Protein quality control and regulated proteolysis in the genome‐reduced organism Mycoplasma pneumoniae
2020; Springer Nature; Volume: 16; Issue: 12 Linguagem: Inglês
10.15252/msb.20209530
ISSN1744-4292
AutoresRaul Burgos, Marc Weber, Sira Martínez, María Lluch‐Senar, Luís Serrano,
Tópico(s)Bacteriophages and microbial interactions
ResumoArticle15 December 2020Open Access Source DataTransparent process Protein quality control and regulated proteolysis in the genome-reduced organism Mycoplasma pneumoniae Raul Burgos Raul Burgos orcid.org/0000-0002-4452-9235 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Marc Weber Marc Weber orcid.org/0000-0001-7920-5655 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Sira Martinez Sira Martinez orcid.org/0000-0002-6642-4514 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Maria Lluch-Senar Maria Lluch-Senar orcid.org/0000-0001-7568-4353 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Luis Serrano Corresponding Author Luis Serrano [email protected] orcid.org/0000-0002-5276-1392 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain ICREA, Barcelona, Spain Search for more papers by this author Raul Burgos Raul Burgos orcid.org/0000-0002-4452-9235 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Marc Weber Marc Weber orcid.org/0000-0001-7920-5655 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Sira Martinez Sira Martinez orcid.org/0000-0002-6642-4514 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Maria Lluch-Senar Maria Lluch-Senar orcid.org/0000-0001-7568-4353 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Luis Serrano Corresponding Author Luis Serrano [email protected] orcid.org/0000-0002-5276-1392 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain ICREA, Barcelona, Spain Search for more papers by this author Author Information Raul Burgos1, Marc Weber1, Sira Martinez1, Maria Lluch-Senar1 and Luis Serrano *,1,2,3 1Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain 2Universitat Pompeu Fabra (UPF), Barcelona, Spain 3ICREA, Barcelona, Spain *Corresponding author. Tel: +34 93 3160101; E-mail: [email protected] Molecular Systems Biology (2020)16:e9530https://doi.org/10.15252/msb.20209530 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 Abstract Protein degradation is a crucial cellular process in all-living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP-dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA-seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half-life measurements also suggest a role for Lon-modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation. SYNOPSIS A minimal protein degradation machinery required for maintaining proteome homeostasis is defined in the genome-reduced bacterium Mycoplasma pneumoniae. Genetic and high-throughput analyses identify substrates and pathways regulated by degradation. All except one of the proteases in M. pneumoniae are essential for cell survival. Phenotypic characterization, protease substrate analysis, and epistasis studies reveal distinct functions for Lon and FtsH, the only two ATP-dependent proteases in M. pneumoniae. Lon degrades misfolded proteins and unassembled subunits of protein complexes, whereas FtsH regulates the quality control of membrane proteins. Introduction Protein degradation is a key biological process that shapes the proteome of cells in response to internal and external signals. During stress conditions, misfolded and damaged proteins may accumulate in the cell with potential harmful consequences. In this scenario, chaperones and proteases play a key role as protein quality control factors assisting and reverting this situation through protein refolding or degradation (Mogk et al, 2011). Apart from maintaining protein homeostasis, protein degradation is also an efficient mechanism to induce changes in cell physiology (Van Melderen & Aertsen, 2009). Indeed, regulated proteolysis also occurs on intact proteins, like transcription factors or regulatory proteins responsible for physiological transitions (Mahmoud & Chien, 2018). Therefore, protein degradation can potentially regulate many biological processes depending on the variety of proteases and the possible substrates existing in a cell system. In bacteria, most intracellular proteolysis is mediated by the ATP-dependent proteases belonging to the AAA+ family proteins (Bittner et al, 2016), including ClpXP, Lon, and FtsH (common to both gram-positive and gram-negative bacteria), ClpAP and ClpYQ (only in gram-negative bacteria), and ClpCP and ClpEP (only in gram-positive bacteria). All of these form oligomeric structures with