chloroplast transit peptide sequence

Green, red, and yellow signals indicate GFP, autofluorescence of chlorophyll, and the overlap between green and red fluorescent signals, respectively. Mol Cells. In vivo targeting and western-blot analysis of double-T block Ala substitution mutations in protoplasts. The T2, T4, and T7 domains have an interesting relationship. C, Western-blot patterns of reporter proteins. Protein extracts from the T3A/T6A:GFP-transformed protoplasts were separated into supernatant and pellet fractions by ultracentrifugation and these fractions were analyzed by western blotting using anti-GFP antibody. These results suggested that the motifs in T3 and T6 may be necessary for initial binding of the reporter protein to the components of the receptor complex. 3B, d). 2A). We present a neural network based method ~ChloroP! The hydrophobic N-terminal region may be necessary for binding of the transit peptide to the chloroplast through the interaction between the hydrophobic region and chloroplast lipid membranes (Perry et al., 1991; van't Hof et al., 1991, 1993; Pilon et al., 1995; Pinnaduwage and Bruce, 1996; Rensink et al., 1998). The 3′ fragments were amplified using the upper primers and the common nos-terminator primer, and thus contained the 18 bps 5′ to the mutated region, the mutated region, the GFP coding region, and the nos terminator. To examine the targeting of these mutants to chloroplasts at the biochemical level, protein extracts were prepared from transformed protoplasts and analyzed by western blotting using an anti-GFP antibody. Further analyses revealed that the N-terminal unfolded region of cargo proteins is critical for their localization capability, and that a length of about 20 amino acids is required to attain the maximum localization efficiency. 5B, j and k), indicating that it was imported efficiently into chloroplasts. As described above, among the individual-T Ala substitutions and deletions, only the T4 substitution or deletion noticeably reduces the targeting efficiency. However, T2, T4, and T7 do not share any sequence similarity, suggesting that the motifs in these regions may bind to different components of the receptor complexes or different sites of the same components. Hydroxylated amino acid residues such as Ser and Thr are highly represented in the transit peptide as a whole and are thought to be important for function (Zhang and Glaser, 2002). The other combinations of double-T mutants were examined for their ability to target the reporter protein to chloroplasts. As a control, protoplasts transformed with GFP were immunostained with anti-GFP antibody. Again, these results suggested that T8, which is the N-terminal region of the mature RbcS, played a role in translocation across the envelope membrane, as observed previously (Comai et al., 1988). Western-blot analysis of protein extracts using an anti-GFP antibody showed that 50% to 60% of the total reporter protein was in the M and the remainder was in the intermediate (Fig. J Biol Chem 271 , 32907–32915 (1996). Sequence alignments revealed that identical or similar sequence motifs are present in the transit peptides of ferredoxin-2 and Asp carbamoyltransferase (ATCase; Fig. 6C, b and e), indicating that LKSSA in T3 or KKFET in T7 completely rescued the targeting deficiency of T3A/T7A. FIG. In addition, the targeting efficiencies of T3A + APFNG/T7A and T3A/T7A + PPIGK were improved over that of T3A/T7A, although not to the extent that T3A + LKSSA/T7A or T3A/T7A + KKFET improved targeting (Fig. Both mutants displayed green fluorescent signals in the cytoplasm and the chloroplast at both time points, indicating that the targeting of these two mutants was less efficient than the wild type (arrows in Fig. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKFET in T7. These data indicated that a large number of Ser residues in T1 were detrimental for targeting. These results strongly suggest that T2, T4, and T7 are important for translocation of protein across the envelope membrane, possibly through interaction with components of the receptor complexes. Soluble aleurain was detected only in the supernatant, whereas a large fraction of γ-COP and all of AtVSR1 were detected in the pellet fraction, confirming that there was no cross contamination of the pellet and supernatant fractions by soluble and membrane proteins, respectively. B, Localization of reporter proteins. It is possible that Ala substitutions in T3 and T6 render the reporter protein susceptible to a protease.  |  However, two trends have emerged: cTPs tend to have a high content of hydroxylated amino acid residues such as Ser, Thr, and Pro, and only a few acidic amino acid residues such as Asp and Glu (von Heijne et al., 1989; Zhang and Glaser, 2002). 4C, l). Figure 1. The authors thank Dr. Hsou-min Li (Academi of Sinica, Nankang, Taiwan) for anti-Toc75 antibody. 1C, b). 5B, h), suggesting that the proteolysis was specific for T4A/T7A:GFP. 4C, a and b), indicating that motifs in T3 and T6 were important for the binding to chloroplasts. doi: 10.7717/peerj.9772. These results indicated that either FP or RK was almost sufficient for restoring the targeting efficiency of T4A to the wild-type level. cTPs are highly divergent in amino acid sequence, length, and organization (von Heijne et al., 1989; Bruce, 2001; Zhang and Glaser, 2002). Found in flowers, petals and fruits. The nature of this putative complex was unclear. The LKSSA sequence is part of a semiconserved motif, F/L/IGLKS/T/A, found in several transit peptides, including ferredoxin, Rubisco activase, plastocyanin, and LHCP-II (Pilon et al., 1995). The underlying cause of the failures remains an open question, and more effective CTPs are needed. Both the 37-kD form and the M were detected in the pellet but not in the supernatant (Fig. Thus, it has been suggested that the cTP is composed of multiple domains that may have different roles and/or overlapping functions that are required for import across the two chloroplast membranes (von Heijne et al., 1989; Pilon et al., 1995; Rensink et al., 1998, 2000). However surprisingly, the M increased only slightly with time, suggesting that Pr was degraded rapidly in the cytoplasm. Plant Cell. Sequence alignment of the transit peptides of RbcS, ATCase, and ferredoxin-2. CA1a is the ancestral state and that loss of a functional chloroplast transit peptide in N. munroi CA1a is associated with the evolution of C4 photosynthesis in Neurachne spp. 3C, d), indicating that restoration of RK to T4A improved the targeting efficiency to near the wild-type level. 7A). This was quite similar to the western-blot pattern of T4A:GFP (Fig. Two glyoxylate reductase isoforms are functionally redundant but required under high photorespiration conditions in rice. Classical examples and original systems (referenced) are shown. In agreement with the image analysis, the intermediate form was detected primarily in the pellet (Fig. 7A). Thus, the LKSSA sequence may be the site of guidance complex formation that is required for the efficient loading of the reporter protein to the receptor complex. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/T7A, T4A/T6A, and T4A/T7A, caused a 50% to 100% loss in targeting ability. These mutant transit peptides were fused to green fluorescent protein (GFP) and introduced into protoplasts by polyethylene glycol-mediated transformation (Jin et al., 2001; Chew et al., 2003; Lee et al., 2003). To further localize the reporter proteins, intact chloroplasts purified from gently lysed protoplasts were treated with thermolysin and analyzed by western blotting using anti-GFP antibody. 18 illustrates the alignment of the EPSPS chloroplast transit peptide sequences of Brassica napus (NCBI Accession No: P17688) and Brassica rapa (NCBI Accession No: AAS80163). One of the most prominent features of the cTP is its unusually long length. ↵[W] The online version of this article contains Web-only data. Analyses showed that RC2 can more effectively target diverse proteins, including EcTSR and EcGCL, into rice chloroplasts. In addition, T1MLM/AAA accumulated more precursor protein than T1ML/AA. T4A + FP:GFP was imported into chloroplasts as efficiently as the wild type (Fig. The amount of RFP increased gradually with time (Fig. See this image and copyright information in PMC. The primers are shown in the supplemental material. Similarly, an alternative processing site is used when transit peptides have mutations in the carboxy-terminal region of the transit peptide (Archer and Keegstra, 1993). 4B, a, b, e, and f). 4B, g). 