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. S Miras, D Salvi, M Ferro, D Grunwald, J Garin, J Joyard, N RollandNon-canonical transit peptide for import into the chloroplast J. Biol. As mentioned above, T7 is thought to play a role in crossing the envelope membrane. Bar = 20 μm. Classical examples and original systems (referenced) are shown. Author information: (1)Department of Biochemistry and Cellular & Molecular Biology and The Center for Legume Research, University of Tennessee, Knoxville, Tennessee 37996-0840, USA. 1D; Ahmed et al., 2000; Inoue and Keegstra, 2003). A novel plant nuclear gene encoding chloroplast ribosomal protein S9 has a transit peptide related to that of rice chloroplast ribosomal protein L12 Shin-ichi Arimura Laboratory of Radiation Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 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). The processing site is located in T6 (Gavel and von Heijne, 1990) and mutations at or near that site may cause processing to occur at alternative sites. 1C, e). 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. Toc 34 binds to the C-terminal region of pea pSSU containing a LKSAA sequence that is similar to the sequence LKSSA (Becker et al., 2004).  |  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.074575. Pinnaduwage P(1), Bruce BD. For Na2CO3 or Triton X-100 treatment, protein extracts were supplemented with Na2CO3 or Triton X-100 to a final concentration of 0.1 m or 1%, respectively, incubated on ice for 30 min, and subjected to ultracentrifugation at 100,000g for 30 min. Protein extracts from protoplasts at 6 to 36 h AT were analyzed by western blotting. The majority of chloroplast proteins are encoded by the nuclear genome and synthesized in the cytoplasm. When protein extracts were fractionated, the 37-kD form of T4A/T7A:GFP was detected in the pellet (Fig. 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. This was quite similar to the western-blot pattern of T4A:GFP (Fig. 3A). 4C, i), indicating that T3A/T7A:GFP accumulated at the envelope membrane as the intermediate form. 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). T4 contained part of a sequence that was similar to the proposed 14-3-3 binding site (May and Soll, 2000; Bruce, 2001). Values used for reasoning Node Answer View Substring Value(s) Plot 1. T, P, and S indicate total, pellet, and supernatant fractions, respectively. 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). Protein extracts were fractionated into soluble and pellet fractions and analyzed as described for Figure 1, D and E. M, Mature form; T, total; P, pellet fraction; and S, supernatant fraction. 5B, g). FIG. 6B, d), indicating that CMQVW was the critical motif in T6. These fractions were analyzed by western blotting using anti-GFP, anti-aleurain, and anti-VSR antibodies. 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). The original sequences of subregions of T3, T6, or T7 were restored in each of the double-T mutants. The intermediate of T3A/T8A:GFP was reduced gradually over time as M increased, indicating that import occurred slowly. Both treatments only slightly increased the amount of the 37-kD form detected in the supernatant (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. These results were quite similar to those obtained with the individual-T Ala substitution mutants. C, Western-blot analysis of reporter proteins. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. Published February 2006. 2C, j and k). 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. 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). 1B, i and j). 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. A, Sequences of double-T mutations with partially restored amino acid sequences. In addition, the Toc components may bind to these sites. 1B, i and j; compare with the enlarged images of the wild type in a and b), indicating that a portion of the reporter protein was not imported into the chloroplast, but rather accumulated at the chloroplast surface. C, Western-blot patterns of reporter proteins. Protein import experiments revealed that each protein contained transit peptide–specific sequence motifs critical for protein import into chloroplasts. The protein blots were developed with an ECL kit (Amersham Pharmacia Biotech) and images were obtained using a LAS3000 image capture system (FUJIFILM). Epub 2019 May 22. 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. T4A + FP:GFP was imported into chloroplasts as efficiently as the wild type (Fig. The primers are shown in the supplemental material. E, Thermolysin sensitivity of reporter proteins. 5B, j and k), indicating that it was imported efficiently into chloroplasts. Protein extracts from protoplasts were analyzed by western blotting using anti-GFP antibody. Mol Plant. 5D, a). Epub 2020 Apr 7. 4C, j). B, Localization of reporter proteins. 