Where is srp located

We also observed that the elongation rate of MsbA was slower than that of FusA Figure 3E and Supplementary Figures 3B—F , which is consistent with the observation that the translation elongation speed of inner membrane proteins is slowed down during targeting, but not that of cytoplasmic proteins Fluman et al. We next examined the translation initiation rate by a computational model called the homogeneous ribosome flow model HRMF , in which the translation elongation rate is assumed to be constant Margaliot and Tuller, The translation initiation rate can be estimated by the measurable translation rate and translation elongation rate Supplementary Table 4.

The translation initiation rate in suppressor cells showed a similar trend to the translation elongation rate.

The initiation rate of FusA was not markedly changed in the suppressor and wild-type cells when grown at the same growth rate Figure 3F. However, the inner membrane protein MsbA had a slower translation initiation rate in suppressor cells than that in wild-type cells when grown at the same growth rate Figure 3F. Thus, in suppressor cells, the translation initiation process was negatively affected, although the formation of the 70S initiation complex was not markedly influenced Figure 3A.

As translation initiation factors play a vital role in translation initiation fidelity Ling and Ermolenko, , we addressed whether the suppressor was detrimental to the fidelity of start codon selection and initiator tRNA binding.

However, the expression level of GFP with the near cognate AUC as the start codon in the suppressor strain was significantly increased relative to that in the wild-type strain Figure 3G.

Thus, the fidelity of translation initiation in the suppressor cells was decreased compared with that in wild-type cells. Taken together, the suppressor cell trades translation speed and accuracy for cell survival in the absence of SRP. To test whether the suppressor mutation could suppress protein targeting defects, we first examined cell morphological changes by SEM and TEM.

SEM images showed that the suppressor strain MY still had typical rod morphology but had a rougher surface relative to the wild-type strain MG Figure 4A and Supplementary Figure 4. TEM images showed that the suppressor strain MY retained cell wall integrity but had damaged inner membrane structure Figure 4A and Supplementary Figure 4.

MY displayed a significant detachment of the inner membrane from the outer membrane Figure 4A and Supplementary Figure 4. Thus, the suppressor mutation partially offsets the negative effects of the loss of the SRP pathway on the inner membrane protein translocation. Figure 4. Suppressor mutation suppresses targeting defects of partial inner membrane proteins. For SEM, the scale bar is 1. For TEM, the scale bar is nM. C FtsQ left and EspP right targeting assay by their biotinylation.

To gain an insight into the localization of inner membrane proteins, we performed proteomic analysis of inner membrane proteins in the wild-type strain MG Zhao et al. According to our previous study, we identified SRP-dependent inner membrane proteins Zhao et al. Our previous study has shown that the inner membrane proteins with a high abundance, such as proteins C 4 -dicarboxylate sensor kinase DcuS and zinc transporter FieF, can be localized to the membrane Zhao et al. This suggested that the high protein abundance can be used as an indicator of protein localization.

We found that the abundance of many identified SRP-dependent inner membrane proteins in both MY and SRP — cells was higher than their abundance in wild-type cells Figure 4B and Supplementary Data Set 1D , indicating that these inner membrane proteins can target to the cytoplasmic membrane in the absence of SRP. This result is consistent with previous studies showing that inhibition of the SRP pathway only partially impedes inner membrane protein targeting Ulbrandt et al.

To examine the targeting level of FtsQ, we used a sensitive method based on protein biotinylation Jander et al. A small biotinylatable peptide Avi-tag was fused to the periplasmic domain of the targeted proteins. The biotinylated proteins would be the untargeted proteins in which the periplasmic domains are exposed in the cytosol. Thus, the protein biotinylation can be used for protein targeting assay. However, in contrast to the prediction of proteomic analysis, the FtsQ targeting showed a slight defect in both the SRP — and MY strains Figure 4C , suggesting that the suppressor mutation played little role in the targeting of FtsQ.

