A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome
2010; Springer Nature; Volume: 29; Issue: 6 Linguagem: Inglês
10.1038/emboj.2010.14
ISSN1460-2075
AutoresRicarda Richter, Joanna Rorbach, Aleksandra Pajak, Paul Smith, Hans J. C. T. Wessels, Martijn A. Huynen, Jan Smeitink, Robert N. Lightowlers, Zofia M. Chrzanowska‐Lightowlers,
Tópico(s)Peptidase Inhibition and Analysis
ResumoArticle25 February 2010Open Access A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome Ricarda Richter Ricarda Richter Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Joanna Rorbach Joanna Rorbach Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Aleksandra Pajak Aleksandra Pajak Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Paul M Smith Paul M Smith Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Hans J Wessels Hans J Wessels Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Martijn A Huynen Martijn A Huynen Center for Molecular and Biomolecular Informatics, NCMLS, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Jan A Smeitink Jan A Smeitink Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Robert N Lightowlers Corresponding Author Robert N Lightowlers Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Zofia M Chrzanowska-Lightowlers Corresponding Author Zofia M Chrzanowska-Lightowlers Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Ricarda Richter Ricarda Richter Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Joanna Rorbach Joanna Rorbach Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Aleksandra Pajak Aleksandra Pajak Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Paul M Smith Paul M Smith Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Hans J Wessels Hans J Wessels Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Martijn A Huynen Martijn A Huynen Center for Molecular and Biomolecular Informatics, NCMLS, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Jan A Smeitink Jan A Smeitink Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Search for more papers by this author Robert N Lightowlers Corresponding Author Robert N Lightowlers Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Zofia M Chrzanowska-Lightowlers Corresponding Author Zofia M Chrzanowska-Lightowlers Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Author Information Ricarda Richter1, Joanna Rorbach1, Aleksandra Pajak1, Paul M Smith1, Hans J Wessels2, Martijn A Huynen3, Jan A Smeitink2, Robert N Lightowlers 1 and Zofia M Chrzanowska-Lightowlers 1 1Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne, UK 2Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 3Center for Molecular and Biomolecular Informatics, NCMLS, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands *Corresponding authors. Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK. Tel.: +44 191 222 8028; Fax: +44 191 222 8553; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2010)29:1116-1125https://doi.org/10.1038/emboj.2010.14 There is a Have you seen ...? (March 2010) associated with this Article. 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 Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes. Introduction Human mitochondria are ubiquitous organelles that are essential for cell viability. Among many crucial functions, mitochondria couple the process of oxidative phosphorylation, where cellular respiration is harnessed to generate ATP. This demanding mechanism requires the synthesis and import of many nucleus-encoded proteins as well as the intramitochondrial production of 13 polypeptides that are encoded by the mitochondrial genome, mtDNA. Consequently, correct maintenance and expression of mtDNA is essential for cell viability. Although we are gradually learning more about the principal factors and mechanisms underlying the maintenance and transcription of mtDNA, the process of mitochondrial translation has proven extremely difficult to be characterised in detail. This is in part because isolated mitochondria lose their capacity to synthesise proteins after solubilisation of the inner membrane, consistent with loss of crucial membrane-associated factors. Furthermore, despite impressive efforts to reconstitute in vitro mitochondrial translation systems (Yasukawa et al, 2001; Takemoto et al, 2009), results have been limited. Invaluable contributions from the laboratories of Spremulli, Watanabe and O'Brien have identified or characterised constituents of both the bovine small 28S (mt-SSU) (Suzuki et al, 2001) and large 39S (mt-LSU) (Koc et al, 2001) mitoribosomal subunits and many proteins involved in translational initiation and elongation (Spremulli et al, 2004), but important factors remain to be unearthed. To improve our understanding of this process, we have started to identify other principal components in mitochondrial protein synthesis. We have focused our efforts on a previous report where tagged mitochondrial ribosome recycling factor (mtRRF) was shown to immunoprecipitate mitoribosomes and associated proteins from mitochondrial lysates (Rorbach et al, 2008). Proteomic analysis uncovered a large number (73) of mitoribosomal proteins (MRPs). In addition, another 94 polypeptides were identified, a number of which have been tentatively identified as nucleoid proteins. Immature colon carcinoma transcript-1 (ICT1) was consistently associated with immunoprecipitation (IP), but it was neither known to be mitochondrial nor have an experimentally verified function, although it is predicted to be a member of the prokaryote/mitochondrial release factor family (uniprot Q14197). This family is intriguing. We have recently been able to show that of the four family members, only mtRF1a is necessary and sufficient to terminate the translation of all 13 mitochondrially encoded polypeptides (Soleimanpour-Lichaei et al, 2007; Temperley et al, 2010). What, therefore, is the function, if any, of the three remaining family members? We report here that a second member of this family, ICT1, is a component of the 39S mt-LSU. It has retained its ribosome-dependent peptidyl-tRNA hydrolase (PTH) activity that is essential for cell viability. Furthermore, this ribosome-dependent PTH activity is codon-non-specific. We speculate that this ribosome-associated activity may be involved in the hydrolysis of peptidyl-tRNAs that have been prematurely terminated and thus in the recycling of stalled mitoribosomes. Results ICT1 is an essential mitochondrial protein To determine the subcellular location of ICT1, western blot analysis was performed using cell lysate and enriched mitochondria. As shown in Figure 1A, a 3- to 5-fold enrichment was seen for the mitochondrial matrix protein mtRF1a along with concomitant increase in ICT1. Crucially, ICT1 was resistant to addition of proteinase-K to intact mitochondria, but was lost on solubilisation of the organelle. To assess whether ICT1 is processed on import into mitochondria, full-length ICT1 (FL) or a truncated form lacking the N-terminal 29 residues (Δ29) were prepared. Both proteins were expressed in Escherichia coli as N-terminal glutathione-S-transferase (GST)-fusion proteins before cleavage and purification as detailed under Materials and methods. Migration of endogenous ICT1 in comparison with recombinant proteins (FL/Δ29) is consistent with cleavage on mitochondrial import, with the loss of ∼30 residues. Figure 1.ICT1 is an essential protein necessary for mitochondrial protein synthesis. (A) Human ICT1 is a mitochondrial protein. Cell lysate (CL 50 μg, lanes 1, 5) or mitochondria (10 μg, lanes 2–4, 6–8) were isolated from HeLa and HEK293T cells and subjected to western blot analysis either immediately (lanes 1, 2; 5, 6) or after treatment with proteinase-K (lanes 3, 7). Mitochondria were lysed with Triton X-100 to confirm the sensitivity of marker proteins to the protease (lanes 4, 8). Mitochondrial release factor-1a (mtRF1a) was used as a mitochondrial matrix marker and ribosomal protein-S6 (S6-RP) as a cytosolic marker. Purified FL (lane 9) and an ICT1 deleted of N-terminal 29 residues (Δ29, lane 10) are shown in comparison with the endogenous protein. (B–E) Depletion of ICT1 inhibits cell growth, impairs mitochondrial protein synthesis and decreases mitochondrial respiratory chain complexes. HeLa cells in standard glucose media were transfected with either of two siRNAs directed to ICT1 transcript (si-ICT1A or B) or a non-targeting control (si-NT), and cell numbers were counted at 3-day intervals. Standard errors were derived from three independent experiments (C). Cell lysates were isolated from non-targeting and ICT1-depleted cells (3 days) and subjected to western blotting for ICT1 (B) and various markers (D). The relative levels for the MRP MRPL3 and respiratory components NDUFB8 or COX2 were quantified (lower panel) with standard errors derived from three independent repeats. (E) After 3-day siRNA-mediated depletion, cells were