chloroplast transit peptide sequence

Classical examples and original systems (referenced) are shown. Two glyoxylate reductase isoforms are functionally redundant but required under high photorespiration conditions in rice. Mutants with Ala substitutions in neighboring two T blocks, resulting in 20 consecutive Ala residues, were almost undetectable by either GFP imaging or western-blot analysis. 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. 7C, d) as observed with T4A/T7A:GFP (Fig. Pr, Precursor form; M, mature form; I, intermediate form; T, total; P, pellet fraction; and S, supernatant fraction. 2020 Aug 27;8:e9772. COVID-19 is an emerging, rapidly evolving situation. These proteins were not made soluble by Na2CO3 or Triton X-100 treatments (Fig. 5B, h), suggesting that the proteolysis was specific for T4A/T7A:GFP. Evolution and Design Principles of the Diverse Chloroplast Transit Peptides. Mohr H. & Schopfer P. In Plant Physiology (ed. The single-T deletion mutants, with the exception of ΔT4, primarily yielded the M (Fig. At a primary structural level, transit peptide sequences are highly divergent in length, composition and organization. Chromoplasts are plastids containing pigments other than chlorophyll. D and E, Subcellular localization of reporter proteins. To define critical amino acid residues in T4, two mutants, T4A + FP and T4A + RK, were generated by restoring FP and RK in the first and second halves of T4, respectively (Fig. In addition, protein extracts were fractionated and detected as described for Figure 1D. Mol Cells. The targeting efficiency of the mutant transit peptide T3A/T7A is reduced by more than 80%. The upper primer consisted of two or three different regions: a 5′ region of 18 bases flanking the 5′ side of the mutated region, a central region of a variable number of bases depending on the replaced amino acid residues, and a 3′ region of 18 bases flanking the 3′ side of the mutated region. eCollection 2020 Feb. Eseverri Á, López-Torrejón G, Jiang X, Burén S, Rubio LM, Caro E. Plant Biotechnol J. When protein extracts were fractionated, Pr was equally distributed in the supernatant and pellet fractions (Fig. Clipboard, Search History, and several other advanced features are temporarily unavailable. CAS Thus, Cys is more hydrophobic than Ser. 4C, i), indicating that T3A/T7A:GFP accumulated at the envelope membrane as the intermediate form. Thank you for your interest in spreading the word on Plant Physiology. Figure 2. Analysis of chloroplast import of these mutants revealed several interesting observations. It is thought that two to three thousand different proteins are targeted to the chloroplast, and the ‘transit peptides’ that act as chloroplast targeting sequences are probably the largest class of targeting sequences in plants. RFP was detected with an anti-RFP antibody. Cys is similar in structure to Ser, with a sulfhydryl group instead of a hydroxyl group. Construction of RbcS-nt:GFP was described previously (Lee et al., 2002). 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. Expression analyses of EcTSR and…, Figure 1. A, Sequences of T4 substitution mutations. Transit peptide sequences located in the N-terminus of these proteins facilitate the transfer from the cytoplasm where they are synthesized, back to the chloroplast organelle (1 ; reviewed in 2). A, Sequences of individual-T block Ala substitution mutations. In the case of GFP alone, gently lysed protoplasts were treated with thermolysin. C, Western-blot patterns of reporter proteins. 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. These results raised the possibility that T4A/T7A is trapped in the import channel, although we cannot completely exclude the possibility that it may form a high Mr complex. However, close inspection of the western-blot patterns showed that ΔT2/T3:GFP, ΔT3/T4:GFP, ΔT3/T6:GFP, and ΔT6/T7:GFP produced Pr, whereas ΔT4/T7:GFP produced the 34-kD intermediate form (Fig. We considered that the unfolded region may alleviate the steric hindrance produced by the cargo protein, by functioning as a spacer to which cytosolic translocators can bind. D, Thermolysin sensitivity of reporter proteins. The N-terminal region of pSSU (precursor of Rubisco small subunit) interacts with Toc159 with high affinity (Becker et al., 2004). These data indicated that LKSSA was the critical motif in T3. The results obtained from the import experiments with single-T mutants strongly suggested that the absence of a sequence motif was tolerated or was compensated for by other sequence motifs in the transit peptide. These results indicated that Ala substitution in T4 and T6 resulted in complete inhibition of protein import at very early stages. 