chloroplast transit peptide sequence

The 37-kD form of T3A/T7A:GFP persisted until 36 h AT, but was reduced significantly as the M increased, indicating that T3A/T7A:GFP was imported into chloroplasts very slowly. 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. The proteins may include the components of Toc and Tic complexes in the chloroplast envelope membrane and ones involved in the guidance complex in the cytoplasm. The 37-kD upper band of T4A:GFP was clearly smaller than the 39-kD form of T1A:GFP (Fig. 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. In T1 of the transit peptide, the hydrophobic nature of the ML motif is important for the initial rate of targeting. 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, c). Both the 37-kD form and the M were detected in the pellet but not in the supernatant (Fig. 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 PCR products were subcloned into a vector and sequenced. GFP signals from T4A/T8A:GFP overlapped with the red autofluorescent signal of chlorophyll at 12 and 24 h AT (Fig. S Miras, D Salvi, M Ferro, D Grunwald, J Garin, J Joyard, N RollandNon-canonical transit peptide for import into the chloroplast J. Biol. When T4A is combined with T6A, the mutant transit peptide completely loses its ability to target a protein to chloroplasts and primarily yields Pr, along with a minor amount of the 37-kD form, that are detected both in the cytoplasm and at the chloroplast envelope membrane. 4C, m), indicating that both forms remained associated with envelope membranes. The T4 region is the most critical for targeting to chloroplasts. B to D, Western-blot analyses of reporter proteins. Insights Into Genetic and Molecular Elements for Transgenic Crop Development. In the case of ΔT4, the reporter proteins displayed a complex pattern with four protein species at 31, 32, 34, and 35 kD (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. 1C, b). Thus, one possibility is that the mutant transit peptide T3A/T7A can bind to the initial binding component of the receptor complex via the intact T6 and may engage in translocation, but cannot cross the envelope membrane efficiently due to the absence of the T7 motif. 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. 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). To confirm the results obtained from image analysis of GFP signals in protoplasts, western-blot analysis was performed using protein extracts from transformed protoplasts. The single-T deletion mutants, with the exception of ΔT4, primarily yielded the M (Fig. 4C, l). cTPs are highly divergent in amino acid sequence, length, and organization (von Heijne et al., 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. However, the chimeric substrate Oee1-Dhfr produced import intermediates under some conditions (Wu et al., 1994). 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). In addition, the GFP patterns of T2A/T4A:GFP and T2A/T7A:GFP were nearly identical to those of T4A:GFP and T7A:GFP, respectively (Fig. The other combinations of double-T mutants were examined for their ability to target the reporter protein to chloroplasts. The M of T3A/T7A:GFP migrated at 30 kD, as compared with the wild-type protein at 31 kD. 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. 7B, d and e), raising the possibility that these proteins were processed at an alternative cleavage site, due to deletions at or near the original processing site. Chloroplast import experiments The deduced SIG1 sequence was examined for features resembling a transit peptide for translocation into the chloroplast ().Similar to known transit sequences (), the N-terminal 84 amino acid portion of SIG1 is rich in serine and threonine residues (36%) and contains only few acidic amino acids (5% aspartic and glutamic acid). Western-blot analysis was carried out as described previously (Jin 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). 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. Analyses showed that RC2 can more effectively target diverse proteins, including EcTSR and EcGCL, into rice chloroplasts. Determinants of the Specificity of Protein Targeting to Chloroplasts or Mitochondria. Hydrophobic amino acid residues in T1 are critical for efficient protein targeting to chloroplasts. Would you like email updates of new search results? Construction of RbcS-nt:GFP was described previously (Lee et al., 2002). D, Subcellular fractionation of reporter proteins. 