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First published online December 29, 2005; 10.1104/pp.105.074575 Plant Physiology 140:466-483 (2006) © 2006 American Society of Plant Biologists
Functional Characterization of Sequence Motifs in the Transit Peptide of Arabidopsis Small Subunit of Rubisco1,[W]Division of Molecular and Life Sciences (D.W.L., S.L., G.L., K.H.L., S.K., I.H.) and Center for Plant Intracellular Trafficking (D.W.L., S.L., G.L., K.H.L., I.H.), Pohang University of Science and Technology, Pohang 790–784, Korea; and Department of Biochemistry, Gyeongsang National University, Jinju 660–701, Korea (G.-W.C.)
The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. However, the sequence information encoded by transit peptides is not fully understood. 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. 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. In addition, we generated mutants that had the original sequence partially restored in single- or double-T-block Ala (A) substitution mutants. Analysis of chloroplast import of these mutants revealed several interesting observations. Single-T-block mutations did not noticeably affect targeting efficiency, except in T1 and T4 mutations. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/T7A, T4A/T6A, and T4A/T7A, caused a 50% to 100% loss in targeting ability. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. 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 T1, the hydrophobicity of ML is important for targeting. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKFET in T7. 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 demonstrate that the transit peptide contains multiple motifs and that some of them act in concert or synergistically.
The majority of chloroplast proteins are encoded by the nuclear genome and synthesized in the cytoplasm. 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). Nuclear-encoded plastid proteins can be divided into two groups depending on whether or not they possess a cleavable transit peptide. In most cases, nuclear-encoded plastid proteins destined for chloroplast localization have a cleavable N-terminal transit peptide that is both necessary and sufficient for targeting to chloroplasts (Keegstra and Cline, 1999; Vothknecht and Soll, 2000; Bruce, 2001), although plastid proteins that lack the classical transit peptide have been recently identified (Rathinasabapathi et al., 1994; Miras et al., 2002). For proteins with a transit peptide, the N-terminal cleavable transit peptide is necessary and sufficient. 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).
Despite numerous studies, the information in the chloroplast-targeted transit peptide (cTP) is not clearly understood. cTPs are highly divergent in amino acid sequence, length, and organization (von Heijne et al., 1989
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
In addition, it has been proposed that the cTP contains a 14-3-3 protein-binding site (May and Soll, 2000 In this study, we identified the sequence motifs in the cTP of RbcS that are critical for chloroplast targeting and investigated the functional relationships among these motifs during protein import. 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.
Ala Substitution of 10-Amino Acid Blocks Identifies Functional Sequences in the RbcS Transit Peptide
To investigate the sequence motifs in the cTPs, Ala substitution mutants of the Arabidopsis (Arabidopsis thaliana) RbcS transit peptide were generated. 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. 1A). These mutant transit peptides were fused to green fluorescent protein (GFP) and introduced into protoplasts by polyethylene glycol-mediated transformation (Jin 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. 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 ). Most of the mutants, with the exceptions of T1A:GFP and T4A:GFP, had western-blot patterns that were nearly identical to the wild type (Fig. 1C), indicating that these mutant transit peptides were able to deliver reporter proteins into chloroplasts.
T1A:GFP and T4A:GFP had different western-blot patterns that were in agreement with the image analyses. Approximately 20% of T1A:GFP accumulated as the Pr at 12 h AT (Fig. 1C, b). However, Pr was no longer observed 24 h or 36 h AT, and, concomitantly, the intensity of M increased. These results suggested that the Pr of T1A:GFP was imported into chloroplasts more slowly than the wild type. T4A:GFP showed a western-blot pattern that differed from the other mutants, as well as from the wild type. T4A:GFP produced two major protein species of 31 and 37 kD (Fig. 1C, e). The 37-kD upper band of T4A:GFP was clearly smaller than the 39-kD form of T1A:GFP (Fig. 1C, j), suggesting that the 37-kD protein species may be an intermediate. 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. 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. 1C, e), indicating that the 37-kD form was imported into chloroplasts and processed correctly. Import intermediates are not usually observed in import assays using the RbcS precursor as the substrate in vitro. However, the chimeric substrate Oee1-Dhfr produced import intermediates under some conditions (Wu et al., 1994
To further confirm the localization of T1A:GFP and T4A:GFP, protein extracts from the transformed protoplasts were separated into supernatant and pellet fractions by ultracentrifugation and these fractions were analyzed by western blotting using anti-GFP antibody. 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. In accordance with this expectation, aleurain in the vacuolar lumen and Toc75 in the chloroplast envelope membrane were detected in the supernatant and pellet fractions, respectively (Fig. 1D; Ahmed et al., 2000
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. 2A). Both mutant transit peptides translocated the reporter proteins into chloroplasts as efficiently as the wild type (Fig. 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. This was not surprising since D1 and D2 both included two Ser, one Met, and one Ala. T1 included 50% hydroxylated amino acid residues. 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
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. 2A). 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. 2A). Protein targeting to chloroplasts with these mutant transit peptides was examined by image and western-blot analyses. 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. 2, B, g–l, C, d–f). These data indicated that a large number of Ser residues in T1 were detrimental for targeting. Surprisingly, the targeting efficiency of T1A + 3C to chloroplasts was similar to that of the wild type (Fig. 2, B, m and n, C, g). Cys is similar in structure to Ser, with a sulfhydryl group instead of a hydroxyl group. Thus, Cys is more hydrophobic than Ser. Finally, targeting of T1A + ML:GFP and T1A + LL:GFP to chloroplasts was as efficient as the wild type (Fig. 2, B, o–r, C, h and i), indicating that the Met and Leu residues in T1 were critical for targeting and that Leu could replace Met. 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. 2A). 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. 2B, s–v). 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. 2C, j and k). In addition, T1MLM/AAA accumulated more precursor protein than T1ML/AA. These results strongly suggested that hydrophobicity in T1 was critical for protein targeting to chloroplasts. Furthermore, T1ML/AA and T1MLM/AAA, which had four Ser residues at the original positions, yielded more precursors than T1A. This result is consistent with the notion that Ser residues are detrimental for targeting to chloroplasts.