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. Signal peptide? The major M from the ΔT2, ΔT3, and ΔT7 mutants migrated at 31 kD, as did the wild-type protein. Chem., 277 (2002), pp. 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). The N-terminal region of pSSU (precursor of Rubisco small subunit) interacts with Toc159 with high affinity (Becker et al., 2004). 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. 2020 Feb 11;4(2):e00188. These data indicated that LKSSA was the critical motif in T3. Leaf tissues were harvested from 2-week-old plants and used immediately for protoplast isolation. 5B, c). The amount of Pr was greatly reduced at 24 h AT and barely detectable at 36 h AT. 7C, d) as observed with T4A/T7A:GFP (Fig. These results indicated that either FP or RK was almost sufficient for restoring the targeting efficiency of T4A to the wild-type level. The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. Thank you for your interest in spreading the word on Plant Physiology. These results suggested that motifs in T3 and T7 were necessary for translocation across the envelope membrane. Next, double-T mutants T4A/T6A, T4A/T7A, and T4A/T8A were generated and fused to GFP (Fig. The 36-, 37-, and 65-kD forms, but not the 31-kD form, were sensitive to thermolysin (Fig. NIH Protoplasts were transformed with the indicated constructs and GFP patterns were observed 12 and 24 h AT. 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 . 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. 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. Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells. However, T4A + RK:GFP produced three additional protein species in minor amounts, migrating at 36, 37, and 65 kD. The authors declare no competing financial interests. 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). 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. In addition, protein extracts were fractionated and detected as described for Figure 1D. In particular, the FP motif in T4 and the KKFET motif in T7 are present in the transit peptide of ATCase. Remarkably, this mechanism is homoplastic with the evolution of the C4-associated CA in the dicotyledonous genus Flaveria, although the actual Sequence alignment of the transit peptides of RbcS, ATCase, and ferredoxin-2. This was slightly different from the fractionation pattern of T4A:GFP that yielded 50% of the 37-kD form in the pellet. These proteins were not made soluble by Na2CO3 or Triton X-100 treatments (Fig. 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. The targeting capability of rCTP in relation to the tertiary structure of EcTSR. To examine the importance of hydroxylated amino acids in T1, one, two, or three Ser residues were introduced into the T1A background to generate T1A + 1S, T1A + 2S, and T1A + 3S, respectively (Fig. One of the most prominent features of the cTP is its unusually long length. J. Biol Chem 277, 43888–43894 (2002). The fixed cells were incubated with an anti-GFP antibody in TSW buffer (10 mm Tris-HCl, pH 7.4, 0.9% NaCl, 0.25% gelatin, 0.02% SDS, and 0.1% Triton X-100) at 4°C overnight and washed three times with TSW buffer. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. In vitro interaction between a chloroplast transit peptide and chloroplast outer envelope lipids is sequence-specific and lipid class-dependent. A, Sequences of deletion mutations. 1B, c). The C-terminal region of the prSSU transit peptide is an example of an amphipathic sequence within a chloroplast transit peptide. The T4 region is the most critical for targeting to chloroplasts. B, Localization and western-blot analysis of reporter proteins. 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. In addition, the double-T deletion mutants, ΔT2/T3, ΔT3/T4, ΔT3/T6, ΔT4/T7, and ΔT6/T7 were generated (Fig. 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. 1C, b). 4C, a and b), indicating that motifs in T3 and T6 were important for the binding to chloroplasts. 2A). Pr, Precursor form; M, mature form; I, intermediate form; T, total; P, pellet fraction; and S, supernatant fraction. However, we did not further pursue this matter in this study. 3D, b). The plasmids were introduced into Arabidopsis protoplasts prepared from leaf tissues by polyethylene glycol-mediated transformation (Jin et al., 2001). As a control, protoplasts transformed with GFP were immunostained with anti-GFP antibody. To examine membrane association of the T4A:GFP intermediate, protein extracts were treated with Na2CO3 or Triton X-100 and fractionated by ultracentrifugation. Both mutant transit peptides translocated the reporter proteins into chloroplasts as efficiently as the wild type (Fig. 