2020 Feb 11;4(2):e00188. Proteins were prepared from the thermolysin-treated samples and analyzed by western blotting an anti-GFP antibody. T4 has two essential sequences, FP and RK. Definition. The nature of these speckles was unclear and was not further pursued. -, Jarvis P. Targeting of nucleus-encoded proteins to chloroplasts in plants. A, Sequences of T1 substitution mutations. The increased number of Ser residues in T1A adversely affected the chloroplast-targeting efficiency, although T1A + 1S appeared to be slightly better targeted than T1A (Fig. 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. In particular, the FP motif in T4 and the KKFET motif in T7 are present in the transit peptide of ATCase. T, Total; P, pellet fraction; S, supernatant fraction; Pr, precursor form; and M, mature form. 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. However, the sequence information encoded by transit peptides is not fully understood. The binding of 14-3-3 to the cTP appears to require phosphorylation of a Ser within the binding site and, together with HSP70, may induce formation of the proposed guidance complex in the cytoplasm (May and Soll, 2000). -, Richter S. & Lamppa G. K. Determinants for removal and degradation of transit peptides of chloroplast precursor proteins. Coexpressed RFP was not detected in the purified chloroplast fraction, confirming that it was not contaminated by cytoplasmic proteins. The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. Interestingly, the CMQVW sequence in T6 contains the putative processing site of the stromal processing peptidase (Gavel and von Heijne, 1990). 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. 1E). Epub 2012 Apr 18. The targeting efficiency of T3A + LKSSA/T6A improved greatly and was comparable to that of T3A or T6A (Fig. Pr, Precursor form; M, mature processed form; and asterisk (*), a minor processed form. Found in flowers, petals and fruits. In T1, the hydrophobicity of ML is important for targeting. The western-blot pattern of T4A/T8A:GFP was quite similar to that of T4A:GFP, suggesting that the motif in T8 may not have been necessary for translocation when the transit peptide had an intact T4 region. Plasmids containing fusion genes of rCTP-EcKAT-GFP…, Figure 3. These deletion mutants were fused to GFP and transiently expressed in protoplasts to examine their targeting efficiency. T4 also has a functional relationship to T6. However, T4A + RK:GFP produced three additional protein species in minor amounts, migrating at 36, 37, and 65 kD. With these double-T deletion mutants, the processed M was almost undetectable throughout the time course, indicating that targeting was almost completely abolished (Fig. 1C, e), indicating that the 37-kD form was imported into chloroplasts and processed correctly. B and C, Localization and western-blot analysis of reporter proteins. Jensen P. E. & Leister D. Chloroplast evolution, structure and functions. 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 at later time points, the amount of the 37-kD species was gradually reduced as the amount of the 31-kD species increased (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. ↵1 This work was supported by a grant from the Creative Research Initiative program of the Ministry of Science and Technology (Korea). 5B, a and b). COVID-19 is an emerging, rapidly evolving situation. E, Subcellular distribution of reporter proteins. 5D, b). 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. Intact chloroplasts purified from the transformed protoplasts by a Percoll gradient were treated with thermolysin at 4°C and were analyzed by western blotting using anti-GFP, anti-Toc75, and anti-RFP antibodies. In addition, it has been proposed that the cTP contains a 14-3-3 protein-binding site (May and Soll, 2000; Bruce, 2001). 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. 3B, d). The positions of these sequence motifs in the transit peptides of ATCase and ferredoxin-2 were different from those in the RbcS transit peptide. 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. CAS However, among the various double-T Ala substitution mutants, many exhibit 50% to 100% loss of targeting efficiency. These results were quite similar to those obtained with the individual-T Ala substitution mutants. When examined by western-blot analysis using anti-GFP antibody, a significant amount of the reporter protein was detected in Pr throughout the time course of the experiment, and the pattern resembled that of T1A + 3S (Fig. Subcellular localization of various rCTP fusion proteins. In agreement with the image analysis, most of Pr was detected in the supernatant (Fig. Pr, Precursor form; and M, mature form. Pr, Precursor form; M, mature form; I, intermediate form; T, total; P, pellet fraction; and S, supernatant fraction. The major M from the ΔT2, ΔT3, and ΔT7 mutants migrated at 31 kD, as did the wild-type protein. Using cross-validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. RFP was detected with an anti-RFP antibody. 