j.1742-4658.2010.07934.x The RNA degradosome is built on the C-terminal half of ribonuclease E (RNase E) which shows high sequence variation, even amongst closely related species. This is intriguing given its central role in RNA processing and mRNA decay. Previously, we have identified RhlB (ATP-dependent DEAD-box RNA helicase)-binding, PNPase (polynucleotide phosphory- lase)-binding and enolase-binding microdomains in the C-terminal half of Vibrio angustum S14 RNase E, and have shown through two-hybrid analy- sis that the PNPase and enolase-binding microdomains have protein-bind- ing function. We suggest that the RhlB-binding, enolase-binding and PNPase-binding microdomains may be interchangeable between Escherichi- a coli and V. angustum S14 RNase E. In this study, we used two-hybrid techniques to show that the putative RhlB-binding microdomain can bind RhlB. We then used Blue Native-PAGE, a technique commonly employed in the separation of membrane protein complexes, in a study of the first of its kind to purify and analyse the RNA degradosome. We showed that the V. angustum S14 RNA degradosome comprises at least RNase E, RhlB, enolase and PNPase. Based on the results obtained from sequence analyses, two-hybrid assays, immunoprecipitation experiments and Blue Native- PAGE separation, we present a model for the V. angustum S14 RNA degradosome. We discuss the benefits of using Blue Native-PAGE as a tool to analyse the RNA degradosome, and the implications of microdomain- mediated RNase E interaction specificity. Structured digital abstract l A list of the large number of protein–protein interactions described in this article is available via the MINT article ID MINT-8049250 Abbreviations BACTH, bacterial adenylate cyclase two-hybrid; CTH, C-terminal half; DMP, dimethyl pimelimidate.2HCl; NTH, N-terminal half; PNPase, polynucleotide phosphorylase; PVDF, poly(vinylidene difluoride); RhlB, ATP-dependent DEAD-box RNA helicase; RNase E, ribonuclease E. FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5161 enzyme RNA degradosome is involved in the steady- state regulation of transcripts in E. coli. Apart from its role in mRNA degradation, the degradosome is also responsible for the processing of 5S ribosomal RNA and tRNAs, as well as the degradation of tmRNAs [4–8]. The principal proteins comprising the degradosome include the 3¢ to 5¢ exoribonuclease polynucleotide phosphorylase (PNPase), the ATP-dependent DEAD- box RNA helicase (RhlB) and the glycolytic enzyme enolase [9–12]. The scaffolding for the degradosome is provided by RNase E. One of the largest proteins found in E. coli, RNase E, is 1061 amino acids in length [10,13,14]. It is defined by two functionally distinct domains of approximately equal size – the N-terminal half (NTH, residues 1–498) and the C-terminal half (CTH, residues 499–1061) [15,16]. The catalytic activity of E. coli RNase E resides within its globular NTH. The sequences within this essential region are highly conserved amongst eubacteria [17]. In contrast, the CTH lacks sequence conservation, has little structural character, has no known catalytic func- tion [17,18] but provides the scaffolding for the recruit- ment of the degradosome through short recognition motifs [19]. The RNase E CTH contains two regions which can bind RNA and interact with substrates such as the 9S precursor for 5S ribosomal RNA [18]. Apart from the canonical degradosome components, polyphosphate kinase, poly(A) polymerase, ribosomal proteins and chaperone proteins GroEL and DnaK have been found to be present in the degradosome, but in substoichiometric amounts [11,20,21]. The molecular interactions of RNase E with other proteins of the RNA degradosome are proposed to be mediated by short microdomains of 15–40 amino acids on RNase E [22]. These microdomains exhibit higher amino acid sequence conservation than the rest of the RNase E CTH. It is through these regions that specific molecular interactions are believed to occur to direct RNase E function, and hence confer adaptive reorgani- zation of protein complex formation [17,22–25]. In order to study RNase E and its interaction part- ners, it would be useful if it could be isolated. The purification and characterization of RNase E have pro- ven challenging [15], and obtaining an amount suffi- cient for analysis is even more difficult. Methods for its purification as part of the RNA degradosome, as well as the reconstitution of an active degradosome through the purification of its individual components, have been developed [11,26–29]. Large-scale prepara- tions have involved purification under denaturing con- ditions; others have involved the overexpression of individual components and subsequent reconstitution of the degradosome. Recently, a method for the preparation of a recombinant degradosome has been described [30]. Co-immunoprecipitation methods have also been used to study the RNA degradosome. This technique is especially useful when studying bacteria in which the introduction of genetic tags is difficult [31]. These methods, however, require a large amount of starting material. Furthermore, as they often require the overexpression and ⁄ or tagging of the RNA degradosome components, the complexes formed may not truly represent what is occurring in the cell. In view of these limitations, we decided to use Blue- Native PAGE (BN-PAGE), a ‘charge shift’ technique employed in the separation of mitochondrial mem- brane proteins and complexes [32,33]. This technique has been successfully employed recently to study the