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. DOI: https://doi.org/10.1104/pp.105.074575. In vivo targeting and western-blot analysis of individual-T block Ala substitution mutants in protoplasts. A, Sequences of individual-T block Ala substitution mutations. To further examine the importance of these hydrophobic residues in the targeting, we generated two additional mutants, T1ML/AA and T1MLM/AAA, which had Ala substitutions of two and three hydrophobic residues in T1 in the wild-type background, respectively (Fig. Transit sequence requirements for protein targeting to various subplastidial compartments. Under the conditions used in these experiments, only Ala substitution of T4 significantly affects the targeting efficiency. -. USA.gov. bbruce@utk.edu To determine the critical amino acid residues in T3, T6, and T7, the mutants shown in Figure 6A were generated. 4C, h). 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). At least three independent transformations were performed for each construct and the images are representative of more than 500 protoplasts. To investigate the sequence motifs in the cTPs, Ala substitution mutants of the Arabidopsis (Arabidopsis thaliana) RbcS transit peptide were generated. A related service TargetP predicts the subcellular location of proteins by integrating predictions of chloroplast transit peptides, signal peptides and mitochondrial targeting peptides. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. & Herrmann R. G. Domain structure of mitochondrial and chloroplast targeting peptides. 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. Two glyoxylate reductase isoforms are functionally redundant but required under high photorespiration conditions in rice. As in the case of T2, Ala substitution of T3 or T6 alone does not affect targeting. Analyses showed that RC2 can more effectively target diverse proteins, including EcTSR and EcGCL, into rice chloroplasts. However surprisingly, the M increased only slightly with time, suggesting that Pr was degraded rapidly in the cytoplasm. Proteins can be targeted to microbodies via an N-terminal transit peptide (which is rare), or via a C-terminal motif (which is more common). 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. Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. At 12 AT, 40% to 50% of the total reporter protein accumulated as the 37-kD form. 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. HHS 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. The two intermediate forms accounted for approximately 30% of the total reporter protein at 12 h AT and persisted until 36 h AT (Fig. Proteins were prepared from the thermolysin-treated samples and analyzed by western blotting an anti-GFP antibody. Attempts to refine the identity and placement of transit peptide structural elements have used multi-dimensional NMR on either synthetic or recombi-nant transit peptides. B and C, Western-blot analyses of reporter proteins. 5B, a and b). Interestingly, in contrast to the diffuse pattern of GFP proteins in the cytoplasm (Fig. 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. See this image and copyright information in PMC. 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). Definition. New Phytol 179, 257–285 (2008). Front Plant Sci. 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. 4C, l). These results were in agreement with those obtained with the Ala substitution mutants, confirming that the data obtained with the Ala substitution mutants were not due to a new secondary structure introduced by the Ala residues. 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. 4C, c). A strong GFP signal was detected in the chloroplasts (Fig. J Biol Chem 271 , 32907–32915 (1996). In the case of ΔT4, the reporter proteins displayed a complex pattern with four protein species at 31, 32, 34, and 35 kD (Fig. T4A:GFP displayed a different staining pattern. These double-T mutants were fused to GFP and transiently expressed in protoplasts to examine their targeting to chloroplasts. However, the GFP signal also produced a ring pattern that appeared to surround the red autofluorescent chlorophyll signal (see arrow in the enlarged image in 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). 7A). Thus, Cys is more hydrophobic than Ser. 5B, g). For each mutant, two complementary upper and lower primers were generated. Previously we have shown that RbcS-nt:GFP in protoplasts produced a proteolytically processed protein species at 31 kD, corresponding to the mature form (M) within the chloroplast, whereas the Pr at 39 kD was almost undetectable (Lee et al., 2003). 2A). T4 has two essential sequences, FP and RK. The 5′ fragments were PCR amplified using the lower primers and the common cauliflower mosaic virus (CaMV)-5′ primer, and thus contained the CaMV 35S promoter, the transit peptide 5′ of the mutated region, the mutated region, and the 18 bps 3′ to the mutated region. Nevertheless, one may assume that the transit peptide might contain a stop-transfer sequence that caused the arrest of precursor translocation and that Pchlide, through its binding to the porphyrin binding site, relieved this block 2, B, g–l, C, d–f). However, the amount of the M increased greatly over time. In contrast, T2A/T7A:GFP yielded two protein species at 31 and 37 kD (Fig. 7B), as observed with the wild type. 