two functional domains: an ATPase domain with unfolding and translocating activities that is dependent on ATP-hydrolysis, and a protease domain with proteolytic activity. The Clp family of proteases is characterized to encode these domains in separate polypeptides, whereas Lon and FtsH have these domains encoded in the same protein sequence. Notably, FtsH is a membrane-anchored protein and the only essential protease in Escherichia coli (Ogura et al, 1999). Protein half-lives range from minutes to days, reflecting distinct degradation rates that generally are determined by the presence of degradation signals that trigger protease engagement. A degradation signal is normally referred to as a degron and can be intrinsically present in a protein or added by post-translational modifications. A common mechanism in both eukaryotes and prokaryotes is the N-end rule pathway, in which the identity of the N-terminal amino acids determines the half-life of the protein (Mogk et al, 2007). In prokaryotes, N-degrons are recognized by the protein adaptor ClpS, which delivers the substrate to the ClpAP protease (Erbse et al, 2006; Schmidt et al, 2009). N-degrons can also be created post-translationally, for example, by exposing destabilizing residues after proteolytic cleavage, or by adding Leu and Phe destabilizing residues to the N-terminus through the leucyl/phenylalanyl-tRNA-protein transferase (Ninnis et al, 2009). Similarly, adding a C-terminal tag by the bacterial tmRNA system promotes degradation of the modified protein (Keiler et al, 1996). Regardless of the mechanism, proteolysis is an energy-expensive and irreversible process and therefore must be strictly regulated. This is especially true for intracellular proteases, for which substrate recognition is very selective. In this respect, protein adaptors modulate substrate specificity, such as SspB, which recognizes and delivers tmRNA-tagged substrates to the ClpXP protease in E. coli (Levchenko et al, 2000; Kuhlmann & Chien, 2017). The accessibility of a degradation motif is also an important determinant factor regulating proteolysis. For instance, hydrophobic residues tend to be buried in the interior of a native protein but become exposed in misfolded proteins. This fact has been proposed to provide a discrimination factor to identify poor-quality proteins, as Lon and other quality control proteins tend to interact with hydrophobic regions (Rudiger, 1997; Chen & Sigler, 1999; Patzelt et al, 2001; Gur & Sauer, 2008). Even though substrate recognition is a common step for proteases, previous studies using substrate trapping methods have revealed that the substrate repertoire of cognate proteases varies within distinct prokaryotic systems and that the nature of the degradation signal seems to be diverse and generally sequence-independent (Liao & van Wijk, 2019). Conversely, there are also examples of substrate overlapping among distinct proteolytic systems, suggesting in some cases common mechanisms of recognition (Kuo et al, 2004; Tsilibaris et al, 2006; Lies & Maurizi, 2008). Most of the studies mentioned above have been performed in complex model organisms such as E. coli, Bacillus subtilis, Staphylococcus aureus, and Caulobacter crescentus (Flynn et al, 2003; Feng et al, 2013; Bhat et al, 2013; Trentini et al, 2016; Arends et al, 2016, 2018). As the complexity of these organisms makes it difficult to have an integrated view of the protein degradation machinery, we chose to study this machinery in the genome-reduced bacterium Mycoplasma pneumoniae, an important human pathogen that causes community-acquired pneumonia, and is considered to be one of the smallest known self-replicating organisms. In the past years, a large effort has been made to obtain and integrate large "omics" datasets to quantitatively understand the biology of this minimal cell model (Güell et al, 2009; Kühner et al, 2009; Maier et al, 2011, 2013; Chen et al, 2016; Trussart et al, 2017). Although major progress has been made in dissecting the distinct regulatory layers at the transcriptional level (Yus et al, 2019), how regulation occurs at the translational level is still poorly understood. To redress this gap of information, we have analyzed in-depth the impact of regulated proteolysis in the biology of M. pneumoniae, which lacks obvious protein adaptors and encodes only two essential ATP-dependent proteases, Lon and FtsH (Himmelreich et al, 1996; Lluch-Senar et al, 2015). To gain insight into the substrate repertoire of these proteases in M. pneumoniae, we used a quantitative proteomics approach combined with RNA expression data to