These results indicated that the SRP suppressor partially contributed to inner membrane protein targeting and allowed for targeting of some SRP-dependent proteins without causing a failure of targeting of SRP-independent proteins.

Additionally, the expression of heat shock response related chaperones and proteases was not upregulated Supplementary Figure 5B and Supplementary Data Set 1B , suggesting that the heat shock response played little role in compensating the loss of SRP, which is consistent with our previous study Zhao et al. This suggested that the component of the Sec translocon SecF may not be involved in the protein targeting process without SRP.

In contrast, the protein abundance of SecY and FtsY in MY was two times higher than that in the SRP — strain, which is likely caused by the effective targeting of inner membrane proteins with the assistance of translational control. Overall, protein transport components were unlikely to play a major role in mediating SRP-dependent protein targeting in the absence of SRP.

Co-translational protein targeting by SRP is an essential and conserved pathway that delivers most inner membrane proteins to their correct subcellular destinations Saraogi and Shan, Our previous work revealed that SRP was not essential in E. Isolation of suppressors is a useful strategy to provide insight into certain molecular mechanisms by suggesting which cellular component is involved in an inefficient process Lee and Beckwith, The SRP suppressors involved in protein translation initiation have been identified before, and these suppressors affect the translation process Zhao et al.

In this study, we obtained an SRP suppressor associated with protein translation too. The regulation of translation may be a general way to mediate the translocation of SRP-dependent proteins in the absence of SRP.

We observed that in suppressor cells, the ribosomal protein expression was upregulated Figure 2B and the 30S and 50S ribosomal subunits accumulated Figure 3A , but the content 70S ribosome complex was not markedly changed relative to those in the wild-type strain Figure 3A. This led us to propose that the increased ribosomes are inactive and accumulate in the cytosol.

Thus, the SRP suppressor and cellular stress responses may play an important role in ribosomal protein synthesis. In suppressor cells, the protein translation initiation was impeded Figure 3F , but the initiation time of translation was constant in wild-type and suppressor cells under different growth rates Supplementary Table 3 , which suggested that the pausing at the start of the initiation can be negligible, and the process of 70S ribosome complex entry into the elongation cycle is slower in suppressor cells.

Thus, the SRP suppressor may be associated with the transition from initiation to elongation. The closely related relationship between translation initiation and elongation Riba et al. Furthermore, we showed that the translation fidelity was decreased in suppressor cells Figures 3D,G. Because the fidelity of translation initiation is modulated by the initiation factors Ayyub et al. We observed that the fidelity of translation elongation was also decreased, implying that suppressor mutation may inactivate the quality control system.

Earlier works revealed that mistranslation could provide a growth advantage in response to stress Gu et al. Hence, the decreased fidelity of translation initiation and elongation may result from the SRP deletion stress response.

Increasing evidence has supported the notion that the translation elongation of nascent polypeptide regulates the targeting of SRP-dependent proteins du Plessis et al. Decreasing the translation elongation rate extends the time window for protein targeting, which plays a critical role in suppressing the loss of SRP Zhao et al.

The maximal SRP binding site is 55 amino acids from the ribosomal peptidyl transferase center in E. Thus, with the help of SRP, most translating ribosomes move to the membrane within this period in E. To get a longer time to find the membrane, the length of translating nascent chains is more likely longer than 55 amino acids. However, the nascent chain cannot exceed a specific length as aggregation would prevent protein from being targeted Siegel and Walter, ; Flanagan et al.

Proteins with fewer transmembrane domains TMDs or longer first loop lengths have a longer critical length Zhao et al. If the targeting time of some SRP-dependent proteins exceeds 10 s, these proteins would not be targeted to the inner membrane in suppressor cells. Taken together, this model shows that SRP greatly shortened the protein targeting time by 8 s, which minimizes the cost of targeting and maintains fast growth. Overall, our data suggest that in response to the deletion of SRP, suppressor cells attenuate translation elongation to give the translating ribosomes more time to find and target to the inner membrane.