2020 May 15;11:509. doi: 10.3389/fpls.2020.00509. Plastid transit peptides-where do they come from and where do they all belong? The plasmids were introduced into Arabidopsis protoplasts prepared from leaf tissues by polyethylene glycol-mediated transformation (Jin et al., 2001). Next, we examined whether the sequence motifs identified in the transit peptide of RbcS are also present in the transit peptides of other proteins. Mohr H. & Schopfer P.) 149–185 (Springer Berlin Heidelberg, Berlin, Heidelberg, 1995). A transit peptide-like sorting signal at the C terminus directs the Bienertia sinuspersici preprotein receptor Toc159 to the chloroplast outer membrane. Transformation with T3A/T7A:GFP produced no GFP signal (data not shown) even though the GFP protein level was comparable to that of the wild type (see western-blot data below). These results suggested that both the FP and RK motifs in T4 were critical for targeting a protein to the chloroplast and that at least one of the motifs was required for efficient targeting. Import intermediates are not usually observed in import assays using the RbcS precursor as the substrate in vitro. With these double-T deletion mutants, the processed M was almost undetectable throughout the time course, indicating that targeting was almost completely abolished (Fig. Samples taken at 12 h AT were fractionated by ultracentrifugation and the supernatant and pellet were analyzed by western blotting using anti-GFP antibody. 4C, c). However, the sequence information encoded by transit peptides is not fully understood. However, the identities of cytoplasmic factors that interact with the transit peptide remain elusive. Gaps were introduced to maximize the alignment. Furthermore, as expected, membrane-associated γ-COP was completely solubilized by 0.1 m Na2CO3 and the integral membrane protein AtVSR1 could only be completely solubilized by 1.0% Triton X-100. The authors declare no competing financial interests. In vitro interaction between a chloroplast transit peptide and chloroplast outer envelope lipids is sequence-specific and lipid class-dependent. 3C, d), which was much larger than Pr, may have been T4A + RK:GFP that was associated tightly with a protein complex, even under SDS/PAGE conditions. bbruce@utk.edu T1A + 3C, T1A+ML, and T1A+LL mutants, which had three Cys, one Met and one Leu, and two Leu, respectively, also were generated in the T1A background (Fig. T4 also has a functional relationship to T6. In contrast, T2A/T7A:GFP yielded two protein species at 31 and 37 kD (Fig. As a control for the fractionation, we detected aleurain and Toc75 using anti-aleurain and anti-Toc75 antibodies, respectively, and found that aleurain and Toc75 were detected only in the supernatant and pellet fractions, respectively. 2020 Jul 29;20(1):357. doi: 10.1186/s12870-020-02568-0. These results were quite similar to those obtained with the individual-T Ala substitution mutants. It is there that a signal peptidase cleaves the stromal transit peptide, only to reveal a second transit peptide sequence underneath; this time directing to the thylakoid membrane. Definition. This was slightly different from the fractionation pattern of T4A:GFP that yielded 50% of the 37-kD form in the pellet. DOI: https://doi.org/10.1104/pp.105.074575. Basso MF, Arraes FBM, Grossi-de-Sa M, Moreira VJV, Alves-Ferreira M, Grossi-de-Sa MF. However, the intermediate disappeared rapidly with no increase in the 31-kD M, indicating that T4A/T7A:GFP was subject to proteolysis, as observed with T3A/T6A:GFP (Fig. Candidate mitochondrial targeting cell Table 3.3. Consistent with our data, a deletion (Δ6-14) in the N-terminal region of ferredoxin that has uncharged amino acids causes the transit peptide to lose its ability to penetrate into the monogalactosyl-diacylglycerol monolayer (Pilon et al., 1995). Previous studies have identified or suggested that components of chloroplast import receptors such as Toc (translocon at the outer envelope of chloroplasts) 159, Toc75, and Toc34 bind to domains or sites in the N-terminal region of the cTP in vitro (Gutensohn et al., 2000; Hinnah et al., 2002; Jarvis and Soll, 2002; Becker et al., 2004; Kessler and Schnell, 2004; Smith et al., 2004). The T4 region is the most critical for targeting to chloroplasts. Various chloroplast transit peptides (CTP) have been used to successfully target some foreign proteins into chloroplasts, but for other proteins these same CTPs have reduced localization efficiencies or fail completely. However, in contrast to T3A/T6A:GFP, the amount of T4A/T6A:GFP was not reduced over time, suggesting that T4A/T6A:GFP was not susceptible to protease despite its localization in the cytoplasm. One possibility was that RK was part of a larger motif and that additional neighboring amino acid residues were necessary for full activity. To further characterize the distribution of T2A/T4A:GFP and T2A/T7A:GFP, protein extracts were separated into supernatant and pellet fractions by ultracentrifugation. 