define proteome changes after Lon and/or FtsH depletion. A total minimum of 62 Lon, and 34 FtsH, candidate substrates were identified, of which, some were validated by immunoblotting and in vivo degradation assays. We found an enrichment of FtsH substrates associated with the membrane, suggesting a key role of this protease in maintaining membrane protein homeostasis. Supporting this, the cell membrane integrity was found to be compromised after FtsH depletion. Lon candidate substrates were found associated with specific biological pathways, including cell division, DNA repair/recombination, and the restriction-modification system. Furthermore, mutational studies of selected Lon substrates identified specific degrons enriched in hydrophobic residues. Additionally, a genome-wide analysis of protein half-lives revealed an enrichment of short-lived proteins among Lon substrates. Overall, these results show that Lon has important regulatory functions in M. pneumoniae and suggest that this minimal organism has evolved to take advantage of the ability of Lon to recognize accessible hydrophobic regions, to regulate precisely the expression of functional native proteins. We also provide evidence that Lon has important roles in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. Finally, by integrating information from proteases and chaperones, we suggest an integrated model of how protein degradation takes place in this bacterium. Results Protease repertoire of Mycoplasma pneumoniae Mycoplasma pneumoniae possesses a small set of intracellular proteases, according to the MEROPS peptidase database (Rawlings et al, 2016) and manual annotation (Yus et al, 2019). This includes the Lon (MPN332) and FtsH (MPN671) ATP-dependent proteases and homologs to intracellular peptidases involved in peptide degradation, such as proline iminopeptidase Pip (MPN022), oligoendopeptidase F PepF (MPN197), X-Pro aminopeptidase PepP (MPN470), and leucine aminopeptidase PepA (MPN572). Several peptidases and proteases implicated in protein processing and maturation are also present, including the methionine aminopeptidase Map (MPN186), ribosomal-processing cysteine protease Prp (MPN326), and the lipoprotein signal peptidase Lsp (MPN293). The genes of all these proteins except for pip (MPN022) are essential for survival based on transposon essentiality studies (Lluch-Senar et al, 2015). Surprisingly, no gene encoding a type I signal peptidase (SPase) has been found in the genome, yet there is evidence supporting the presence of SPase-like activity in M. pneumoniae (Catrein et al, 2005). Out of all the intracellular peptidases and proteases found in M. pneumoniae; Lon is the protease that exhibit larger transcriptional changes and is more commonly affected in its expression upon different perturbations (Yus et al, 2019), whereas PepP, Lsp, and PepF are the ones that exhibit less variability in expression, suggesting a housekeeping-like behavior (Fig EV1A and B). Among the perturbations tested, glucose starvation was the condition that negatively disturbed the most the expression level among peptidases and proteases genes, except for lon (Fig EV1A). Genes encoding Map, Prp, Lon, and to a lesser extent Pip and FtsH were also induced by cold shock, suggesting that these proteases/peptidases could play a role in cold stress adaptation. Lon and PepA are encoded in operons regulated by the heat-shock transcription factor HrcA (MPN124), while Pip is in an operon with DnaJ (MPN021) that contains a degenerated motif for HrcA. According to this gene organization, all three genes correlate well with transcriptional changes affecting chaperones regulated by HrcA, including DnaJ (MPN021), DnaK (MPN434), ClpB (MPN531), GroEL (MPN573), and GroES (MPN574) (Fig EV1C). Click here to expand this figure. Figure EV1. Variability of transcriptional changes across perturbations for chaperons and proteases A–C. Analysis of transcriptional changes of proteases, peptidases, and chaperones across a set of 35 environmental and genetic perturbations taken from Yus et al (2019). (A) Fold changes in mRNA levels for proteases and peptidases. Genes and perturbations were clustered based on the similarity of their transcriptional change pattern. (B) Distribution (upper plot) and standard deviation (lower plot) of the log2 of mRNA fold changes for proteases, peptidases, and chaperones. (C) Pearson correlation coefficient of mRNA fold changes between pairs of genes in the chaperones, proteases, and peptidases functional groups. Genes were clustered based on the similarity of their correlation pattern. Download figure Download