E. coli complexome [34]. It requires far less starting material and, when used in conjunction with immuno- blotting and ⁄ or mass spectrometric analysis, may provide a better view of the dynamic nature of the RNA degradosome. In this study, we characterized the RNA degradosome from an environmental species: the marine heterotro- phic, Gram-negative bacterium, Vibrio angustum S14 [35]. V. angustum S14 is a model organism for the study of starvation, as it exhibits remarkable physio- logical changes and increased mRNA stability during carbon starvation [36,37]. We have demonstrated pre- viously, using two-hybrid screening, that RNase E from V. angustum S14 contains sites for interaction with enolase and PNPase [38]. Here, we use a combi- nation of proteomic techniques, such as BN-PAGE, co-immunoprecipitation and tandem MS to identify the components of the RNA degradosome in this organism. We also show, using two-hybrid analysis, that the CTH of RNase E from V. angustum S14 inter- acts with RhlB. Results RhlB binds to V. angustum S14 RNase E and PNPase Previously, we predicted that the RNase E CTH from V. angustum S14 possessed interaction sites for PNPase and enolase, and demonstrated these interactions through two-hybrid analysis [38]. We also predicted that RhlB should bind to RNase E at residues 719– 753. Here, we undertook two-hybrid analysis to test this; the results of this analysis and our previous analy- sis [38] are shown in Table 1. This two-hybrid system introduces two proteins of interest fused to the T18 and T25 domains of Bordetella pertussis adenylate cyclase into E. coli cya ) on plasmids. When physically RNA degradosome complex in Vibrio angustum S14 M. A. Erce et al. 5162 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS Table 1. Escherichia coli two-hybrid analysis results. Incubations were at 25 °C. The horizontal axis indicates the protein fused with the T18 domain and the vertical axis indicates the pro- tein fused with the T25 domain. Empty cells indicate crosses not carried out in this study. ‘++’ denotes strong interactions, ‘+’ denotes moderate interactions and ‘)’ denotes weak ⁄ no interactions. pKT25 pUT18 ⁄ pUT18C 1 2 3 4 5 6 7 8 9 10111213 E. coli RhlBsite 684–784 V. S14 RhlBsite 714–758 V. S14 CTH 526–1094 E. coli PNPsite 844–1061 V. S14 PNPsite 1015–1094 E. coli Enosite 833–851 V. S14 Enosite 885–909 E. coli PNPase V. S14 PNPase E. coli RhlB V. S14 RhlB E. coli enolase V. S14 enolase T18 a T18C b T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C A E. coli RhlBsite )) )) ++ ++ ++ + )) )) B V. S14 RhlBsite )) )) ++ + ++ ++ )) )) C V. S14 CTH + c ++ c ) c ) c ) c + c ++ ++ ++ ++ ) c + c ++ c ++ c D E. coli PNPsite ) c + c ) c ) c )) )) E V. S14 PNPsite ) c ) c ) c + c )) )) ) c ) c ) c ) c F E. coli Enosite ) c ) c ) c ) c )) )) + c + c + c + c G V. S14 Enosite ) c ) c ) c ) c )) )) + c ++ c + c + c H E. coli PNPase )) )) + c ) c ++ c ) c + c ) c ) c ) c ) c ) c ) c ++ c ) c ++ c ++++) I V. S14 PNPase )) )) ++ c ) c + c ) c + c ) c ) c ) c ) c ) c ) c ++ c ) c ++ c + ) + ) J E. coli RhlB ++ ++ ) ++ ++ ++ )) )) )) )) +++)) ++ ++ ++ + K V. S14 RhlB + ))++ ++ ++ ) ) )) )) )) )) )) )+ )) L E. coli enolase )) )) ++ c ) c )) ) c ) c ) c ) c + c + c ++ c ++ c ++ c + c M V. S14 enolase )) )) ++ c ++ c )) ) c ) c + c + c ++ c + c ++ c ++ c ++ c ++ c a Two-hybrid result between bait fused with T25 on its N-terminus and prey fused with T18 on its C-terminus (pUT18). b Two-hybrid result between bait fused with T25 on its N-terminus and prey fused with T18 on its N-terminus (pUT18C). c Two-hybrid result that has been published previously [38]. M. A. Erce et al. RNA degradosome complex in Vibrio angustum S14 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5163 separated, the T18 and T25 domains are inactive, but the interaction of the hybrid proteins results in the functional complementation of adenylate cyclase in E. coli cya ) and the subsequent expression of the lac operon [39]. RhlB demonstrated positive interactions with the CTH of V. angustum S14 RNase E (grids 3K and 11C). In order to better define the region of interac- tion of RNase E with RhlB, we tested the putative RhlB-binding domain of V. angustum S14 RNase E (residues 719–753, plus five amino acid residues flank- ing on either side); it was found to interact with RhlB (grid 2K and its reciprocal cross in 11B). Interest- ingly, this region was also capable of interacting with E. coli RhlB (grid 2J and its reciprocal cross in 10B). It should be noted, however, that, when the T18 domain was fused to the C-terminus of the V. angustum S14 RNase E RhlB-binding site (residues 714–758), the interaction with RhlB was negative, probably because of a difference in conformation (grid 2K) or because the T18 domain occluded the RhlB-binding site. Previous reports have suggested that RhlB and PNPase can interact independently of RNase E in E. coli [16]. Our two-hybrid analysis fur- ther confirmed this (grid 8J and its reciprocal cross in 10H). We also observed an interaction between V. angustum S14 PNPase and RhlB; however, this was weaker than that seen in E. coli (grid 11I, C-terminal fusion of the T18 domain). We did not expect PNPase to interact with the RhlB-binding site, and we observed negative interactions for this (grids 1H, 1I, 2H, 2I, 8A, 8B, 9A and 9B). Negative interactions were also seen between RhlB and the PNPase-binding site of RNase E (grids 4J, 4K, 5J, 5K, 10D, 10E, 11D and 11E), which was expected. These, together with the negative interaction of PNPase with the