1A). Furthermore, the transit peptide of ferredoxin-2 was found to contain a copy of the LKSS motif of T3 present in the RbcS transit peptide. Bar = 20 μm. To confirm that this putative proteolytic degradation was specific for T3A/T6A:GFP, RFP and T3A/T6A:GFP were cotransformed into protoplasts and RFP was detected by western-blot analysis using anti-RFP antibody. J. Biol Chem 277, 43888–43894 (2002). T3A/T6A:GFP produced diffuse GFP signals in the cytoplasm, but not in the chloroplasts, at both 12 and 24 h AT (Fig. Transit sequence requirements for protein targeting to various subplastidial compartments. Subcellular localization of various rCTP fusion proteins. 47770-47778 View Record in Scopus Google Scholar 5C, a). 4C, d), confirming that the proteolysis was specific for T3A/T6A:GFP. 6B, e), whereas the targeting efficiency of T3A/T6A + GGRVN improved marginally, to 30% to 40% of that of T3A (Fig. There are at least three ways for the protein to go through the thylakoid membrane: through a ATP-hydrolyzing Type II secretion system , through a SecY translocon, or through the Tat/VSP pathway . Protein extracts from protoplasts were analyzed by western blotting using anti-GFP antibody. In addition, the Toc components may bind to these sites. Leaf tissues were harvested from 2-week-old plants and used immediately for protoplast isolation. Mol Plant. These results suggested that the restoration of RK to T4A rescued T4A just enough to translocate the reporter protein to the point where the stromal processing peptidase could cleave the transit peptide, but the M still remained in the channel. Coexpressed RFP was not detected in the purified chloroplast fraction, confirming that it was not contaminated by cytoplasmic proteins. To investigate the sequence motifs in the cTPs, Ala substitution mutants of the Arabidopsis (Arabidopsis thaliana) RbcS transit peptide were generated. The original sequences of subregions of T3, T6, or T7 were restored in each of the double-T mutants. 1C), indicating that these mutant transit peptides were able to deliver reporter proteins into chloroplasts. Bar = 20 μm. The transit peptides of seven representative proteins were analyzed by the ChloroP algorithm to predict the cleavage sites, which are indicated by arrowheads. We do not capture any email address. Determinants of the Specificity of Protein Targeting to Chloroplasts or Mitochondria. However, the chimeric substrate Oee1-Dhfr produced import intermediates under some conditions (Wu et al., 1994). T4A:GFP displayed a different staining pattern. The PCR products were subcloned into a vector and sequenced. ΔT3/T4:GFP and ΔT3/T6:GFP were detected primarily in the supernatant fraction, whereas ΔT4/T7:GFP and ΔT6/T7:GFP yielded significant amounts of reporter protein in the pellet (Fig. Images were taken with a cooled CCD camera and a Zeiss Axioplan fluorescence microscope (Zeiss), and presented in pseudocolor format (Jin et al., 2001; Kim et al., 2001). In a previous study, the LKSS motif in the transit peptide of ferredoxin-2 was shown to be part of the binding site of a 14-3-3 protein (Pilon et al., 1995). They also contain the potential amphiphilic a-helix characteristic of mitochondrial presequences. Next, we examined accessibility of T4A/T7A:GFP to thermolysin and found that the 37-kD form of T4A/T7A:GFP was thermolysin sensitive (Fig. This semiconserved motif forms the most stable structure in a transit peptide and plays a critical role in protein targeting (Pilon et al., 1995; Rensink et al., 1998; Wienk et al., 2000). In agreement with the image analyses, the western-blot patterns of T2A/T5A:GFP, T2A/T6A:GFP, and T2A/T8A:GFP were nearly identical to that of the wild type (data not shown). 5B, g). 