PowerPoint Construction of conditional Lon and FtsH mutants in Mycoplasma pneumoniae Lon and FtsH can target folded proteins for unfolding and subsequent digestion via their ATPase unfoldase activity (Sauer & Baker, 2011); in contrast, the other peptidases and proteases present in M. pneumoniae can only digest peptides or unfolded proteins. Thus, Lon and FtsH seem to be the main proteases of M. pneumoniae that have the capacity to control protein function through protein degradation. Isolation and characterization of null mutants has so far been hampered by the fact that Lon and FtsH are essential for cell growth. To study the cellular functions of these proteases, we overcame these difficulties by generating the first conditional mutants in M. pneumoniae. We used genome-editing tools (Piñero-Lambea et al, 2020) based on the phage recombinase GP35 (Sun et al, 2015) to control Lon and FtsH expression through a Tet-inducible system (see Materials and Methods and Appendix Fig S1). To determine the effect of the absence of both proteases, we also constructed a Lon/FtsH double mutant, by performing the same genome editing within the lon locus in the FtsH-inducible mutant. Lon and FtsH expression monitored by RNA-seq and Western blot assays under inducing and depleting conditions, showed good repression-induction transcriptional pattern, which correlated with protein expression (Fig 1A–C). Compared to the wild-type strain, the inducible system supported Lon and FtsH protein levels slightly below and above, respectively. Proteome-wide measurements of protein half-life revealed protein turnovers for Lon and FtsH of 13 and 52 h (Table EV1). Accordingly, Western blot analysis showed that complete depletion of Lon was not observed until 48 h after removing the inducer from the medium, whereas FtsH was significantly reduced after 72 h of depletion. Quantitative MS analysis indicated a 4-fold (log2) reduction for both proteases under these depleting conditions (Table EV2). Thus, unless otherwise indicated, depletion experiments for Lon and FtsH were performed at 48 and 72 h, respectively. Figure 1. Construction of Lon and FtsH conditional mutants in Mycoplasma pneumoniae A–C. RNA-seq transcriptional profiles across the modified locus, as well as immunoblots assessing protein expression of Lon and FtsH, are shown for ΔIndLon (A), ΔIndFtsH (B), and ΔIndLon_FtsH (C) grown under inducing or depleting conditions (48 and 72 h of depletion for Lon and FtsH, respectively). Symbols +/− indicate inducing or depleting conditions. LC, loading control. WT, wild-type. A schematic representation of the DNA rearrangements in the lon and ftsH locus is also shown for each strain. The ftsH-inducible platform inserted by transposon delivery is shown for ΔIndFtsH and ΔIndLon_FtsH strains. The Pxyl/tetO2-inducible promoter is highlighted with a red bent arrow and the terminator sequence used to isolate the promoter is represented by a hairpin structure. The tetR repressor gene and the resistance markers cat and tetM are indicated in purple, blue, and red, respectively. Download figure Download PowerPoint Phenotypic characterization of Lon and FtsH mutants To gain insights into the cellular functions of Lon and FtsH, we first assessed the effect of depletion of Lon and FtsH in cell growth. Consistent with their reported essentiality, depletion of either of both proteases inhibited growth based on pH (Appendix Fig S2) and cell biomass measurements (DNA and protein) along the growth curve (Fig 2A). Slow down of DNA replication was further confirmed by pulse-chase experiments using the analog bromodeoxyuridine (BrdU) (Appendix Fig S3). Figure 2. Phenotypic characterization of Lon and FtsH conditional mutants in Mycoplasma pneumoniae Cell growth assessment of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red). Growth was monitored by measuring DNA and protein biomass over time. The average from two independent biological replicates is shown. Protein aggregation in ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red, 48 h, and 72 h of depletion for Lon and FtsH, respectively). Protein aggregates in Triton X-100 insoluble fractions of untreated or heat-shock (15 min at 45°C)-treated cells were measured by Thioflavin (ThT) staining. Bars represent the mean of three biological replicates (dots). Significance of comparisons was assessed by two-sided independent t-test (exact P-values are shown). Assessment of cell membrane integrity of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red, 72 h of depletion for both, Lon and FtsH). Membrane integrity of untreated cells or after