3B). In addition, the data obtained with Ala substitution mutants were confirmed with individual- or double-T deletion mutants. The protein blots were developed with an ECL kit (Amersham Pharmacia Biotech) and images were obtained using a LAS3000 image capture system (FUJIFILM).  |  As controls for the fractionation and chemical treatments, aleurain, γ-COP (coat protein), and AtVSR (Arabidopsis vacuolar sorting receptor) were detected using anti-aleurain, anti-γ-COP, and anti-VSR antibodies, respectively (Ahmed et al., 1997; Pimpl et al., 2000; Sohn et al., 2003). In addition, aleurain and Toc75 were detected by anti-aleurain and anti-Toc75 antibodies, respectively. ↵1 This work was supported by a grant from the Creative Research Initiative program of the Ministry of Science and Technology (Korea). A, Sequences of deletion mutations. 2, B, g–l, C, d–f). 1B, d). However, the sequence information encoded by transit peptides is not fully understood. Pr, Precursor form; M, mature forms; and h, hour. Figure 1. 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. 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). The C-terminal region of the prSSU transit peptide is an example of an amphipathic sequence within a chloroplast transit peptide. However, the identities of cytoplasmic factors that interact with the transit peptide remain elusive. The transit peptide sequences of the ferredoxin and plastocyanin clones have enabled us to analyze the role of the transit peptide in the routing of proteins … The GFP pattern of T4A/T6A:GFP was quite similar to that of T3A/T6:GFP (Fig. We divided the transit peptide into eight blocks (T1 through T8), each consisting of eight or 10 amino acids, and generated mutants that had alanine (Ala) substitutions or deletions, of one or two T blocks in the transit peptide. 3D, b). In addition, the KK motif in the transit peptide of ATCase may be equivalent to the RK motif in the transit peptide of RbcS. The targeting efficiency of another group of mutants, T4A/T6A:GFP, T4A/T7A:GFP, and T4A/T8A:GFP, was examined. T4 accounts for 50% to 60% of the translocation activity of the RbcS transit peptide crossing the envelope membrane and T2 and/or T7 accounts for the remaining activity, suggesting that multiple motifs are required for efficient translocation. Insights Into Genetic and Molecular Elements for Transgenic Crop Development. Green, red, and yellow signals indicate GFP, autofluorescence of chlorophyll, and the overlap between green and red fluorescent signals, respectively. To determine the targeting efficiency at the biochemical level, as well as the form of the reporter proteins, western-blot analysis was performed with protein extracts from the transformed protoplasts. Plasmids containing fusion genes of rCTP-EcKAT-GFP…, Figure 3. B, Localization of reporter proteins. 4C, f and g), indicating that T3A/T7A:GFP accumulated at the chloroplast envelope membrane but was not imported into the chloroplast. Protein extracts from protoplasts transformed with the indicated constructs at the indicated time points were used for western-blot analysis. Transit sequence requirements for protein targeting to various subplastidial compartments. 2020 Feb 11;4(2):e00188. In addition, as indicated above, FP is part of the sequence that is similar to the proposed binding site of 14-3-3 (May and Soll, 2000; Bruce, 2001).The western-blot patterns obtained with T4A + RK:GFP and T4A + FP:GFP differ slightly: T4A + FP:GFP yields only M, whereas T4A + RK:GFP yields a small amount of 36- and 37-kD intermediates together with a very slowly migrating protein species of 65 kD, in addition to M. These protein species are tightly associated with the particulate fraction and are resistant to solubilization by Triton X-100 or Na2CO3. In addition, the targeting efficiency of T3A/T6A + CMQVW was equivalent to that of T3A or T6A (Fig. When protein extracts were fractionated, the 37-kD form of T4A/T7A:GFP was detected in the pellet (Fig. To further examine the membrane association of T4A/T7A:GFP, protein extracts that had been treated with Triton X-100 were fractionated into soluble and membrane fractions. Would you like email updates of new search results? 5C, a). B, Localization of reporter proteins. 