subjected to metabolic labelling of mitochondrial proteins for 15 min after inhibition of cytosolic protein synthesis. Aliquots (50 μg) were separated by 15% SDS–PAGE and exposed to a PhosphorImager. Proteins are identified by comparison against those reported by Chomyn (1996). A section of the gel stained with Coomassie blue (CBB) following exposure is shown to indicate even loading of cell lysate. Download figure Download PowerPoint To assess whether ICT1 serves an essential function in mitochondria, siRNAs were designed to target the transcript. Two siRNAs were highly efficient in depleting ICT1 from cells (Figure 1B), causing a morphological alteration and a reduction in cell number even when grown on standard, mainly glycolytic media (Figure 1C). To confirm that effects were specific and not off-target, cells lacking mtDNA (rho0 cells) were also transfected with ICT1-specific siRNA and control siRNAs. The rationale being, as rho0 cells lack mitochondrial gene expression and are still able to grow on glucose media supplemented with uridine and pyruvate, depletion of any protein involved solely in mitochondrial gene expression should have minimal effect. Accordingly, siRNA-mediated depletion of β-actin or HSP70 severely compromised the growth of rho0 cells, whereas growth was unaffected by depletion of ICT1 or the mitochondrial translation factor mtEF-Tu (Supplementary Figure S1). These data are strongly indicative that ICT1 functions in mitochondrial gene expression. After only 3 days of ICT1 depletion, a decrease in the markers of the highly stable mitochondrial respiratory chain complex-I (NDUFB8) and IV (COX2) was confirmed by western blotting of HeLa lysates (Figure 1D). Interestingly, a similar decrease was also noted for the MRP MRPL3, indicating that levels of mitoribosome and possibly rates of mitochondrial protein synthesis may be compromised. Therefore, de novo synthesis of mitochondrial translation products was assessed by in vivo metabolic labelling. Figure 1E shows that in the ICT1-depleted cells 35S-met incorporation is indeed reduced. ICT1 is a member of the large mitoribosomal subunit Why does loss of ICT1 lead to reduction in mitochondrial protein synthesis? To investigate this question and to determine what components of the mitochondrial matrix associated with ICT1, a FLAG-tagged ICT1 was inducibly expressed in human HEK293T cells, facilitating IP. As shown in Figure 2A, silver staining uncovered a large number of proteins, similar to a previous profile where tagged mtRRF had immunoprecipitated the mitoribosome and associated proteins (Rorbach et al, 2008). Western blot analysis (Figure 2B) confirmed the presence of numerous MRPs and the predicted trace amounts of mtRRF. From proteomic data of the complete eluate, more than 200 mitochondrial proteins were identified (Supplementary Table S1), the MRPs being the most abundant (Supplementary Table S2); consistent with ICT1 interacting with entire mitoribosomes. As a second method to determine whether ICT1 was a component of the mitoribosome, complexes from untransfected cells were separated by isokinetic sucrose density gradients and fractions were subjected to blotting (Figure 2C). ICT1 co-sediments with MRPL3 and MRPL12, both components of the 39S mt-LSU. As has been found in other reports (Nolden et al, 2005; Williams et al, 2005), the mitochondrial monosome is not easily identified in cell or mitochondrial lysates by western blot after density-gradient centrifugation. This was also evidenced here by the lack of detectable small subunit marker DAP3 in the more dense fractions (Figure 2C, fractions 7–10). Therefore, to resolve this issue, we pre-concentrated samples by first immunoprecipitating mt-LSU and monosomes from mitochondrial lysates using FLAG-ICT1 and subjected the entire eluate to an identical gradient centrifugation. Fractions were then assessed by silver staining and western blotting. A similar distribution profile is still seen for ICT1 and MRPL3 (excluding free ICT1 caused by overexpression) (Figure 2D). Crucially, however, DAP3 is now visible in fractions 7–9, defining the monosomal fractions. Therefore, ICT1 behaves as an integral member of the 39S mt-LSU and a component of the intact 55S monosome. Figure 2.ICT1 is an integral component of the mitoribosome. (A, B) FLAG-tagged ICT1 immunoprecipitates mitoribosomes. HEK293T cells expressing FLAG-tagged ICT1 or mitochondrially localised luciferase (mtLuc-FLAG) were induced for 3 days; mitochondria were isolated, lysed and subjected to IP as detailed. The eluate and mitochondrial lysate before IP (IP-input) were separated by 15% SDS–PAGE and visualised by silver staining. * designates the FLAG protein. (B) Aliquots of the eluates were also subjected to western blot analysis with the indicated antibodies: MRPL3, MRPL12, MRPS6 and DAP3 as mitoribosomal markers; mtRRF, mitoribosome recycling factor; SDH, 70-kDa component of complex-II. (C) ICT1 co-sediments with the large mitoribosomal subunit. HeLa cells were lysed (600 μg), separated through a 10–30% sucrose gradient and fractionated as detailed (HeLa and HEK293T lysates gave identical separations). Components of the 39S mt-LSU (MRPL3, MRPL12) and 28S mt-SSU (DAP3) mitoribosomal subunits were visualised by western blotting. On immediate lysis, mtRRF is used as a matrix-soluble marker. (D) ICT1 also co-sediments with the intact monosome. Mitochondria (3 mg) of ICT1-FLAG-expressing HEK293T cells were subjected to FLAG IP; the entire eluate was separated by isokinetic density gradients and fractions were blotted as detailed above or visualised by silver staining (lower panel). Mitochondrial SSU (DAP3) and mt-LSU (MRPL3) MRPs are visualised. The approximate indicators for 28S mt-SSU, 39S mt-LSU and 55S monosome are shown and were determined as described under Materials and methods. (E) ICT1 is an integral member of 39S mt-LSU. Cell lysates (600 μg) from ICT1-depleted (si-ICT1B) or non-targeted control cells (si-NT) were separated by isokinetic gradients and proteins were visualised in the fractions by western blotting as described. Sedimentation markers were identified as above. (F) Loss of ICT1 causes depletion of the monosome. Cells expressing MRPS27-FLAG were treated with si-NT or si-ICT1B, after which IP was performed. To assess monosome formation, levels of MRPL3 and MRPL12 were quantified by western blotting of three individual experiments (right panel; MRPL3 P=0.001, MRPL12 P<0.001, MRPS27 P=0.3). (G) ICT1's association with mitoribosomes is not FLAG-dependent. Mitochondria from cells expressing MRPL20-FLAG were subjected to FLAG IP and the eluate was analysed by western blotting after isokinetic density gradients as described in panel D. Download figure Download PowerPoint Further support for ICT1 being an integral component of the 39S mt-LSU, was obtained from ICT1-depleted HeLa cells. Lysate was subjected to a similar isokinetic gradient, showing that the mt-LSU marker MRPL3, although present, was shifted into less dense fractions as compared with that in cells treated with non-targeting control siRNA (si-NT; Figure 2E). By contrast, the profile of the 28S mt-SSU protein DAP3 was unchanged, implying that only assembly of the mt-LSU and not the mt-SSU is affected on ICT1 depletion. To confirm that the decrease in mt-LSU assembly observed in ICT1-depleted cells caused a concomitant decrease in the level of intact monosome, we used a cell line that expresses a FLAG-tagged component of the mt-SSU, MRPS27, that was able to IP both mt-SSU and entire monosome. After 3 days of ICT1 depletion and MRPS27-FLAG induction, FLAG-IP was performed and eluates were blotted (Figure 2F). Levels of DAP3 were unaffected by ICT1 depletion. However, anti-MRPL3 and MRPL12 antibodies showed a 60% reduction of each of these proteins in the ICT1-depleted cells, consistent with a decrease in the monosome formation, presumably due to decreased mt-LSU assembly. This was also consistent with the decreased 35S metabolic labelling of mitochondrial proteins as shown in Figure 1E. Finally, to show that mitoribosomal association of ICT1 was not simply mediated by the FLAG tag, similar isokinetic gradients were used to separate the immunoprecipitated eluate from cells expressing MRPL20-FLAG. When correctly assembled, this FLAG-tagged protein was able to IP mt-LSU, assembly intermediates and intact monosome. As show in Figure 2G, ICT1 was clearly associated with mt-LSU and monosome. These data confirm that ICT1 is an important component of the complete monosome. ICT1 is a ribosome-bound, codon-independent PTH ICT1 is an integral component of the mitoribosome, but on the basis of homology it is predicted to be a ribosome-dependent PTH/translation release factor. This is surprising, as to our knowledge, no other PTH has been shown to be an integral ribosomal component. There are now four proteins classified