7B, c). As controls for the fractionation and chemical treatments, we detected aleurain, γ-COP, and AtVSR1 using anti-aleurain, anti-γ-COP, and anti-VSR antibodies, respectively. Mohr H. & Schopfer P. In Plant Physiology (ed. Pr, Precursor form; M, mature form; T, total; P, pellet fraction; and S, supernatant fraction. In this study, we investigated sequence motifs in the transit peptide of the small subunit of the Rubisco complex by examining the ability of various mutant transit peptides to target green fluorescent protein reporter proteins to chloroplasts in Arabidopsis (Arabidopsis thaliana) leaf protoplasts. The subcellular localization of the reporter proteins in protoplasts showed that most mutants (T2A, T3A, T5A, T6A, T7A, and T8A) had GFP patterns that were nearly identical to that of the wild type (Fig. In agreement with the image analysis, most of Pr was detected in the supernatant (Fig. The targeting efficiency of T3A + LKSSA/T6A improved greatly and was comparable to that of T3A or T6A (Fig. 7E). These data indicated that T1A:GFP was imported into chloroplasts slowly in comparison to the wild type. 4B, c). Therefore, as an alternative approach, serial deletion mutants, ΔT2 through ΔT7, with a single-T deletion were generated (Fig. A related service TargetP predicts the subcellular location of proteins by integrating predictions of chloroplast transit peptides, signal peptides and mitochondrial targeting peptides. To determine which amino acid residues in T1 played a critical role in chloroplast targeting, T1 was subdivided into two regions of five amino acids each, D1 and D2, and each region was replaced with five Ala residues (Fig. A, Sequences of individual-T block Ala substitution mutations. T4A/T6A:GFP produced a diffuse GFP pattern in the cytoplasm, together with a ring pattern around the chloroplasts, at 12 and 24 h AT (Fig. To examine the thermolysin sensitivity of the reporter proteins, intact chloroplasts were purified from gently lysed protoplasts and were treated with thermolysin, as described previously (Li et al., 1991). RFP was detected with an anti-RFP antibody. 3D, b). Next, double-T mutants T4A/T6A, T4A/T7A, and T4A/T8A were generated and fused to GFP (Fig. Figure 2. In this study, the RbcS transit peptide was divided into eight blocks of eight or 10 amino acids, T1 through T8, and each block was replaced with the corresponding number of Ala residues to generate the mutants T1A through T8A (Fig. Additionally, the transit peptide residues upstream of the signal peptide peptidase cleavage site in many cases is insufficient to target GFP efficiently to the chloroplast, but the addition of some downstream sequence restores; ; ; ; for identifying chloroplast transit peptides and their cleavage sites. These data indicated that the sequence motifs in T2, T4, and T7 were important for crossing the chloroplast envelope membrane. These mutant transit peptides fused to GFP were transiently expressed in protoplasts and their localization was examined. Again, GFP signals in the chloroplasts were much stronger at 24 h AT than at 12 h AT, indicating that T4A:GFP was imported slowly. -. Ala substitutions inevitably introduced a new secondary structure into the transit peptide that could affect targeting efficiency. Green, red, and yellow signals indicate GFP, autofluorescence of chlorophyll, and the overlap between green and red fluorescent signals, respectively. In the cases of T4A/T7A:GFP and GFP alone, protoplasts were fixed and immunostained with anti-GFP antibody followed by FITC-labeled anti-rabbit IgG. The data herein clearly demonstrate that the transit peptides have multiple sequence elements with complex relationships. γ-COP, a component of the COPI vesicle, is peripherally associated with the Golgi membrane, and AtVSR1, a vacuolar sorting receptor, is an integral membrane protein. To address this question, double-T mutants were generated in which two 10-amino acid blocks in the transit peptide were substituted with the corresponding number of Ala residues (Fig. In contrast, a majority of outer envelope membrane proteins are targeted without a cleavable transit peptide (Lee et al., 2001, 2004; Tu et al., 2004; Hofmann and Theg, 2005). It is predicted from the genome sequence of Arabidopsis thaliana that up to 14% of nuclear genes encode precursor proteins with a transit peptide ( 1 , 2 ). In T1 of the transit peptide, the hydrophobic nature of the ML motif is important for the initial rate of targeting. Author information: (1)Department of Biochemistry and Cellular & Molecular Biology and The Center for Legume Research, University of Tennessee, Knoxville, Tennessee 37996-0840, USA. NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. 