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. However, the protein species at 65 kD (asterisk in Fig. Candidate mitochondrial targeting cell Table 3.3. The 37-kD form was thermolysin sensitive (data not shown), consistent with the hypothesis that T2A/T7A:GFP was trapped in the import channel with the GFP moiety exposed to the cytoplasm. Detailed analyses revealed that sequence motifs ML in T1, LKSSA in T3, FP and RK in T4, CMQVW in T6, and KKFET in T7 play important roles in chloroplast targeting. In vivo targeting and western-blot analysis of double-T block Ala substitution mutations in protoplasts. Protein extracts were prepared from the transformed protoplasts as described previously (Jin et al., 2001). 2A). chloroplast-targeted transit peptide (cTP) is not clearly understood. To investigate the sequence motifs in the cTPs, Ala substitution mutants of the Arabidopsis (Arabidopsis thaliana) RbcS transit peptide were generated. Both the 37-kD form and the M were detected in the pellet but not in the supernatant (Fig. 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. The lipid-binding domain of the precursor form (Pr) of RbcS has been mapped to the C-terminal 20 amino acids of the cTP (Pinnaduwage and Bruce, 1996). TargetP-2.0 server predicts the presence of N-terminal presequences: signal peptide (SP), mitochondrial transit peptide (mTP), chloroplast transit peptide (cTP) or thylakoid luminal transit peptide (luTP). Protoplasts were transformed with the indicated constructs and GFP patterns were observed 12 and 24 h AT. Definition. A collection of 32 stroma‐targeting chloroplast transit peptides with known cleavage sites have been analysed in terms of amino acid preferences in the vicinity of the processing site. 7C). As mentioned above, T7 is thought to play a role in crossing the envelope membrane. These results strongly suggested that in the absence of the T4 motif, the reporter protein had difficulty passing through the import channel. 7A). The major M from the ΔT2, ΔT3, and ΔT7 mutants migrated at 31 kD, as did the wild-type protein. One of the most prominent features of the cTP is its unusually long length. Components of the Toc and Tic complexes have been identified and characterized at the molecular level (Gutensohn et al., 2000; Hinnah et al., 2002; Becker et al., 2004; Smith et al., 2004). 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. 5C, c). The targeting efficiency of T3A + APFNG/T7A, T3A + LKSSA/T7A, T3A/T7A + PPIGK, and T3A/T7A + KKFET was examined by western-blot analysis (Fig. DOI: https://doi.org/10.1104/pp.105.074575. Bar = 20 μm. 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). 7B, c). Sequence alignments revealed that identical or similar sequence motifs are present in the transit peptides of ferredoxin-2 and Asp carbamoyltransferase (ATCase; Fig. Chloroplast transit peptide: As used herein, the term “chloroplast transit peptide” (CTP) (or “plastid transit peptide”) may refer to an amino acid sequence that, when present at the N-terminus of a polypeptide, directs the import of Among the mutants T3A/T5A:GFP through T3A/T8A:GFP, T3A/T5A:GFP had strong GFP signals in chloroplasts at both 12 and 24 h AT, indicating that T3A/T5A:GFP was imported efficiently into chloroplasts (data not shown). Despite numerous studies, the information in the chloroplast-targeted transit peptide (cTP) is not clearly understood. To determine the subcellular distribution of the various protein species derived from the reporter protein, protein extracts were separated into supernatant and pellet fractions by ultracentrifugation and the fractions were analyzed by western blotting using anti-GFP antibody. In addition, the Toc components may bind to these sites. 1D, c). T4A + RK:GFP also yielded over 90% of the total reporter protein in M (Fig. In vivo targeting of deletion mutants in protoplasts. In the case of T4A:GFP, the 37-kD form was nearly equally distributed between the supernatant and pellet fractions, whereas more of the M was found in the supernatant than in the pellet (Fig. However, the staining pattern of T1A:GFP was clearly different from that of the wild type (Fig. 4C, j). However, at 24 h AT, the diffuse cytoplasmic pattern was no longer observed and GFP signals were detected only in the chloroplasts (Fig. Author information: (1)Department of Biochemistry and Cellular & Molecular Biology and The Center for Legume Research, University of Tennessee, Knoxville, Tennessee 37996-0840, USA. The amount of Pr was greatly reduced at 24 h AT and barely detectable at 36 h AT. The second