Figure 5. The SRP suppressor extends the time window for protein targeting. The suppressor cells with a slower elongation rate extended the time window for protein targeting to 2—10 s. As expected, the suppressor mutation can partially offset the defective targeting of inner membrane proteins Figure 4B , which is consistent with the previous result Zhao et al. However, proper localization of these proteins cannot bypass the requirement of SRP Phillips and Silhavy, We speculated that the proteins that could be correctly located in the suppressor strain MY but not in SRP depletion strain SRP — may be responsible for cell survival.

We hypothesized that specific membrane protein targeting defects could block the essential cellular process, which would be responsible for the loss of cell viability. Among these localization defective proteins, only one protein PgsA is essential for E. PgsA catalyzes the step in the synthesis of the acidic phospholipids that are considered to be indispensable in multiple cellular processes Gopalakrishnan et al.

We inferred that mislocalization of PgsA inhibited cell growth. More studies are needed to investigate the targeting of some proteins that determine whether cells can survive without SRP. The SRP-dependent delivery pathway is essential for membrane protein biogenesis.

Previously, we reported that SRP was non-essential in Escherichia coli , and slowing translation speed played a critical role in membrane protein targeting. Here, we identified a novel SRP suppressor that is also involved in translation. We found that translation speed and accuracy regulate membrane protein targeting. A slowdown of translation speed extended the time window for protein targeting.

Meanwhile, a moderate decrease in translation fidelity ensured a suitable translation speed for better cell growth. These results argued that translation control could be a practical way to compensate for the loss of SRP. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. LZ and YC performed the experiments. LZ and DZ wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Dr. Akopian, D. Signal recognition particle: an essential protein-targeting machine. Allen, T. Phylogenetic analysis of L4-mediated autogenous control of the S10 ribosomal protein operon.

Ayyub, S. Contributions of the N- and C-Terminal domains of initiation Factor 3 to its functions in the fidelity of initiation and antiassociation of the ribosomal subunits.

Google Scholar. Bernstein, H. Physiological basis for conservation of the signal recognition particle targeting pathway in Escherichia coli. Functional substitution of the signal recognition particle kDa subunit by its Escherichia coli homolog. Bornemann, T. Signal sequence-independent membrane targeting of ribosomes containing short nascent peptides within the exit tunnel.

Caban, K. A conformational switch in initiation factor 2 controls the fidelity of translation initiation in bacteria. Callister, S. Normalization approaches for removing systematic biases associated with mass spectrometry and label-free proteomics.

Proteome Res. Chartron, J. Cotranslational signal-independent SRP preloading during membrane targeting. Nature , — Chen, I. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Methods 2, 99— Dai, X. Slowdown of translational elongation in Escherichia coli under hyperosmotic stress. Reduction of translating ribosomes enables Escherichia coli to maintain elongation rates during slow growth.

The Sec translocase. Egea, P. Targeting proteins to membranes: structure of the signal recognition particle. Elvekrog, M. Dynamics of co-translational protein targeting. Flanagan, J. Signal recognition particle binds to ribosome-bound signal sequences with fluorescence-detected subnanomolar affinity that does not diminish as the nascent chain lengthens.

Fluman, N. Gopalakrishnan, A. Structure and expression of the gene locus encoding the phosphatidylglycerophosphate synthase of Escherichia coli. Gu, C. FEBS Lett. Hasona, A. Membrane composition changes and physiological adaptation by Streptococcus mutans signal recognition particle pathway mutants.

Hecht, A. Measurements of translation initiation from all 64 codons in E. Nucleic Acids Res. Hughes, P. A novel role for cAMP in the control of the activity of the E. Cell 55, — Ishihama, A.