RhlB- binding site, served as negative controls for the exper- iment (grids 1H, 1I, 2H, 2I, 8A, 8B, 9A and 9B). A series of cross-species’ interactions was also tested here. We observed strong interactions for E. coli RhlB and the CTH of V. angustum S14 RNase E (grids 3J and 10C). Weaker interactions were seen for other cross-species’ crosses involving RhlB and the RhlB- binding site (grids 1K, 2J, 10B and 11A). Further, we observed the self-interaction of E. coli RhlB (grid 10J), suggesting that this protein can self-interact [40]. PNPase and RhlB copurify with V. angustum S14 RNase E Having shown that PNPase and RhlB can interact with RNase E microdomains as well as the RNase E CTH and with each other in a two-hybrid assay, we investigated whether they interacted in vivo. First, we determined whether V. angustum S14 proteins can be detected by antisera against E. coli RNase E, PNPase and RhlB (Fig. 1A). Following that, we then carried out several immunoprecipitation experiments to deter- mine whether V. angustum S14 RNase E forms a complex with V. angustum S14 PNPase and RhlB, and to determine whether other possible interaction partners were present. V. angustum S14 PNPase and RhlB were found to coprecipitate with RNase E when antiserum against RNase E was used for immuno- precipitation (Fig. 1B). Similarly, when antiserum against PNPase was used for immunoprecipitation, V. angustum S14 RhlB and RNase E were found to associate with V. angustum S14 PNPase (Fig. 1C). As enolase comigrated in the same region in the gel as RNase E PNPase RhlB 188 98 62 49 MW (kDa) RhlB RNase E PNPase 250 148 64 50 MW (kDa) RNase E PNPase IgG RhlB 250 148 64 50 Mw (kDa) A B C Fig. 1. Immunoprecipitation of RNase E and PNPase in V. angu- stum S14 by antisera against E. coli RNase E and PNPase. (A) V. angustum S14 lysate probed with antisera against RNase E, PNPase and RhlB. (B) Immunoprecipitation using RNase E antise- rum. The RNase E antiserum was incubated with V. angustum S14 lysate and then isolated by protein G-conjugated Sepharose. The eluted fraction was separated by SDS ⁄ PAGE and analysed by immunoblotting using a series of antisera. PNPase and RhlB were found to copurify with V. angustum S14 RNase E. The antibody heavy chain is indicated. Composite image obtained when the PVDF membrane was probed serially with antisera against RNa- se E, PNPase and RhlB. (C) Immunoprecipitation using PNPase antiserum. Antiserum against PNPase was incubated with V. angu- stum S14 lysate and then isolated by protein G-conjugated Sepha- rose. Following SDS ⁄ PAGE separation, the immunoprecipitates were analysed by western blot and probed in a serial fashion with antisera against RNase E, PNPase and RhlB. RNase E and RhlB were found to copurify with PNPase. RNA degradosome complex in Vibrio angustum S14 M. A. Erce et al. 5164 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS the IgG heavy chain, any signal from probing with enolase antiserum was masked by IgG at that posi- tion in the gel. Identification of the RNA degradosome in V. angustum S14 using BN-PAGE Previously, we predicted that V. angustum S14 RNa- se E contains interaction sites for RhlB, PNPase and enolase [38]. Using two-hybrid analysis here and in a previous study, we demonstrated the interactions of V. angustum S14 RNase E microdomains and the CTH of RNase E with RhlB, enolase and PNPase. From the results of our immunoprecipitation experi- ments, we found that RhlB and PNPase associate with RNase E in V. angustum S14. Together, these results strongly suggest, but do not prove, that all of these proteins form a degradosome complex. To ascertain that these proteins all co-associate into a degrado- some-like complex in V. angustum S14, we separated the cell lysate using BN-PAGE (Fig . 2A). This revealed a range of protein complexes of various sizes up to 1.2 MDa. To identify the RNA degradosome amongst all these complexes, the proteins were transferred onto a poly(vinylidene difluoride) (PVDF) membrane and probed with antisera against RNase E, PNPase and RhlB (Fig. 2B). On probing with RNase E-specific antibodies, RNase E was detected in two high-molecular-mass bands above 1 MDa and in bands corresponding to approximately 480 and 650 kDa. The latter is consistent with RNase E’s ho- motetrameric state (Fig. 2B). It should be noted that the true molecular mass of RNase E is 116 kDa, but its apparent molecular mass is approximately 180 kDa [41]. PNPase was observed in the same two high- molecular-mass bands (above 1 MDa) as detected by antiserum against E. coli RNase E. PNPase was also observed at approximately 750 kDa (a potential trimer of trimers), as well as in a band running at approxi- mately 240 kDa, which is consistent with its trimeric form (Fig. 2B). The presence of RhlB was detected in the same two high-molecular-mass complexes above 1 MDa, which also contained RNase E and PNPase (Fig. 2B), but was not seen below 1 MDa. The detec- tion of lower mass bands containing RhlB may have been reduced as the proteins were in their native rather than denatured state, making the epitopes more difficult to recognize by the antiserum. It is interesting to note that RNase E, PNPase and RhlB are part of the high-molecular-mass complexes, but did not co-associate in other lower mass heteromultimers. Probing with antiserum against enolase proved to be uninformative as enolase is present in high abundance in the cell and comigrates with other protein complexes. Antibodies were not available for the identification of other possible interactor