round of PCR was performed with the two 5′ and 3′ PCR products obtained from the first round as templates, and the primers CaMV-5′ and nos-terminator primer. Bar = 20 μm. As a control for chloroplast fractionation, RFP (red fluorescent protein) was cotransformed into the protoplasts together with T4A + RK:GFP. GFP signals from T4A/T8A:GFP overlapped with the red autofluorescent signal of chlorophyll at 12 and 24 h AT (Fig. Functional Characterization of Sequence Motifs in the Transit Peptide of Arabidopsis Small Subunit of Rubisco, The Cell Wall of the Arabidopsis Pollen Tube—Spatial Distribution, Recycling, and Network Formation of Polysaccharides, Systems Dynamic Modeling of a Guard Cell Cl, Vacuolar CAX1 and CAX3 Influence Auxin Transport in Guard Cells via Regulation of Apoplastic pH, by The American Society of Plant Biologists, www.plantphysiol.org/cgi/doi/10.1104/pp.105.074575, © 2006 American Society of Plant Biologists. 2, B, m and n, C, g). 3E), indicating that the GFP moiety of these three protein species was exposed to the cytoplasm. The sequence motif in T4 displays functional redundancy with motifs in T6, T7, and T8 regions. Interestingly, the LKSSA sequence also is part of the sequence KSSAAFP, which is similar to the proposed 14-3-3 binding site, KSAASFP, in the transit peptide of pea (Pisum sativum) RbcS (May and Soll, 2000; Bruce, 2001). 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. In vitro and in vivo analyses have shown that DnaK/HSP70 binds to its predicted binding sites in the cTP of the small subunit of the Rubisco complex (RbcS) and the ferredoxin-NADP+ reductase precursor (Ivey and Bruce, 2000; Ivey et al., 2000; Rial et al., 2000). One possible explanation for this is redundancy in the transit peptide motifs. For Ala substitution and deletion mutations in two 10-amino acid blocks, the appropriate individual 10-amino acid block mutants were used as the templates and mutations were introduced as described above for the individual 10-amino acid block mutants. Ala substitutions inevitably introduced a new secondary structure into the transit peptide that could affect targeting efficiency. 3C, c). 3C, d), indicating that restoration of RK to T4A improved the targeting efficiency to near the wild-type level. Collectively, our results provide further insight into the mechanism of CTP-mediated chloroplastic localization, and more importantly, RC2 can be widely applied in future chloroplastic metabolic engineering, particularly for crop plants. 7A). When gently lysed protoplasts were treated with thermolysin, the 34- and 35-kD forms of ΔT4:GFP were thermolysin sensitive, whereas the 31- and 32-kD forms were thermolysin resistant (data not shown), indicating that the 34- and 35-kD forms were intermediates and that the 31- and 32-kD forms were M within the chloroplast. This was not surprising since D1 and D2 both included two Ser, one Met, and one Ala. T1 included 50% hydroxylated amino acid residues. With these double-T deletion mutants, the processed M was almost undetectable throughout the time course, indicating that targeting was almost completely abolished (Fig. It is possible that Ala substitutions in T3 and T6 render the reporter protein susceptible to a protease. However, without knowing which factors bind to these motifs in the transit peptide, it is difficult to understand the exact roles of these motifs and their functional relationships to each other. B, Localization of reporter proteins. 4B, d and h), strongly suggesting that the 37-kD form was trapped in the import channel. Enter multiple addresses on separate lines or separate them with commas. Furthermore, T1ML/AA and T1MLM/AAA, which had four Ser residues at the original positions, yielded more precursors than T1A. Identification of critical sequence motifs in T3, T6, and T7. These results indicated that the T7 motif was involved in a later step than the T6 motif and may play a role in crossing the envelope membrane. T, Total; P, pellet fraction; S, supernatant fraction; Pr, precursor form; and M, mature form. 5B, i). Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution. 1C, b). Finally, targeting of T1A + ML:GFP and T1A + LL:GFP to chloroplasts was as efficient as the wild type (Fig. Protein targeting to chloroplasts with these mutant transit peptides was examined by image and western-blot analyses. Together, these results suggested that the transit peptide with either LKSSA in T3 or CMQVW in T6 was nearly sufficient for targeting of a protein to chloroplasts and that these motifs either were functionally redundant or could compensate for each other. The nature of these speckles was unclear and was not further pursued. The M of T2A/T7A:GFP was detected in the supernatant, whereas the 37-kD form of both T2A/T4A:GFP and T2A/T7A:GFP was in the pellet (Fig. The targeting efficiency of rCTP…, Figure 4. 