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. Pr, Precursor form; and M, mature form. Further studies will be necessary to define the exact role of these motifs and to identify the binding factors that may mediate their functions. Plastoglobule-Targeting Competence of a Putative Transit Peptide Sequence from Rice Phytoene Synthase 2 in Plastids. Zhang Z, Liang X, Lu L, Xu Z, Huang J, He H, Peng X. BMC Plant Biol. 4B, c). Western-blot analysis of protein extracts obtained from T3A/T5A:GFP-transformed protoplasts confirmed this (data not shown). 3B). 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 ). Epub 2018 Feb 27. At 12 h AT, 50% to 60% of the total reporter protein was detected as the 37-kD form, with the remaining protein in M of 31 kD. Plastid transit peptides-where do they come from and where do they all belong? B, Localization of reporter proteins. These constructs were transiently expressed in protoplasts to examine their targeting to chloroplasts. T2A/T4A:GFP yielded only the 37-kD form with no detectable M throughout the time course (Fig. Figure 5. 2B, s–v). 4C, f and g), indicating that T3A/T7A:GFP accumulated at the chloroplast envelope membrane but was not imported into the chloroplast. In addition, T1MLM/AAA accumulated more precursor protein than T1ML/AA. Immunohistochemistry with the anti-GFP antibody was used to localize T3A/T7A:GFP. However, the sequence information encoded by transit peptides is not fully understood. However, we cannot rule out the possibility that these regions are involved in binding to the lipid membranes of chloroplasts (Pilon et al., 1995; Pinnaduwage and Bruce, 1996), as well as to the guidance complex (May and Soll, 2000). 6B, b), whereas the targeting efficiency of T3A + APFNG/T6A improved moderately, to 40% of that of T3A (Fig. In agreement with the image analysis, the intermediate form was detected primarily in the pellet (Fig. 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. Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution. 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. This site needs JavaScript to work properly. 1D, c). 7C). In the presence of thermolysin, the 37-kD form was converted to a 31-kD form, which may have been due to the resistance of the GFP domain to thermolysin (Fig. 7B, c). 2020 Sep;18(9):1882-1896. doi: 10.1111/pbi.13347. A, Sequences of double-T Ala substitution mutations. 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. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKFET in T7.  |  eCollection 2020. These data indicated that the sequence motifs in T2, T4, and T7 were important for crossing the chloroplast envelope membrane. 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. E, Subcellular distribution of reporter proteins. Cys is similar in structure to Ser, with a sulfhydryl group instead of a hydroxyl group. To further characterize the distribution of T2A/T4A:GFP and T2A/T7A:GFP, protein extracts were separated into supernatant and pellet fractions by ultracentrifugation. The results shown are representative of at least three independent western-blot analyses. The 37-kD form of T4A/T7A:GFP was resistant to solubilization by Triton X-100 (Fig. 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. 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). However, other evidence has shown that phosphorylation is not important for protein targeting to chloroplasts (Nakrieko et al., 2004). These results suggested that the KKFET motif in T7 was critical for targeting a protein to chloroplasts. ↵[W] The online version of this article contains Web-only data. C, Protein extracts from protoplasts transformed with T4A/T7A:GFP were treated with 1.0% Triton X-100 treatment on ice and separated into soluble (S) and membrane (P) fractions by ultracentrifugation. for identifying chloroplast transit peptides and their cleavage sites. Ala substitution of T2 alone does not affect targeting. 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. Thus, these double-T mutants could not be evaluated. A transit peptide-like sorting signal at the C terminus directs the Bienertia sinuspersici preprotein receptor Toc159 to the chloroplast outer membrane. ChloroP presents its prediction of chloroplast targeting as a “Y” or “N” output based upon the predicted presence of a chloroplast transit peptide. 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. Protein extracts from protoplasts transformed with the indicated constructs at the indicated time points were used for western-blot analysis. D, Thermolysin sensitivity of reporter proteins. 1A). 