Autogenous and post-transcriptional regulation of RNA polymerase synthesis. Jander, G. Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion. Clin Proteomics Kikuchi, S. Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin. Kremer, B. Characterization of the sat operon in Streptococcus mutans : evidence for a role of Ffh in acid tolerance.

Lee, C. Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis. Lee, H. The targeting pathway of Escherichia coli presecretory and integral membrane proteins is specified by the hydrophobicity of the targeting signal. Ling, C. Initiation factor 2 stabilizes the ribosome in a semirotated conformation. Malecki, M. Characterization of the RNase R association with ribosomes.

BMC Microbiol. Margaliot, M. On the steady-state distribution in the homogeneous ribosome flow model. Mason, N. Elongation arrest is a physiologically important function of signal recognition particle.

EMBO J. Miller, J. Experiments in Molecular Genetucs. Mohler, K. Translational fidelity and mistranslation in the cellular response to stress. Mutka, S. Multifaceted physiological response allows yeast to adapt to the loss of the signal recognition particle-dependent protein-targeting pathway. Cell 12, — Naaktgeboren, N. The effect of initiation factor IF-1 on the dissociation of S ribosomes of Escherichia coli.

Newitt, J. The structure of multiple polypeptide domains determines the signal recognition particle targeting requirement of Escherichia coli inner membrane proteins. Ng, D. Signal sequences specify the targeting route to the endoplasmic reticulum membrane. Cell Biol.

Decoding fidelity at the ribosomal A and P sites: influence of mutations in three different regions of the decoding domain in 16S rRNA. Pechmann, S. The ribosome as a hub for protein quality control. Cell 49, — Phillips, G. Pool, M. Signal recognition particles in chloroplasts, bacteria, yeast and mammals review. Powers, T. Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor.

Prabhakar, A. Post-termination ribosome intermediate acts as the gateway to ribosome recycling. Cell Rep. Quan, S. Regalia, M. Prediction of signal recognition particle RNA genes. Riba, A. Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates.

Rodnina, M. Translation in prokaryotes. Cold Spring Harb. Saraogi, I. Co-translational protein targeting to the bacterial membrane. The malfunctioning of this mutant in membrane targeting was supported by the fluorescence microscopy Fig. Interestingly, mutation of the cysteine residue into the more hydrophobic valine restored membrane targeting indicating that not the cysteine residue but the hydrophobicity is critical for SRP binding Fig.

Finally, the positively charged residues at 22, 24 and 26 were investigated for membrane targeting and SRP binding. The sfGFP fusion protein showed that the fluorescence was evenly distributed in the cytoplasm suggesting that the binding to SRP is affected. This shows that the positively charged residues play an important role for the interaction with SRP. Taken together, this study shows that a SRP signal sequence is not restricted to a transmembrane segment but can be localized in a cytoplasmic region.

The results obtained with the mutants of the signal sequence underline the importance of the positively charged N-terminal part and the hydrophobic C-terminal part of the signal sequence to allow the interaction with the M-pocket of SRP and membrane targeting. For Ffh depletion, E. Media preparation and bacterial manipulations were performed according to standard methods All oligonucleotides used in this study are listed in the Supplementary Table S1 and the used plasmids in the Supplementary Table S2.

The coding regions of all constructs were verified by DNA sequence analysis. This results in RNCs with exposing a short nascent chain of about 13 amino acids. The plasmid encoding the first 50 amino acids of the cytoplasmic protein firefly luciferase was generated by amplification from plasmid pUCT7-Luc 50 kindly provided by Shu-ou Shan; Caltech. Beckmann, Munich. Emission was detected with filters specific for GFP.

Analysis was done by using the AxioVision Software Zeiss. Before cell disruption 0. After the addition of 0.

To get a functional SRP the purified Ffh protein was reconstituted with the in vitro synthesized 4. Therefore, plasmid pUC First, the plasmid was linearized with Bam HI and gel purified. Ffh and 1.