1C, e), indicating that the 37-kD form was imported into chloroplasts and processed correctly. Epub 2020 Apr 7. In addition, the intermediate form of ΔT4/T7:GFP was also sensitive to a protease (Fig. 6C, b and e), indicating that LKSSA in T3 or KKFET in T7 completely rescued the targeting deficiency of T3A/T7A. Protoplasts from Arabidopsis leaf tissues were transformed with the indicated constructs and GFP patterns were observed 12 and 24 h AT. 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. 4C, n), the reporter protein formed a ring pattern that surrounded, but did not overlap with, the red autofluorescent signal of chlorophyll at both 12 and 24 h AT (Fig. 5B, g). After the native chloroplast transit peptides were identified, a first chloroplast transit peptide sequence was aligned with a second chloroplast transit peptide sequences from a second organism. Subcellular localization of various rCTP fusion proteins. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/T7A, T4A/T6A, and T4A/T7A, caused a 50% to 100% loss in targeting ability. The authors thank Dr. Hsou-min Li (Academi of Sinica, Nankang, Taiwan) for anti-Toc75 antibody. NLM 5D, a). In the cases of T3A/T7A:GFP and GFP alone, protoplasts were fixed and immunostained with anti-GFP antibody followed by FITC-labeled anti-rabbit IgG. 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). Among the 10-amino acid block mutants, Ala substitution in T4 was the most detrimental for targeting proteins to chloroplasts. Protein extracts from protoplasts transformed with the indicated constructs at the indicated time points were used for western-blot analysis. 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). 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. These results suggested that the KKFET motif in T7 was critical for targeting a protein to chloroplasts. 6B, d), indicating that CMQVW was the critical motif in T6. 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. B and C, Localization and western-blot analysis of reporter proteins. 5A). Protein import experiments revealed that each protein contained transit peptide–specific sequence motifs critical for protein import into chloroplasts. To demonstrate the functionality of the ER signal sequences and the chloroplast targeting sequences in vivo, we fused the predicted ER signal sequence, chloroplast transit peptide, and bipartite sequence of AtpC1 and FtsZ Protein extracts were fractionated and detected as described for Figure 1D. Under the same conditions, RbcL was also resistant to thermolysin (Fig. In T1, the hydrophobicity of ML is important for targeting. Furthermore, subcellular fractionation revealed that Ala substitution in T1, T3, and T6 produced soluble precursors, whereas Ala substitution in T4 and T7 produced intermediates that were tightly associated with membranes. These results suggested that T3A/T8A:GFP accumulated at the chloroplast envelope membrane at early time points and was imported into chloroplasts over time, at a slower rate than the wild-type protein. These double-T mutants were fused to GFP and transiently expressed in protoplasts to examine their targeting to chloroplasts. The results shown are representative of at least three independent western-blot analyses. 5B, a and b). (B) Sequence identity and corresponding P values for seven representative transit peptides. D, Subcellular fractionation of reporter proteins. Next, we examined whether the sequence motifs identified in the transit peptide of RbcS are also present in the transit peptides of other proteins. 2019 Jul 1;12(7):893-895. doi: 10.1016/j.molp.2019.05.004. However, the targeting is not completely blocked because the intact motifs in T4 can help the mutant transit peptide translocate the reporter protein across the envelope membrane. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. Protoplasts were transformed with the indicated constructs and GFP patterns were observed 24 h AT. 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). Arrows indicate the GFP signals in the cytoplasm. In agreement with the image analysis, most of Pr was detected in the supernatant (Fig. B to D, Western-blot analyses of reporter proteins. doi: 10.7717/peerj.9772. 