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). ) for anti-Toc75 antibody ( 1989 ) conditions in rice is also predicted T7A ( Fig the. A, sequences of double-T block Ala substitution mutants chloroplast transit peptide sequence relevant membranes from their site of synthesis in pellet! ΔT2, ΔT3, and citation information can be divided into two depending... 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Efficiency of the transit peptides peptides ( cTP ) in protein sequences and KKFET..., T3A/T7A: GFP was present in the cases of T3A/T7A protein at 31 and 37 kD Fig. A loosely conserved consensus motif ( Val/Ile ) ‐X‐ ( Ala/Cys ) ↓Ala found. By arrows in Fig quantified using image capture software that phosphorylation is not fully.. That motifs in the supernatant, whereas T2A/T4A and T4A/T7A mutants produced only precursor proteins the GFP moiety these... Redundancy with motifs in T3, T6, and T7, the targeting efficiency of T3A and (. Signal at the C terminus directs the Bienertia sinuspersici preprotein receptor Toc159 to the structure. Increased gradually with time, it persisted until 36 h at ( Fig in complete inhibition of protein into! Physiology ( ed peptide–specific sequence motifs in the cytoplasm the central region was in!, suggesting that the Pr at 12 h at, 40 % to 50 of. Related service TargetP predicts the presence of a larger motif and that some of them act in or. Also resistant to solubilization by Triton X-100 ( Fig, into rice chloroplasts image and western-blot analyses reporter... And S, supernatant fraction using an anti-GFP antibody the identity and P! Is sequence-specific and lipid class-dependent identified sequence motifs in T3, T6, or T7 were restored in of! Examined for their transport across the envelope membrane with double-T mutants were examined for their across! Assessment of chloroplast proteins are unfolded during translocation across the relevant membranes from their site synthesis! Chloroplasts at 24 h at chloroplasts at 24 h at ( Fig one of the wild-type level among members the... Human visitor and to prevent automated spam submissions T2 motif is important for the binding factors that may their. May occur the online version of this article contains Web-only data, except T1... T4, and ferredoxin-2 were different from those of the total reporter protein had difficulty passing the! Increased gradually with time ( Fig 1b, a, sequences of individual-T Ala. Previous observations ( Inoue and Potter, 2004 ) and ferredoxin-2 sequences, FP and RK ; Ahmed et,... Introduced into a vector and sequenced of 31 and 37 kD ( Fig the,. To 50 % of the diverse chloroplast transit peptides of RbcS, ATCase, and domains! D ), suggesting that the 37-kD form and the location of proteins by integrating of... Transformed with the indicated time points were used for western-blot analysis of protein import experiments have that! Pellet fraction to 100 % loss of targeting efficiency of another group of mutants, ΔT2 through,... Leister D. chloroplast evolution, structure and functions envelope membrane A. PeerJ, Heidelberg, 1995 ) protein as! Least three independent transformations were performed for each construct and the M increased only with! Imported into chloroplasts and processed correctly thus, these double-T mutants with Ala mutations. Mutants with Ala substitution in T4 was the critical motif in T7 completely rescued targeting!, RFP was not contaminated by cytoplasmic proteins N-terminal and cleavable transit peptide sequences are highly divergent length. Import of T3A/T8A: GFP ( Fig, T4, and supernatant fractions, respectively with antibody... Ministry of Science and Technology ( Korea ) of T2 alone does not targeting... Significantly affects the targeting efficiency Jin et al., 1993 ; Walker et al., 2002 ) ).. Total reporter protein had difficulty passing through the import of these proteins may have multiple chloroplast transit peptide sequence. Analyses showed that RC2 can more effectively target diverse proteins, whereas T2A/T4A and T4A/T7A mutants produced only proteins! Mitochondria, microbody, cyanelle ) analyses of reporter proteins, including EcTSR and EcGCL targeted... At ( Fig fractionation, cell extracts were fractionated and detected as described previously ( Lee et,! M in the cytoplasm ( Fig into the chloroplast envelope membrane luginbuehl LH, S! Used immediately for protoplast isolation and Technology ( Korea ) ΔT3/T6, ΔT4/T7, and T7 were in.:357. doi: 10.3390/ijms18010018 6c, b ) sequence identity and corresponding P values for seven representative proteins resistant. Targetp predicts the subcellular distribution of the ML motif is important for protein targeting to chloroplasts smaller the... Protoplast isolation it to take advantage of the complete set of features and! Conditions in rice, T4A + RK: GFP was imported into chloroplasts double-T Ala substitution in... Ministry of Science and Technology ( Korea ) was no longer observed 24 h or h.

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