proteins; we therefore used MS to analyse Bands A and B (Fig. 2A). This verified the presence of enolase and PNPase, but not RNase E (Table 2). This was not unexpected because of the low abundance of RNase E in the cell. The amino acid sequence of RNase E is arginine-rich, giving rise to peptides that are either too short or too long for frag- mentation when subjected to digestion by trypsin. This may have occluded the identification of RNase E in the mass spectrometric analysis of Bands A and B. However, RNase E was detected by MS of immuno- precipitates. Ribosomal protein L4 was detected in Bands A and B, together with GroEL, a large number of ribosomal proteins and a high abundance of meta- bolic proteins (data not shown). It is unclear whether these ribosomal proteins are truly complexed with the RNA degradosome in V. angustum S14 or whether parts of the ribosome or, indeed, other protein com- plexes co-electrophorese with the degradosome; this is likely when one considers the abundance of these pro- teins within the cell and the large complexes which comprise the ribosome. The proteins identified, which have been described to associate with the RNA MW (kDa) 1236 1048 1236 MW (kDa) RNase E PNPase RhlB Band A Band B 720 1048 480 720 242 480 146 66 242 A B Fig. 2. Separation of protein complexes through BN-PAGE. (A) Analytical analysis of protein complexes in V. angustum S14. V. angustum S14 lysate (17 lg) was separated by BN-PAGE and silver stained. (B) Western blot of V. angustum S14 RNA degrado- some components. V. angustum S14 lysate (200 lg) was sepa- rated through BN-PAGE and electroblotted onto a PVDF membrane. Bands were detected by probing serially with antisera against RNase E, PNPase and RhlB. Molecular mass markers are shown in kDa. M. A. Erce et al. RNA degradosome complex in Vibrio angustum S14 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5165 degradosome in E. coli, are shown in Table 2. Only one peptide was identified for PNPase and ribosomal protein L4, but these have been found previously to be associated with RNase E [42]. Fragmentation spectra for both of these showed a strong series of y-ions and highly significant identification scores (Fig. 3). Two-dimensional BN-SDS ⁄ PAGE analysis To better understand the subunit composition of the degradosome protein complex, we used two-dimen- sional BN-SDS ⁄ PAGE. In this technique, protein com- plexes can be separated into subunits in an SDS ⁄ PAGE second dimension, following first dimen- sion BN-PAGE. We excised six bands from the first dimension BN-PAGE separation (Fig. 4A), and ran these bands separately in a second dimension reducing SDS ⁄ PAGE to separate their constituent proteins according to size (Fig. 4B). As a control, cleared lysate was loaded (Fig. 4B, lane CL). It can be seen that the composition of each of the bands excised from the BN-PAGE separation varied (Fig. 4B, lanes 1–6) and was different from the control. It was evident that each band contained proteins and complexes apart from the RNA degradosome. This is to be expected as BN- PAGE was separating a whole-cell lysate. After second dimension SDS ⁄ PAGE separation, the proteins were electroblotted onto a PVDF membrane and serially probed with antisera against RNase E, PNPase, RhlB and enolase (Fig. 4C). We found that all BN-PAGE bands excised between 900 kDa and 1.2 MDa contained RNase E (Fig. 4C, lanes 3–5). However, RNase E was not present in Band 6. In all instances in which RNase E was present, PNPase, RhlB and eno- lase were also present, but in varying quantities. Although the antisera used have different affinities for their target protein, Band 4 showed more RNase E and PNPase than RhlB and enolase. By contrast, Bands 1, 2, 3 and 5 showed roughly equivalent amounts of each of these proteins in the four samples. Interestingly, Band 6, which corresponded to a band excised in the vicinity of the 380-kDa region of the first dimension BN-PAGE separation, only contained PNPase, enolase and RhlB. There was no full-length RNase E. However, there was evidence of a band migrating at approximately 80 kDa, which migrated above PNPase. This corresponds to a fragment of RNase E which is seen in one-dimensional SDS ⁄ PAGE analyses and immunoblots of whole-cell lysate of V. angustum S14 RNase E (Figs 1B and 4D), and which has been observed previously to migrate in this region [18]. This suggests that RhlB, enolase and PNPase may be found together in association with a fragment of RNase E containing its CTH. This would be consistent with the known positions of the microdo- mains in RNase E. Although more unlikely, we cannot rule out that, if this fragment of RNase E is its NTH, and not its CTH, RhlB, enolase and PNPase may be able to form an association independent of RNase E. Discussion Through sequence analysis, we have shown previously that the CTH of V. angustum S14 RNase E contains binding sites for RhlB (residues 714–758), enolase (resi- dues 885–909) and PNPase (residues 1015–1094). We Table 2. Identification of V. angustum S14 RNase E-associated proteins through mass spectrometric analysis. V. angustum S14 protein Mass (Da) Score Peptide Position Individual peptide score a Band A Enolase (Q1ZNA5) b 45920 123 LNQIGSLTETLAAIK 345–359 90 SGETEDATIADLAVGTAAGQIK 374–395 53 GroEL (Q1ZKN3) b 57302 72 NAGDEESVVANNVK 457–470 20 EVASQANDAAGDGTTTATVLAQAIIAEGLK 76–107 70 50S Ribosomal protein L4 (Q1ZJA7) b 21707 63 LIVVDNFALEAPK 117–129 63 Band B Enolase (Q1ZNA5) b 45920 106 GITNSILIK 336–344 54 LNQIGSLTETLAAIK 345–359 76 PNPase (Q1ZJW2) b 