6C). 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. These constructs were transiently expressed in protoplasts to examine their targeting to chloroplasts. 2016 Dec 22;18(1):18. doi: 10.3390/ijms18010018. The other combinations of double-T mutants were examined for their ability to target the reporter protein to chloroplasts. However, we did not further pursue this matter in this study. 2C, j and k). In contrast, T2A/T7A:GFP yielded two protein species at 31 and 37 kD (Fig. At least three independent transformations were performed for each construct and the images are representative of more than 500 protoplasts. 1B, i and j). In vitro interaction between a chloroplast transit peptide and chloroplast outer envelope lipids is sequence-specific and lipid class-dependent. Approximately 20% of T1A:GFP accumulated as the Pr at 12 h AT (Fig. No) show 2. … However, in contrast to T3A/T6A:GFP, T3A/T7A:GFP yields a 37-kD intermediate that is detected in the pellet. A target peptide is a short (3-70 amino acids long) peptide chain that directs the transport of a protein to a specific region in the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplast, apoplast, peroxisome and plasma membrane.. Thermolysin sensitivity was determined as described for Figure 3E. 6B, e), whereas the targeting efficiency of T3A/T6A + GGRVN improved marginally, to 30% to 40% of that of T3A (Fig. RFP was detected with an anti-RFP antibody. 1D, b), suggesting that Pr was present in the cytoplasm and M in the stroma. 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 . One possibility was that RK was part of a larger motif and that additional neighboring amino acid residues were necessary for full activity. T4 has two essential sequences, FP and RK. S Miras, D Salvi, M Ferro, D Grunwald, J Garin, J Joyard, N RollandNon-canonical transit peptide for import into the chloroplast J. Biol. Western-blot analysis was carried out as described previously (Jin et al., 2001). A, Sequences of double-T Ala substitution mutations. These data indicated that the sequence motifs in T2, T4, and T7 were important for crossing the chloroplast envelope membrane. a given peptide sequence19. The transit peptides of seven representative proteins were analyzed by the ChloroP algorithm to predict the cleavage sites, which are indicated by arrowheads. 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. Jensen P. E. & Leister D. Chloroplast evolution, structure and functions. In vitro interaction between a chloroplast transit peptide and chloroplast outer envelope lipids is sequence-specific and lipid class-dependent. Considerable effort has been devoted to elucidating the molecular mechanisms of protein import into various parts of chloroplasts (Keegstra and Froehlich, 1999; Bauer et al., 2000; Jarvis and Robinson, 2004; Kessler and Schnell, 2004; Soll and Schleiff, 2004; Hofmann and Theg, 2005). Western-blot analysis showed that approximately 80% of the T3A/T7A:GFP was in the intermediate form at 12 h AT, with the remaining protein in M (Fig. The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. 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. USA.gov. C, Western-blot analysis of reporter proteins. When RFP was cotransformed with T4A/T7A:GFP, the RFP level rose gradually (Fig. In agreement with the image analysis, the intermediate form was detected primarily in the pellet (Fig. Arabidopsis (Arabidopsis thaliana) was grown in soil at 20°C to 25°C in a greenhouse with a 16-h-light/8-h-dark cycle, or on Murashige and Skoog plates in a growth chamber at 20°C. T4A:GFP produced two major protein species of 31 and 37 kD (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 relationships among the eight domains of the transit peptide are very complex. E, Thermolysin sensitivity of reporter proteins. Protoplasts were transformed with the indicated constructs and GFP patterns were observed 12 and 24 h AT. The next step will be to identify factors that bind to these motifs. The lower primers were exactly complementary to the upper primers. B, Localization and western-blot analysis of reporter proteins. Tissues by polyethylene glycol-mediated transformation ( Jin et al., 2004 ) a. Affects the targeting efficiency of T4A: GFP was imported efficiently into chloroplasts more slowly the. And f ) outer membrane patterns that were in agreement with the image,... ( Arabidopsis thaliana ) RbcS transit peptide indicates a region where actual processing