4C, c). Pr, Precursor; M, mature form; and asterisk (*), unknown 65-kD form. T4A:GFP showed a western-blot pattern that differed from the other mutants, as well as from the wild type. The amount of Pr was greatly reduced at 24 h AT and barely detectable at 36 h AT. This site needs JavaScript to work properly. Here we demonstrate that the cTP of RbcS consists of a complex set of sequence motifs and that some of these sequence motifs display complex functional relationships during protein targeting to chloroplasts. The targeting capability of rCTP…. Expression analyses of EcTSR and…, Figure 1. & Herrmann R. G. Domain structure of mitochondrial and chloroplast targeting peptides. These motifs likely serve as binding sites for proteins or membranes at various steps during translocation (Richter and Lamppa, 1999; Gutensohn et al., 2000; Rial et al., 2000; Hinnah et al., 2002; Zhang and Glaser, 2002; Becker et al., 2004; Smith et al., 2004). These results strongly suggested that hydrophobicity in T1 was critical for protein targeting to chloroplasts. The protein blots were developed with an ECL kit (Amersham Pharmacia Biotech) and images were obtained using a LAS3000 image capture system (FUJIFILM). Identification of critical sequence motifs in T3, T6, and T7. Protein extracts from protoplasts transformed with the indicated constructs were separated into soluble and pellet fractions by ultracentrifugation, before or after treatment with 0.1 m Na2CO3 or 1% Triton X-100. Under the same conditions, aleurain and Toc75 were detected in the supernatant and pellet fractions, respectively, confirming that the fractionation experiment had been performed properly. To confirm the results obtained from image analysis of GFP signals in protoplasts, western-blot analysis was performed using protein extracts from transformed protoplasts. In vivo targeting experiments using various Ala substitution mutants show that sequence motifs for protein targeting to chloroplasts are distributed throughout the entire transit peptide. The targeting efficiency of rCTP…, Figure 4. In vivo targeting of deletion mutants in protoplasts. 6B, b), whereas the targeting efficiency of T3A + APFNG/T6A improved moderately, to 40% of that of T3A (Fig. It is possible that the presence of the T3 motif may allow a portion of the precursor proteins to bind to the chloroplasts, but the absence of T4 motifs may prevent the mutant transit peptide from translocating through the import channel. Thus, these double-T mutants could not be evaluated. 2020 Aug 27;8:e9772. Plant Direct. In certain cases, RFP was cotransformed into protoplasts together with GFP constructs. Under these conditions, reporter proteins present in the cytoplasm and the stroma of chloroplasts should be detected in the supernatant, whereas the reporter proteins accumulated in envelope membranes via interaction with lipid membranes or receptor components, and trapped in the import channels should be detected in the pellet. Interestingly, the CMQVW sequence in T6 contains the putative processing site of the stromal processing peptidase (Gavel and von Heijne, 1990). In T1, the hydrophobicity of ML is important for targeting. HHS C, Western-blot patterns of reporter proteins. Under the same conditions, the integral membrane AtVSR1 protein was completely solubilized (Fig. In vivo import experiments in protoplasts reveal the importance of the overall context but not specific amino acid residues of the transit peptide during import into chloroplasts. The 36- and 37-kD protein species that migrated faster than the precursor may have been intermediate forms. Chromoplasts are plastids containing pigments other than chlorophyll. With these primers, the first round of PCR was performed to generate 5′ and 3′ fragments, using as the template RbcS-nt:GFP in an expression vector. tion, chloroplast transit peptides from C. reinhardtii are more similar to mitochondrial targetting peptides than to chloroplast transit peptides from higher plants. The intermediate of T3A/T8A:GFP was reduced gradually over time as M increased, indicating that import occurred slowly. Values used for reasoning Node Answer View Substring Value(s) Plot 1. RFP was detected with an anti-RFP antibody. -, Heijne G. V., Steppuhn J. These deletion mutants were fused to GFP and transiently expressed in protoplasts to examine their targeting efficiency. In addition, the data obtained with Ala substitution mutants were confirmed with individual- or double-T deletion mutants.

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