76671 31 IAELAEAK 245–251 31 50S Ribosomal protein 21707 116 SILSELVR 106–113 30 L4 (Q1ZJA7) b AIDPVSLIAFDK 173–184 75 GADALTVSETTFGR 7–20 61 Immunoprecipitation RNase E (Q1ZS71) b 122521 54 AALSTLDLPQGMGLIVRT 153–169 54 a For this analysis, peptides with scores > 10 are considered to be statistically significant. b Swiss-Prot accession number. RNA degradosome complex in Vibrio angustum S14 M. A. Erce et al. 5166 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS have also shown, using two-hybrid analysis, that the enolase- and PNPase-binding sites are capable of interacting with enolase and PNPase [38]. Here, we report that the putative V. angustum S14 RNase E RhlB-binding site (residues 714–758) interacts with RhlB. Further, results from our BN-PAGE analysis and immunoprecipitation experiments confirmed that RNase E complexes with RhlB, enolase and PNPase in V. angustum S14. V. angustum S14 PNPase exists as a trimer and, through two-hybrid experiments, we showed that enolase and PNPase in V. angustum S14 can self-associate. Based on our observations, we present a model for the RNA degradosome complex in V. angustum S14 which shows the positions of the microdomains in the CTH of RNase E, the proteins that bind to them, as well as their dimerization state (Fig. 5). We have predicted previously that there are five regions of increased structural order in the V. angustum S14 RNase E CTH: the first corresponds to Segment A, which is involved in membrane binding (residues 565–584); the second is a putative RNA-bind- ing domain (residues 604–661); the third has a function # b Seq y # 1 114.0913 L13 2 227.1754 I 1315.7256 12 3 326.2438 V 1202.6416 11 4 425.3122 V 110.3.5732 10 5 540.3392 D 1004.5047 9 6 654.3821 N 889.4778 8 7 801.4505 F 775.4349 7 8 872.4876 A 628.3664 6 9 985.5717 L 557.3293 5 10 1114.6143 E 444.2453 4 11 1185.6514 A 315.2027 3 12 1282.7042 P 244.1656 2 13 K 147.1128 1 1 114.0913 I8 2 185.1285 A 731.3934 7 3 314.1710 E 660.3563 6 4 427.2551 L 531.3137 5 5 498.2922 A 418.2296 4 6 627.3348 E 347.1925 3 7 698.3719 A 218.1499 2 # b Seq y # 8K147.1128 1 A B 0 200 L L y(1) a(2) b(2) y(2) y(4) y(5) y(6) y(7) y*(4)++ ,y*(2) b(6)++ a(2) , a(4)++ b(2) , y*(2) b(3) y(4) y(6) y(7) y(8) y(9) y(10) y(11) 400 600 800 1000 1200 0 200 400 600 800 1000 1200 y*(6)++ Fig. 3. Fragmentation spectra for peptide sequences from V. angustum S14 proteins identified in Bands A and B. (A) Fragmentation spectra for peptide sequence LIVVDNFALEAPK from ribosomal protein L4 identified in Band A. For this peptide, the theoretical ion mass was 1427.802 Da and the experimental ion mass was 1427.767 Da. (B) Fragmentation spectra for peptide sequence IAELAEAK from PNPase identified in Band B. For this peptide, the theoretical ion mass was 843.470 Da and the experimental ion mass was 843.454 Da. The boxes illustrate the fragment ions for these peptide sequences as predicted by Mascot. Fragments that match between our data and the predicted peptide fragments are shown in red. The fragment ions for both peptide sequences include a near-complete y-ion series and two from the b-ion series, indicating a good match of the peptide fragments to the predicted sequence. M. A. Erce et al. RNA degradosome complex in Vibrio angustum S14 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5167 which has yet to be described (residues 792–797); the fourth corresponds to the enolase-binding microdo- main (residues 885–909); and the fifth corresponds to the region which binds PNPase (residues 1015–1094). The RhlB-binding site, which has been shown here to be capable of binding RhlB, did not correspond to a region of increased structural order (residues 714–758). From the results obtained in all of our analyses, it would be reasonable to conclude that the core of the V. angustum S14 RNA degradosome comprises RNase E, PNPase, RhlB and enolase, all of which are also components of the so-called canonical degradosome in E. coli. This is not surprising as V. angustum S14 belongs to the same subset of c-proteobacteria as E. coli. Organisms belonging to this group have previously been classified to have Type A RNase E homologues [22]. The interactions observed in the immunoprecipita- tion experiments and two-hybrid analyses are pairwise interactions; therefore, we sought to prove that these proteins actually form a single protein complex using BN-PAGE. Previous techniques to isolate the RNA AB C D Fig. 4. Analysis of the V. angustum S14 RNA degradosome complex through two-dimensional BN-SDS ⁄ PAGE. (A) Coomassie-stained gel of the preparative separation of V. angustum S14 protein complexes in the first dimension by BN-PAGE. Bands 1–6 were excised and sepa- rated in a second dimension SDS ⁄ PAGE. Bands were chosen for excision based on the results obtained from our BN-PAGE blot (Fig. 2B). We chose to excise Band 1 as we suspected that it consisted mainly of protein aggregation. Band 2 was chosen as we wanted to deter- mine whether the excision of a band slightly higher than Bands 3 and 4 (where RNA degradosome components were detected previously) would also contain degradosome proteins. Bands 3 and 4 were excised as they corresponded to the high-molecular-mass bands, Bands A and B in Fig. 2A, where the degradosome components were detected previously (Fig. 2B). Band 5 was excised to determine whether the degradosome components seen in Fig. 2B were still present across the mass range. Band 6 was chosen on the basis of the apparent migra- tion of the PNPase trimer on the BN-PAGE blot. (B) Coomassie-stained 4–12% Bis-Tris