The data of three independently pipetted measurements were merged and analyzed using the software MO. Affinity Analysis v2. All data generated or analysed during this study are included in this published article and its Supplementary Information Files or are available from the corresponding author on reasonable request. Akopian, D. Cell Biol. Kuhn, A. Targeting and insertion of membrane proteins. EcoSal plus 7 , 1—27 Article Google Scholar.

Seluanov, A. FtsY, the prokaryotic signal recognition particle receptor homologue, is essential for biogenesis of membrane proteins. Doudna, J. Structural insights into the signal recognition particle. Luirink, J. SRP-mediated protein targeting: structure and function revisited. Acta , 17—35 Jomaa, A. Structure of the E. Denks, K. The signal recognition particle contacts uL23 and scans substrate translation inside the ribosomal tunnel.

Nature Microbio. Zopf, D. The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence. EMBO J. Peluso, P. Role of 4. Science , — Egea, P. Substrate twinning activates the signal recognition particle and its receptor. Nature , — Shan, S. Conformational changes in the GTPase modules of the signal reception particle and its receptor drive initiation of protein translocation.

Connolly, T. Requirement of GTP hydrolysis for dissociation of the signal recognition particle from its receptor. Keenan, R. The signal recognition particle. Welte, T. Mol Biol Cell 23 , — Valent, Q.

Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor. Mol Microbiol 25 , 53—64 Lee, H. The targeting pathway of Escherichia coli presecretory and integral membrane proteins is specified by the hydrophobicity of the targeting signal.

USA 98 , — Peterson, J. Basic amino acids in a distinct subset of signal peptides promote interaction with the signal recognition particle. Batey, R. Crystal structure of the ribonucleoprotein core of the signal recognition particle. Maier, K. An amphiphilic region in the cytoplasmic domain of KdpD is recognized by the signal recognition particle and targeted to the Escherichia coli membrane. Facey, S. The sensor protein KdpD inserts into the E.

Jung, K. Truncation of amino acids causes deregulation of the phosphatase activity of the sensor kinase KdpD of Escherichia coli. Heermann, R. The N-terminal input domain of the sensor kinase KdpD of Escherichia coli stabilizes the interaction between the cognate response regulator KdpE and the corresponding DNA-binding site.

Hainzl, T. Signal-sequence induced conformational changes in the signal recognition particle. Ullers, R. Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome.

Bornemann, T. Signal sequence-independent membrane targeting of ribosomes containing short nascent peptides within the exit tunnel. Zhang, X. Sequential checkpoints govern substrate selection during cotranslational protein targeting. Science , —60 Biogenesis of bacterial inner-membrane proteins. Life Sci. Pross, E. Membrane targeting and insertion of the C-tail protein SciP. Loh, B. The transmembrane morphogenesis protein gp1 of filamentous phages contains Walker A and Walker B motifs essential for phage assembly.

Viruses 9 , 73 Wertman, K. Gene 49 , —62 Seitl, I. The C-terminal regions of YidC from Rhodopirellula baltica and Oceanicaulis alexandrii bind to ribosomes and partially substitute for SRP receptor function in Escherichia coli. Mol Microbiol 91 , — Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Press Studier, F.

Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Calhoun, K. Total amino acid stabilization during cell-free protein synthesis reactions. Balzer, D. Acid Res. Functional and structural studies of C-terminally extended YidC. Dissertation University of Hohenheim opus Download references. We are grateful to Katja Maier and Sandra Facey who had initiated this project.

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Subjects Cellular microbiology Chaperones. Abstract KdpD is a four-spanning membrane protein that has two large cytoplasmic domains at the amino- and at the carboxyterminus, respectively. Results Membrane targeting of NsfGFP depends on SRP The integral sensor protein KdpD consists of two large cytoplasmic domains, located at the N- and C-terminus 1— and — , which are separated by four closely spaced transmembrane segments — Figure 1.

Full size image. Figure 2. Figure 3. Figure 4.