exposure during 30 min to 0.001% Triton X-100 was assessed by trypan blue exclusion staining. Bars represent the mean of three biological replicates (dots). Significance of comparisons was assessed by two-sided independent t-test (exact P-values are shown). Role of Lon and FtsH under heat-shock stress conditions. ΔIndLon and ΔIndFtsH mutants were grown under inducing (Lon+ or FtsH+) or depleting conditions (Lon- or FtsH-, 60 h of depletion for both, Lon and FtsH), and then exposed at 45°C during 0, 5, 10, 15, or 20 min. Then, growth after the heat treatment was monitored over time under inducing conditions by the 430/560 absorbance rate index that shows pH changes in the medium. The average from two independent biological replicates is shown for each condition. Download figure Download PowerPoint Next, we examined whether Lon or FtsH depletion resulted in increased protein aggregation, as a consequence of possible accumulation of misfolded proteins. For this, we obtained insoluble fractions after Triton X-100 solubilization from mutants grown under inducing or depleting conditions, and we stained them with Thioflavin-T (ThT), a commonly used fluorescent dye to monitor protein aggregation (Morell et al, 2008). As shown in Fig 2B, a significant increase in ThT fluorescence intensity was detected after Lon depletion, which was magnified when Lon mutants were exposed to heat stress conditions. In contrast, no significant differences were observed after FtsH depletion, suggesting that increased protein aggregation is a Lon KO-specific phenotype. We also explored the roles of Lon and FtsH in the maintenance of the cell membrane using a dye-exclusion assay. We found that depletion of FtsH, but not Lon, compromises the membrane integrity under normal and mildly membrane disruptive conditions (Fig 2C). In fact, we were unable to regrow FtsH mutant cells following FtsH depletion, suggesting important cellular damage (Fig 2D). This was not the case for the Lon-depleted mutant, which allowed us to assess the role of Lon under proteotoxic stress conditions. Consistent with a role in stress tolerance, Lon-depleted cells exhibited increased sensitivity to heat stress as compared to Lon-expressing cells (Fig 2D). Identification of Lon and FtsH candidate substrates To identify Lon and FtsH candidate substrates in M. pneumoniae, we performed label-free quantitative mass spectrometry to compare the proteomes of Lon and FtsH mutants grown in inducing or depleting conditions. A protein was considered as detected if at least one common unique peptide was found in the two biological replicates (see Materials and Methods). Using this criterion, we identified 494–547 proteins, which correspond to a proteome coverage of 67.1–74.3% with respect to all mutants and conditions analyzed. A total of 73 and 43 proteins were significantly upregulated after Lon or FtsH depletion, respectively, with protein fold changes higher than 2-fold (log2(protein_FC) ≥ 1), while 40 and 20 proteins were downregulated after Lon or FtsH depletion, respectively (log2(protein_FC) ≤ −1) (Appendix Fig S4). To define Lon and FtsH candidate substrates, we focused our attention on upregulated proteins, which we classified into differentially detected (DD) or differentially expressed (DE) (see Materials and Methods). As changes in protein levels could result from transcription regulation rather than a decrease in the degradation rate, we integrated RNA-seq data from the protease mutants grown in inducing or depleting conditions into the analysis (Fig 3A and Table EV2). To predict candidate substrates, we applied a cut-off criterion in which we selected DD proteins with fold changes of mRNA levels lower than 2-fold (log2(mRNA_FC) ≤ 1) (Appendix Fig S5), and DE proteins with protein/mRNA fold changes higher than 2-fold (log2(protein_FC/mRNA_FC) ≥ 1). As a result, we obtained a minimum of 62 and 34 candidate substrates for Lon and FtsH, respectively (Table EV3). After applying similar cut-off criteria, downregulation of 19 and 11 proteins could not be explained by decreased mRNA expression of their respective genes upon Lon or FtsH depletion, respectively. Intriguingly, one third of these 30 proteins represented lipoproteins (Table EV2). Figure 3. Identification and analysis of Lon and FtsH candidate substrates Transcriptional and protein abundance changes between inducing (+) and depleting (−) conditions (48 and 72 h of depletion for Lon and FtsH, respectively) in ΔIndLon and ΔIndFtsH mutant strains. Differentially expressed (DE) Lon/FtsH candidate substrates were identified as proteins with a significant increase