gel of the second dimension SDS ⁄ PAGE separation of the excised bands (Bands 1–6). Cleared lysate (CL) is shown for comparative purposes. There is a clear difference in proteins present in each band, indicating that the bands excised from the BN-PAGE gel are composed of different proteins. (C) Western blot of the second dimension SDS ⁄ PAGE separation of the excised bands. The blot was probed simultaneously with a combination of RNase E, PNPase, RhlB and enolase antisera. Bands 1 and 3 are more enriched in the components of the degradosome than the others. Band 6 does not contain full-length RNase E. Cleared lysate (CL) is again shown for comparison. The lower half of the blot is not shown as there was no signal detected below the 40-kDa region. A band migrating at approximately 80 kDa (*) cross-reacts with the RNase E antiserum and may be the RNase E CTH. (D) Western blot of V. angustum S14 lysate probed with RNase E antiserum. Molecular mass markers are shown in kDa. CTHNTH RNase E RhlB (714–758) Putative RNA-binding domain ? (792–797) “Segment A” (565–581) Enolase (885–909) PNPase (1015–1094) (604–661) Fig. 5. V. angustum S14 RNA degradosome model. The CTH of V. angustum S14 RNase E contains sites for interaction with RhlB, enolase and PNPase. In addition, we have identified regions with sequence homology to ‘Segment A’ and the RNA-binding domain in E. coli.A remaining region (residues 792–797) which may be involved in interactions has yet to be characterized. Figure not drawn to scale. RNA degradosome complex in Vibrio angustum S14 M. A. Erce et al. 5168 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS degradosome have required large amounts of starting material, or have involved the denaturing purification of individual components followed by their reconstitu- tion into the RNA degradosome. As emerging evi- dence points to the RNA degradosome as a dynamic complex with many possible components, these tech- niques provide limited avenues for truly assessing what is occurring in vivo. We report the use of BN- PAGE, a technique commonly employed to separate protein complexes, two-dimensional BN-SDS ⁄ PAGE and immunoprecipitation to analyse the RNA de- gradosome from V. angustum S14. As little as 17 lg of protein from whole-cell lysate is sufficient to visu- alize the separation of protein complexes through BN-PAGE. Our results from the BN-PAGE separa- tion and subsequent immunodetection indicate that PNPase and RhlB associate with RNase E in a com- plex above 1.2 MDa, but that these proteins also co- associate in smaller complexes of approximately 900 kDa. This is in agreement with current findings that the RNA degradosome may be an assembly which can range from 500 kDa to possibly in excess of 4 MDa [30]. Mass spectrometric analysis of the bands at 1.1 and 1.2 MDa identified GroEL and ribosome proteins as possible interaction partners. This was not surprising as ribosomal proteins have been found in association with RNase E and the de- gradosome, and play a role in its regulation, espe- cially in times of stress [42]. However, it could also be that components of the ribosome are comigrating with the RNA degradosome in the native gels. Together with ribosomal proteins, we identified eno- lase and PNPase in these high-molecular-mass com- plexes, confirming the results obtained in our previous E. coli two-hybrid analysis and immunopre- cipitation experiments [38]. It remains to be eluci- dated whether the multiple locations of degradosome components observed in BN-PAGE was a result of a heterogeneous population of RNA degradosomes in the cell, or whether the degradosome subunits dissociated in preparation steps preceding BN-PAGE separation. We have identified sequence homologues for the degradosome proteins in V. angustum S14 and showed that they are capable of interacting with the CTH of V. angustum S14 RNase E as well as their respective binding sites ([38] and this work). Previously, we have shown that the RNase E enolase-binding and PNPase- binding sites from V. angustum S14 and E. coli may be interchangeable. Enolase and PNPase from V. angustum S14 and E. coli can bind to RNase E microdomains (used in two-hybrid analysis) in V. an- gustum S14 RNase E and E. coli RNase E. Here, we have expanded on these results and shown that RhlB can interact with the CTH of V. angustum S14 RNa- se E and the predicted RhlB-binding microdomain. Interestingly, we found that, despite low sequence identity between the CTH of E. coli and V. angu- stum S14 RNase E (28% identity) and the high varia- tion in sequences flanking the microdomains, E. coli RhlB, enolase and PNPase are able to bind to the V. angustum S14 RNase E CTH. These results suggest that specific protein interactions with RNase E can occur despite the disordered nature and variability of the sequences flanking the microdomains. This high- lights the importance of microdomain sequence conser- vation; the cross-species’ setting of the two-hybrid experiments lends further strength to this. The fact that the CTH of RNase E appears to adopt little struc- ture probably plays a role in providing the flexibility that is required by many molecular interactions [18,38]. Further, there is decreased evolutionary con- straint, allowing the sequences to adapt to the specific requirements of the organism. Future experiments may be aimed at the further investigation of how microdo- mains direct the specificity of RNase E and how they influence the assembly of different types of degrado- somes in the cell. Experimental