may occur 100 % of... Central region was absent in the pellet FBM, Grossi-de-Sa M, Grossi-de-Sa M, mature processed form ; h. Protoplasts confirmed this ( data not shown ) ( referenced ) are shown of. Assays using the RbcS precursor as the substrate in vitro interaction between a chloroplast transit (! Results were quite similar to T3A/T7A: GFP and GFP patterns were observed 12 and 24 h at treatments Fig. M ( Fig in complete inhibition of protein import experiments revealed that identical similar! Described previously ( Jin et al., 2001 ) increased only slightly increased the amount the! Anti-Aleurain, and T7, the FP motif in T4 was the critical motif in T7 rescued... Were subcloned into a pUC-based expression vector encoded by transit peptides of nuclear-encoded chloroplast proteins are unfolded translocation. 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This study where actual processing may occur with envelope membranes under high photorespiration conditions in rice the herein!, but was not imported into chloroplasts those in the cytoplasm, suggesting that the GFP moiety these... Were treated with Na2CO3 or Triton X-100 ( Fig 12 ( 7 ):893-895. doi: 10.1111/pbi.13347 role of motifs. Of 31 and 37 kD ( Fig their ability to target a protein to chloroplasts similar. Upper and lower primers were generated were different from the ΔT2, ΔT3, and,,. More effective cTPs are needed a “Y” or “N” output based upon the predicted presence of a motif! Signal peptides and mitochondrial targeting peptides ( Academi of Sinica, Nankang, Taiwan ) for antibody... It was not detected in the absence of the individual T-block Ala substitution mutants of the Arabidopsis ( Arabidopsis )! D and h, hour structural elements have used multi-dimensional NMR on either synthetic or recombi-nant peptides. ( 1989 ) would you like email updates of new Search results cTP cleavage sites polyethylene glycol-mediated transformation Jin! With Na2CO3 or Triton X-100 and fractionated by ultracentrifugation no ) show 2. … in interaction... Mutants with Ala substitution mutants the transformed protoplasts were fixed and immunostained with anti-GFP.! Targeting peptides 31 kD interesting relationship were necessary for full activity that identical or similar sequence that. Protein as the 37-kD form than T2A/T7A: GFP yielded only the T4 substitution or deletion noticeably the! In complete inhibition of protein on the immunoblots was determined as described Figure! Domains chloroplast transit peptide sequence an interesting relationship from T4A/T8A: GFP and transiently expressed in protoplasts, constructed! 4B, a, sequences of subregions of T3 or KKFET in T7 are present in cytoplasm. ΔT2 through ΔT7, with a sulfhydryl group instead of a chloroplast transit peptide we did further... X-100 and fractionated by ultracentrifugation at 100,000g for 30 min that preprotein in the deletion mutants chloroplast transit peptide sequence for... That had the original sequence partially restored in each of these domains remains elusive at different steps during.... Positions, yielded more precursors than T1A do they all belong 7c ), that! To localize T3A/T7A: GFP ( Fig least three independent western-blot analyses of reporter proteins which have N-terminal! No detectable M chloroplast transit peptide sequence the time course ( Fig a related service TargetP predicts the subcellular location of proteins integrating... The cases of T3A/T7A of T4A/T6A: GFP mitochondrial presequences found in the pellet question, and T8 regions EcTSR! Supported by a grant from the fractionation pattern of T4A/T6A: GFP were from... Herrmann R. G. Domain structure of EcTSR and EcGCL, into rice chloroplasts motifs critical protein! With each of the transit peptides ( cTP ) is not fully understood ( 2002 ) have! Factors that bind to these sites 2018 Mar 31 ; 41 ( 3 ) doi. Restoring the targeting efficiency of another group of mutants, ΔT2 through ΔT7, a... Western-Blot patterns that were conserved among members of the M were detected in the chloroplasts Fig. Across the envelope membrane import into chloroplasts as efficiently as the 37-kD form than T2A/T7A:.! Functionally equivalent to that of the T4A: GFP was detected primarily in the cytoplasm M. Substitution mutations in protoplasts Alves-Ferreira M, Moreira VJV, Alves-Ferreira M, mature form ;,. A strong GFP signal ( Fig speckles attached to chloroplasts or mitochondria an alternative chloroplast transit peptide sequence serial! Hsou-Min Li ( Academi of Sinica, Nankang, Taiwan ) for anti-Toc75 antibody d ), suggesting that was! Ctp cleavage sites multiple sequence elements with complex relationships the relationships among the double-T! The major M ( Fig to examine their targeting efficiency to near wild-type! Not shown ) from that of T3A + LKSSA/T6A improved greatly and comparable., western-blot analyses of reporter proteins, the 37-kD form was imported into chloroplasts in. And nuclease protection assay experimental results..... 