in abundance upon protease depletion that could not be attributed to an increase in mRNA levels, i.e. log2(protein_FC/mRNA_FC) ≥ 1 (upper dashed line). Differentially detected (DD) candidates were identified as proteins not detected in the induced condition and detected in the depleted condition, and whose mRNA level did not increase more than 2-fold (vertical dashed line). Overlap of substrate candidates between the two mutant strains. Proportion of membrane and cytoplasmic proteins among candidate substrates. Distribution of half-lives measured by a SILAC time course experiment (average from 2 to 4 biological replicates) for Lon/FtsH candidate substrates as compared to the other proteins. Lon candidate substrates showed significantly shorter half-lives (MWW two-sided test, ***P = 6.53 × 10−4). Box shows the quartiles of the distribution, line shows the median, whiskers extend to 1.5 times the inter-quartile range past the low and high quartiles and define the limits for outliers, shown as points. Changes in the protein to mRNA fold changes ratio, log2(protein_FC/mRNA_FC), in the double mutant strain ΔIndLon_FtsH as compared to the predicted ratio, computed as the sum of the log2 ratios of both individual mutants. Proteins that showed a significant change in protein level in at least one of the individual mutants are shown (374 proteins). Proteins whose ratio deviated significantly from the predicted one (upper and lower dashed diagonal lines) reveal epistasis effects. Functional classification of candidate substrates. Validated substrates are highlighted in bold. Asterisks indicate candidate substrates classified as pseudogenes or truncated gene variants. Download figure Download PowerPoint Regarding the candidate substrates, only the ortholog of the TsrB glycosyltransferase (MPN028), which has a large cytoplasmic catalytic domain and two C-terminal transmembrane helices, was identified as a common substrate for both Lon and FtsH (Fig 3B), indicating that some intracellular proteins with transmembrane domains can be targeted by both proteases. The low degree of substrate overlapping suggests that Lon and FtsH perform distinct functions in M. pneumoniae protein homeostasis, probably due to their different cell location (cytoplasmic for Lon and membrane anchored for FtsH). Supporting this, 82.3% of the FtsH candidates were membrane-associated proteins, in contrast to 17.7% of the Lon substrates (Fig 3C; half of the Lon membrane-associated substrates have a predicted cytoplasmic domain larger than 25 amino acids). We also determined M. pneumoniae protein turnover rates by SILAC-based proteomics (Table EV1). Lon candidate substrates exhibited significant lower protein half-lives as compared to the average [Mann–Whitney–Wilcoxon (MWW) two-sided test, P = 6.53 × 10−4; Fig 3D and Table EV3]. We found that Lon targets were involved in several functional pathways, including cell division, the restriction-modification system, or DNA repair/recombination (Fig 3F). Substrate candidates were found particularly enriched in genes of the functional category "defense mechanisms" (two-sided Fisher test with multiple test correction, family-wise false discovery rate 5%). A putative toxin–antitoxin system, putative transporters, enzymes associated with different metabolic pathways, and proteins of unknown function complete the list. For the FtsH substrates, we identified three components of the Sec secretion pathway including SecD (MPN396), SecY (MPN184), and SecE (MPN068), proteins related to metabolism, some putative transporters, and numerous proteins of unknown function. Validation of Lon and FtsH substrates Our comparative proteomic analysis suggested that Lon regulates proteins associated with different cellular pathways, including cell division and the restriction-modification system. To further validate the potential substrates involved in these processes, we performed time course depletion experiments and in vivo degradation assays on a subset of candidate substrates. Mycoplasma pneumoniae ftsA (mpn316) and ftsZ (mpn317) cell division-related genes are transcribed in a single transcriptional unit together with mraZ (mpn314) and mraW (mpn315). Even though these genes are transcriptionally expressed at similar levels, the protein abundance of FtsA and FtsZ is significantly reduced as compared to MraZ and MraW (Fig 4A). While transcript levels remained unaffected, the protein abundances after Lon depletion of FtsA and FtsZ increased significantly, and doubled in the case of MraW. A similar situation was found
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