procedures Bacterial strains and plasmids Vibrio angustum S14 was used to prepare protein samples for BN-PAGE analysis and co-immunoprecipitation of RNase E. E. coli strain DHM1 was used in E. coli two- hybrid assays to study protein–protein interactions as described below. Media and growth conditions Vibrio angustum S14 was grown in high-salt Luria–Bertani broth (LB20) (10 gÆL )1 tryptone, 5 gÆL )1 yeast extract, 20 gÆL )1 NaCl). E. coli strains were grown in Luria–Bertani broth (LB10) (10 gÆL )1 tryptone, 5 gÆL )1 yeast extract, 10 gÆL )1 NaCl). Solid medium was made by the addition of 15 gÆL )1 of agar. Where appropriate, isopropyl thio-b-d- galactoside (IPTG), the lac promoter inducer, was added to a final concentration of 0.5 mm, and 5-bromo-4-chloro- 3-indolyl-b-d-galactopyranoside (X-gal), a substrate of b-galactosidase, was added to a final concentration of 40 mgÆL )1 . Liquid bacterial cultures were inoculated with day-old single colonies and grown in the appropriate medium on a rotary shaker at 180 r.p.m. V. angustum S14 was grown at 25 °C and E. coli cultures were grown at 37 °C, unless M. A. Erce et al. RNA degradosome complex in Vibrio angustum S14 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5169 otherwise specified. Where appropriate, the medium was supplemented with antibiotics (ampicillin, kanamycin and nalidixic acid at 100, 50 and 30 mgÆL )1 , respectively). E. coli two-hybrid analysis of interactions of RNase E with RhlB The Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system [39] was used to test for an interaction between RhlB and RNase E. All genes and gene fragments were cloned into the BACTH parental plasmids, whereby the T25 domain was fused to the N-terminus (pKT25) of the fragment and the T18 domain was fused to both the N- (pUT18C) and C-termini (pUT18) (see Table S1 for a com- plete list of strains and constructs, and Table S2 for the primers used). The genes used included V. angustum S14 RhlB (Swiss-Prot Accession No. Q1ZNA5) and V. angu- stum S14 PNPase (Q1ZJW2). Gene fragments included the V. angustum S14 RNase E CTH (residues 526–1094) and the V. angustum S14 RNase E putative RhlB-binding micr- odomain (residues 714–758). Experimental details and posi- tive and negative controls were the same as in a previous study [38]. The negative interaction of RhlB with the RNase E PNPase-binding site also served as a negative control. Briefly, reciprocal crosses were performed where appropriate by cotransforming chemically competent E. coli DHM1 cells and plating on LB–X-gal plates supplemented with appropriate antibiotics. The transformants were grown at 30 °C for 26 h before being picked and patched onto new plates. The patched transformants were then grown separately at 25 °C for 48 h. The BACTH patch assay results were scored by a ‘++’ for strong interactions, ‘+’ for moderate interactions and ‘)’ for very weak or no inter- actions, based on colour intensity. Analysis of protein complexes Vibrio angustum S14 cell pellets were resuspended in 1· Native PAGE buffer (50 mm Bis-Tris, 6 m HCl, 50 mm NaCl, 10% w ⁄ v glycerol, 0.001% Ponceau S, 0.8% Triton X-100, pH 7.2) supplemented with Roche Complete EDTA-free protease inhibitor cocktail (one tablet per 50 mL of solution; Roche Diagnostics, Mannheim, Germany) and lysed by sonication (Branson sonifier, Bran- son Ultrasonics Corporation, Danbury, CT, USA). The resulting lysate was clarified by centrifugation (22 000 g, 4 °C, 30 min). Samples were loaded onto 3–12% Native- PAGE Novex Bis-Tris gels (Invitrogen Life Techonologies, Carlsbad, CA, USA). Gels were run at a constant voltage of 100 V using 1· NativePAGE running buffer (50 mm Bis- Tris, 50 mm Tricine, pH 6.8) at the anode and a light blue cathode buffer (50 mm Bis-Tris, 50 mm Tricine, 0.02% Coomassie G-250, pH 6.8) until the dye front migrated to the end of the gel. The apparent molecular mass of protein complexes was estimated by comparison with very high- molecular-mass markers of range 20–1236 kDa (Invitrogen Life Techonologies). Protein bands were visualized either by silver staining for analytical gels or Coomassie blue staining for preparative gels. Bands of interest were excised and analysed through mass spectrometric analysis. For subsequent analysis in a second dimension via SDS ⁄ PAGE, following first dimension separation, the BN- PAGE gel was equilibrated in 1· Mes buffer (Invitrogen Life Technologies) for 10 min. Bands of interest were excised and placed into the wells of a 4–12% Novex Bis- Tris gel (Invitrogen Life Technologies). Electrophoresis was performed at a constant voltage of 150 V until the dye front migrated to the end of the gel. Protein bands were visualized with Bio-Safe Coomassie (Bio-Rad Laboratories, Hercules, CA, USA). Western blotting and immunodetection For western blot analysis, proteins were separated on a 3–12% NativePAGE Novex Bis-Tris gel, 4–12% Novex Bis-Tris gel, or both (Invitrogen Life Technologies), and transferred onto PVDF membranes (Millipore, Bedford, MA, USA) using the Invitrogen XCell II blot apparatus in 1· NativePAGE Transfer buffer (25 mm Bicine, 25 mm Bis- Tris, 1 mm EDTA, pH 7.2) for native gels and 1· Native- PAGE transfer buffer in 20% methanol (v ⁄ v) for Bis-Tris gels. Transfer was carried out at a constant 600 mA for 90 min. After blocking with 5% (w ⁄ v) dried skimmed milk in NaCl ⁄ Pi with 0.1% Tween 20 (v/v) overnight, mem- branes were probed by incubation with a 1 : 5000 dilution of primary antisera (against E. coli RNase E, RhlB, enolase or PNPase, which were generous