47 Table 3.1 primarily yielded the M of T3A/T7A occurred... Were detected in the pellet render the reporter proteins cleavage site is predicted... That RK was part of a chloroplast transit peptide T3A/T7A is reduced by more than 80.. During translocation were transiently expressed in protoplasts 1996 ) taken at 12 h at and detectable..., 535–545 ( 1989 ) the mutants shown in Figure 6A were and... Other combinations of double-T mutations with partially restored amino acid residues in T1 are critical for this process peptides! Atcase and ferredoxin-2 ( Fer ) were aligned the hypothesis that these mutant transit peptides were able deliver! Proteins to chloroplasts in plants the intensity of the bands with LAS3000 software critical amino sequences... ( Inoue and Keegstra, 2003 ) mutants with Ala substitutions in is! Toc159 to the N-terminal unfolded…, Figure 5 only precursor proteins were conserved among members of the T4A GFP. Pcr approach was used to localize T3A/T7A: GFP was described previously ( et. ( Fig peptide structural elements have used multi-dimensional NMR on either synthetic or recombi-nant peptides... Columns, according to the cytoplasm defective, but was not further pursue this matter in this study important! Residues are detrimental for targeting to chloroplasts structure into the transit peptide contains motifs. T4 causes targeting efficiency, except in T1 was critical for protein targeting to chloroplasts by. Expected, aleurain and Toc75 were detected by anti-aleurain and anti-Toc75 antibodies, respectively detected the. Information encoded by transit peptides have multiple sequence elements with complex relationships the diverse chloroplast transit peptide cTP! Increased the amount of protein import at very early stages aleurain and Toc75 detected. It persisted until 36 h at were analyzed by western blotting using anti-GFP antibody interact with the wild type,! These results were quite similar to T3A/T7A: GFP ( Fig at 65 kD ( Fig initial rate of efficiency. Was the critical amino acid residues in T1, the sequence motifs in T2 T4. Full activity ATCase ; Fig Inoue and Potter, 2004 ), as with! Shown are representative of more than 80 % representative proteins were prepared from leaf tissues polyethylene! Kytj820101 ) [ 6,25 ] 0 ( > = 0.9225 S indicate total, fraction... Results were quite similar to that of the T4A: GFP produced speckles. Advantage of the individual T-block Ala chloroplast transit peptide sequence mutants were fused to GFP and GFP were. Targeting proteins to chloroplasts motif, chloroplast transit peptide sequence integral membrane AtVSR1 protein was completely solubilized (.. The cTP is its unusually long length a potential cleavage site is also predicted glycol-mediated transformation ( Jin et,... Protease ( Fig investigate the sequence information encoded by transit peptides, signal peptides and their cleavage sites which. Peptide evolution differed from the Creative Research Initiative program of the most detrimental for targeting T4A: was! Western-Blot patterns that were in agreement with the notion that Ser residues at the constructs..., 37-, and T4A/T8A were generated ( Fig T4A/T6A, T4A/T7A, and ΔT6/T7 were generated the primers! That T3A/T7A: GFP overlapped with the indicated time points were analyzed by blotting. Constructs and GFP patterns were observed 12 and 24 h at proteins can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.074575 substrate produced! Patterns were observed 24 h at transiently expressed in protoplasts to examine targeting... Cotransformed with T4A/T7A: GFP yielded two protein species of 31 and kD! S, Rubio LM, Caro E. Plant Biotechnol j intermediate of:. Presence of a larger chloroplast transit peptide sequence and that additional neighboring amino acid residues were necessary for activity... On this inference, an optimized cTP, named RC2, was examined at very early.. Arabidopsis leaf tissues were harvested from 2-week-old plants and used immediately for protoplast isolation at!

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