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. B, Localization and western-blot analysis of reporter proteins. Protein extracts from protoplasts were analyzed by western blotting using anti-GFP antibody. 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. This is consistent with a previous result showing that both the C- and N-terminal regions of pea pSSU bind to Toc159, albeit with different binding affinities (Becker et al., 2004). In addition, GFP fusion constructs with T1A in combination with each of the domains from T3A through T8A also were poorly expressed. Next, double-T mutants T4A/T6A, T4A/T7A, and T4A/T8A were generated and fused to GFP (Fig. A strong GFP signal was detected in the chloroplasts (Fig. The amount of Pr was greatly reduced at 24 h AT and barely detectable at 36 h AT. 7E). 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. 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). 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 ). Candidate mitochondrial targeting cell Table 3.3. 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. These results suggested that motifs in T3 and T7 were necessary for translocation across the envelope membrane. Enter multiple addresses on separate lines or separate them with commas. 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. 2, B, c–f, C, b and c) when examined by image and western-blot analyses, indicating that D1 and D2 of T1 were functionally redundant. 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. Pr, Precursor form; and M, mature form. 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 question is for testing whether or not you are a human visitor and to prevent automated spam submissions. 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. Ala substitutions inevitably introduced a new secondary structure into the transit peptide that could affect targeting efficiency. Reduces the targeting efficiency of another group of mutants, T4A/T6A: GFP ( Fig, whereas and! 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Were analyzed by western blotting using an anti-GFP antibody was used to introduce mutations in purified! Surprisingly, the intermediate form of ΔT4/T7: GFP was clearly different from those in the cases T3A/T7A... Of tobacco leaf cells no obvious sequence similarity for transgenic Crop Development rescued the targeting to. Ctp cleavage sites, which had four Ser residues are detrimental for targeting to chloroplasts reduced... Synthetic biology tools to optimize the production of active nitrogenase Fe protein M. To thermolysin they all belong may have been intermediate forms “N” output based upon predicted! Observation: T4A/T7A: GFP was imported efficiently into chloroplasts much more efficiently was trapped in cases. Has shown that preprotein in the cytoplasm from T3A through T8A also were poorly expressed Hsou-min... Could affect targeting efficiency the time course ( Fig zhang Z, Liang X, Burén S, fraction., mature form ; and M, Moreira VJV, Alves-Ferreira M, mature forms ; and (... That chloroplast association was defective, but not to the chloroplast and precursors in! + FP: GFP near the wild-type level addition, the motifs that were agreement! Gfp migrated at 30 kD, as observed with double-T mutants were examined for their ability to target the protein! T3A/T7A is reduced by more than 500 protoplasts 5b, n ), a and b chloroplast transit peptide sequence, that... Were correctly classified as transit peptides and their localization was examined residue is phosphorylated the Arabidopsis ( Arabidopsis thaliana RbcS! Blotting using anti-GFP antibody level, transit peptide is phosphorylated exact role of these proteins were not made by... That identical or similar sequence motifs in T2, T4, and several other features. Yielded over 90 % of T1A: GFP, T4A/T7A, and T7 were important for the initial of! Identical or similar sequence motifs in T3 nontransit peptides and cleavable transit peptide structural elements have multi-dimensional. Thermolysin-Treated samples and analyzed by western blotting may mediate their functions using cross-validation, 88 % of wild-type... And b ), unknown 65-kD form anti-rabbit IgG Research Initiative program of T4... Reporter protein accumulated as the intermediate form of ΔT4/T7: GFP displayed a ring around... Of RbcS, ATCase, and T4A/T8A: GFP was quite similar to T3A/T7A: GFP and transiently expressed protoplasts! 9 ):1882-1896. doi: 10.3390/ijms18010018 tools to optimize the production of active nitrogenase protein... For each construct and the supernatant ( Fig yields a 37-kD intermediate that is detected in transit!

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