gifts from Dr A. J. Carp- ousis, CNRS-Universite ´ Paul Sabatier, Toulouse) in NaCl ⁄ Pi with 0.1% Tween 20 (v/v), followed by incubation with a 1 : 5000 dilution of anti-rabbit IgG conjugated to horseradish peroxidase (GE Healthcare, Little Chalfont, Buckinghamshire, UK) in NaCl ⁄ Pi with 0.1% Tween 20 (v/v). Protein bands recognized by each specific antibody were then detected using chemiluminescence (GE Health- care) and visualized using a Fujifilm LAS 3000 imager (Fuijifilm, Tokyo, Japan). Immunoprecipitation Escherichia coli RNase E antiserum (a generous gift from Dr A. J. Carpousis) was crosslinked to Dynabeads Protein A (Invitrogen Dynal, Oslo, Norway) using dimethyl pim- elimidate.2HCl (DMP, Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Briefly, the Dynabeads were incubated with antibodies for 4 h on a rotating wheel at 4 °C. Excess antibodies were washed off with NaCl ⁄ P i (0.16 mm Na 2 HPO 4 .H 2 O, 5.51 mm NaH 2- PO 4 .2H 2 O, 140 mm NaCl, pH 7.4), followed by 200 mm triethanolamine, pH 8.0. The antibody-bound beads were incubated with DMP (20 mm DMP, 200 mm RNA degradosome complex in Vibrio angustum S14 M. A. Erce et al. 5170 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... for 15 min at 55 °C or boiling the beads in 1· SDS gel loading buffer for 10 min Following SDS ⁄ PAGE separation, the proteins were visualized by silver staining, or processed for western blot analysis and immunodetection The bands of interest were excised and subjected to mass spectrometric analysis In a parallel experiment, V angustum S14 RNase E was immunoprecipitated using either E coli RNase E... (Branson sonifier) The resulting lysate containing 5 mg of protein was clarified by centrifugation at 22 000 g for 30 min at 4 °C and mixed with 100 lL of Dynabeads Protein A crosslinked to an equal volume of antiserum raised against E coli RNase E The mixture was rotated at 4 °C for 2 h The beads were washed three times using NaCl ⁄ Pi and the target protein was eluted by incubating the beads in 1· SDS buffer... employing the Protein G Immunoprecipitation Kit (Sigma-Aldrich, Castle Hill, NSW, Australia) following the manufacturer’s instructions RNA degradosome complex in Vibrio angustum S14 2 3 4 5 6 7 8 9 Mass spectrometric analysis The bands of interest were excised, digested with trypsin and analysed by LC-MS-MS, as described previously [43] Searches were performed using the Mascot search engine [44] employing... starvation by the marine Vibrio sp S14 FEMS Microbiol Ecol 74, 129–140 37 Albertson NH, Nystrom T & Kjelleberg S (1990) Functional mRNA half-lives in the marine Vibrio sp S14 during starvation and recovery J Gen Microbiol 136, 2195–2199 38 Erce MA, Low JKK, March PE, Wilkins MR & Takayama KM (2009) Identification and functional analysis of RNase E of Vibrio angustum S14 and two-hybrid analysis of its interaction... degrade doublestranded RNA independent of the Degradosome- assembling region of RNase E J Biol Chem 277, 41157– 41162 17 Kaberdin VR, Miczak A, Jakobsen JS, Lin-Chao S, McDowall KJ & von Gabain A (1998) The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly... Carpousis AJ, Robinson CV, Symmons MF et al (2004) Studies of the RNA degradosome- organizing domain of the Escherichia coli ribonuclease RNase E J Mol Biol 340, 965– 979 19 Vanzo NF, Li YS, Py B, Blum E, Higgins CF, Raynal LC, Krisch HM & Carpousis AJ (1998) Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome Genes Dev 12, 2770–2781 20 Butland G, Peregrin-Alvarez JM,... (2006) The RNA degradosome: life in the fast lane of adaptive molecular evolution Trends Biochem Sci 31, 359–365 23 Morita T, Kawamoto H, Mizota T, Inada T & Aiba H (2004) Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phosphosugar stress in Escherichia coli Mol Microbiol 54, 1063–1075 24 Chandran V & Luisi BF (2006) Recognition of. .. (1999) Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3¢ exonuclease and a DEAD-box RNA helicase Genes Dev 13, 2594– 2603 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5171 RNA degradosome complex in Vibrio angustum S14 M A Erce et al 16 Liou G-G, Chang H-Y, Lin C-S & Lin-Chao S (2002) DEAD box RhlB RNA helicase physically... 2632–2641 44 Perkins DN, Pappin DJC, Creasy DM & Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 20, 3551–3567 RNA degradosome complex in Vibrio angustum S14 Supporting information The following supplementary material is available: Table S1 List of bacterial strains and plasmids used Table S2 List of oligonucleotide... (1999) RNase G (CafA protein) and RNase E are both required for the 5¢ maturation of 16S ribosomal RNA EMBO J 18, 2878–2885 Lin-Chao S, Wei CL & Lin YT (1999) RNase E is required for the maturation of SsrA RNA and normal SsrA RNA peptide-tagging activity Proc Natl Acad Sci USA 96, 12406–12411 Lee K, Bernstein JA & Cohen SN (2002) RNase G complementation of rne null mutation identifies functional interrelationships . commonly employed in the separation of membrane protein complexes, in a study of the first of its kind to purify and analyse the RNA degradosome. We showed that the V. angustum S14 RNA degradosome comprises. its role in mRNA degradation, the degradosome is also responsible for the processing of 5S ribosomal RNA and tRNAs, as well as the degradation of tmRNAs [4–8]. The principal proteins comprising the degradosome include. (ATP-dependent DEAD-box RNA helicase)-binding, PNPase (polynucleotide phosphory- lase)-binding and enolase-binding microdomains in the C-terminal half of Vibrio angustum S14 RNase E, and have shown