as members of the mitochondrial release factor family, namely mtRF1a, mtRF1, ICT1 and another uncharacterised protein C12orf65 (Figure 3B). Curiously, both ICT1 and C12orf 65 have lost the two domains involved in codon recognition (α5 and PXT/SPF domain), potentially resulting in the dangerous situation of release factors that lack codon specificity. However, none of our previous IPs identified C12orf65 as a co-precipitant with mitoribosome components (data not shown). To determine whether ICT1 could promote hydrolysis of the ester bond between the growing peptide and the P-site tRNA, we used a well-established assay using isolated E. coli ribosomes, tritiated fmet-tRNAMet, synthetic codons and purified ICT1 (Caskey et al, 1971; Tate and Caskey, 1990; Soleimanpour-Lichaei et al, 2007). In comparison to the exquisite codon selectivity of mtRF1a, ICT1 promiscuously promoted the release of formylmethionine from its P-site tRNA irrespective of the codon sequence used to programme the assay and indeed even in the absence of codons in the A-site (Figure 3C). To determine whether ICT1 possessed a direct ribosome-independent PTH activity, or whether it functioned specifically to promote ribosome-dependent hydrolysis, purified ICT1 was incubated with f[3H]met tRNAmet in the absence of ribosomes before extraction and estimation of standard release factor activity. No significant increase in counts was noted over background, confirming that no significant hydrolysis of the substrate occurred in the absence of the ribosome (Figure 3C). Figure 3.ICT1 is a codon-independent PTH. (A) Structural comparisons between ICT1 and members of RF1 or RF2. Only limited structure is available for murine ICT1 (centre, PDB 1J26 unpublished structural genomics output), but this can be superimposed using Topmatch (Sippl and Wiederstein, 2008) onto either RF family (left T. maritima PDB 1RQ0 (Shin et al, 2004); right T. thermophilus PDB 2IHR (Zoldak et al, 2007)), making it unclear as to its evolutionary origin. The arrows show the ICT 1GGQ motif and the boxes enclose the codon recognition domains (Ito et al, 2000; Laurberg et al, 2008). (B) Primary sequence comparisons of ICT1 and translation release factor members. Representatives of the release factor families are shown; RF1 (human mtRF1a), RF2 (T. thermophilus) aligned with human sequences for ICT1 and a fourth member of the mitochondrial release factor family, C12orf65. Three regions are highlighted; the GGQ motif conferring PTH activity, and α-5/tripeptide domains that are implicated in codon recognition. The latter two domains are absent in ICT1 and C12orf65. There are two other families of PTHs, PTH1 and PTH2 (reviewed by Das and Varshney, 2006), which are predicted to be represented in the human mitochondrion by PTRH1 and PTRH2 (uniprot Q86Y79 and Q9Y3E5, respectively). These protein families function independently of the ribosome and on the basis of comparison of their three-dimensional structures (data not shown), are not homologous to the ribosome-dependent PTH family that contains ICT1. (C) ICT1 has codon-independent and ribosome-dependent translation release factor activity. E. coli ribosomes were programmed with tritiated P-site fmet-tRNAMet and A-site codons as indicated (detailed under Materials and methods). Activity was measured as hydrolysis of f[3H]met from its cognate tRNAMet and is represented as pmol f[3H]met released. Non-limiting amounts of protein (50 pmol) and RNA triplet (400 pmol) were used in the assay where required, with mtRF1a as a positive control. Activities are also evident where ribosomes were programmed with no codon or were absent from the assay, entirely. Reactions lacking 70S ribosomes contained the UAA triplet. Standard errors were calculated from a minimum of eight repeats; ***P<0.001. Download figure Download PowerPoint A mutation of the GGQ domain of ICT1 causes loss of cell viability ICT1 is an essential MRP with a ribosome-dependent yet codon-independent PTH activity, in vitro. Is it possible that ICT1 needs to maintain this activity when it is assembled into the mitoribosome, in vivo? PTH activity of release factors is mediated by the domain containing the tripeptide motif GGQ (Frolova et al, 1999; Figure 3A and B). It has been shown that mutations in either of the two glycine residues completely abolishes in vitro PTH activity while retaining the structural integrity of the protein (Frolova et al, 1999). To determine whether the GGQ domain is critical for ICT1's function, we reproduced two of these GGQ mutants by site-directed mutagenesis. First, recombinant ICT1GSQ and ICT1AGQ were purified, monodispersion was confirmed by dynamic light scattering, and then they were subjected to the release assay described above (Figure 4A). Each retained ∼1% of peptide-release activity (GSQ 1.1%, AGQ 0.8% residual activity). Having confirmed the requirement of the GGQ domain for the PTH activity of ICT1, it was then possible to assess the importance of the PTH activity in vivo. If PTH activity is crucial then replacement of wild-type ICT1 with ICT1GSQ would be predicted to compromise mitochondrial gene expression, leading to reduced growth on galactose. Consequently, an HEK293T cell line was engineered to express a FLAG-tagged version of ICT1GSQ and compared with the wild-type ICT1-FLAG expressor. On induction, the ICT1 mutant was incorporated into mitoribosomes, which were assembled at levels similar to that in the wild-type transfected control (Figure 4B and C), but induction resulted in a substantial overexpression of ICT1GSQ such that the majority of the protein remained free and not mitoribosome-bound (Figure 4C). A similar overexpression was also noted on induction of wild-type ICT1-FLAG (Figure 4C, upper panels), which in itself produced a mild growth phenotype. To reduce the levels of the overexpressed protein to that of the endogenous untransfected controls and concomitantly deplete the endogenous ICT1, serial dilutions of si-ICT1B were used. A concentration of siRNA was achieved that resulted in levels of FLAG-tagged ICT1 equivalent to that of the untransfected controls (10 nM; Supplementary Figure S2). Using this strategy, growth rates of these various cell lines were compared, uncovering a slower doubling time in the ICT1GSQ mutant, confirming that ICT1 requires a functional GGQ domain to maintain cell viability (Figure 4D, 3 cf. 4). Figure 4.Mutations of the GGQ domain can affect cell viability. (A) GGQ-mutant derivatives of ICT1 have lost PTH activity. Wild type and mutant derivatives (AGQ, GSQ) of Δ29 ICT1 were expressed as GST-fusion proteins, cleaved and assayed for f[3H]met release as described in Figure 3. All assays were performed with UAG codons and purified proteins, all equally monodispersed as assessed by dynamic light scattering (data not shown); ***P<0.001. (B, C) Mutated ICT1 is assembled into the mitoribosome. (B) FLAG-tagged wild-type (GGQ) and mutant (GSQ) ICT1 were expressed in HEK293T cells and the eluate from FLAG IP was subjected to silver staining (left panel) or western blotting (right panels) after denaturing gel electrophoresis. Molecular weight markers are indicated. The western blots of mitochondrial lysates shown are those before (IP-input) and after (Elution) FLAG IP of the wild type and mutated ICT1 derivatives. (C) Cell lysates were subjected to isokinetic gradient analysis before fractionation and western blotting, as described. The upper three panels are from wild-type ICT1-FLAG, the middle panels from mutated GSQ ICT1-FLAG and the lower panels from control mtLuc-FLAG. (D) A mutation of the GGQ domain affects cell growth. Non-targeting si-RNA-treated cells served as negative control (1, WT si-NT). Cells with only endogenous ICT1 (WT), or overexpressing normal (GGQ) or mutated (GSQ) ICT1 were treated for 4 days with 10 nM si-ICT1B to deplete endogenous ICT1 (2–4), whereas lane-2 represented the fully depleted control (WT si-ICT1B). Growth rates were compared by counting populations after 3 days of siRNA treatment; 3 versus 4: P<0.01; 1 versus 4: P<0.001. Western blots of lysates (4 days of siRNA treatment) after interrogation with the indicated antibodies are shown to the right. Download figure Download PowerPoint Discussion Following stringent purification methods, isolation of bovine mt-SSU, mt-LSU and the 55S monosome has allowed characterisation of many constituents and a low-resolution structure (Sharma et al, 2003). These data are in agreement that the mammalian mitoribosome differs substantially from other ribosomes that have been characterised. In particular, the 30:70 (w:w) protein-to-RNA ratio is reversed, with only one reduced rRNA component for each subunit and an increased number of protein factors, as determined by initial LCMS, resulting in a larger, more porous structure (O'Brien, 2002). We
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