Among the 10-amino acid block mutants, Ala substitution in T4 was the most detrimental for targeting proteins to chloroplasts. T4 contained part of a sequence that was similar to the proposed 14-3-3 binding site (May and Soll, 2000
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. T4A + FP:GFP was imported into chloroplasts as efficiently as the wild type (Fig. 3C, c). T4A + RK:GFP also yielded over 90% of the total reporter protein in M (Fig. 3C, d), indicating that restoration of RK to T4A improved the targeting efficiency to near the wild-type level. However, T4A + RK:GFP produced three additional protein species in minor amounts, migrating at 36, 37, and 65 kD. The 36- and 37-kD protein species that migrated faster than the precursor may have been intermediate forms. However, the protein species at 65 kD (asterisk in Fig. 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. The nature of this putative complex was unclear.
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. All of the 36-, 37-, and 65-kD forms, together with approximately 50% of the M, were detected in the pellet fraction (Fig. 3D, a). These proteins were not made soluble by Na2CO3 or Triton X-100 treatments (Fig. 3D, b). As controls for the fractionation and chemical treatments, we detected aleurain,
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. As a control for chloroplast fractionation, RFP (red fluorescent protein) was cotransformed into the protoplasts together with T4A + RK:GFP. Coexpressed RFP was not detected in the purified chloroplast fraction, confirming that it was not contaminated by cytoplasmic proteins. The 36-, 37-, and 65-kD forms, but not the 31-kD form, were sensitive to thermolysin (Fig. 3E), indicating that the GFP moiety of these three protein species was exposed to the cytoplasm. As controls for thermolysin treatment, Toc75 and the large subunit of Rubisco (RbcL) were detected by anti-Toc75 antibody and Coomassie Blue staining, respectively. In agreement with previous observations (Inoue and Potter, 2004
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. 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. 4A). These double-T mutants were fused to GFP and transiently expressed in protoplasts to examine their targeting to chloroplasts. 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. In addition, GFP fusion constructs with T1A in combination with each of the domains from T3A through T8A also were poorly expressed. Thus, these double-T mutants could not be evaluated.
The other combinations of double-T mutants were examined for their ability to target the reporter protein to chloroplasts. First, the targeting of double-T mutants with combinations of T2A and each domain from T4A through T8A, T2A/T4A to T2A/T8A, was examined by image analysis of GFP fluorescence. The GFP patterns of T2A/T5A:GFP, T2A/T6A:GFP, and T2A/T8A:GFP were nearly identical to that of the wild type (data not shown). 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. 4B, a, b, e, and f). 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. 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). However, the western-blot pattern of T2A/T4A:GFP and T2A/T7A:GFP were different from those of the individual T-block Ala substitution mutants. T2A/T4A:GFP yielded only the 37-kD form with no detectable M throughout the time course (Fig. 4B, c). In contrast, T2A/T7A:GFP yielded two protein species at 31 and 37 kD (Fig. 4B, g). At 12 AT, 40% to 50% of the total reporter protein accumulated as the 37-kD form. Although the amount of the 37-kD form declined gradually over time, it persisted until 36 h AT. This was quite similar to the western-blot pattern of T4A:GFP (Fig. 1C, e), although T4A:GFP yielded slightly more of the 37-kD form than T2A/T7A:GFP. These data indicated that the sequence motifs in T2, T4, and T7 were important for crossing the chloroplast envelope membrane. 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 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. 4B, d and h), strongly suggesting that the 37-kD form was trapped in the import channel. 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. 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.
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). Western-blot analysis of protein extracts obtained from T3A/T5A:GFP-transformed protoplasts confirmed this (data not shown). T3A/T6A:GFP produced diffuse GFP signals in the cytoplasm, but not in the chloroplasts, at both 12 and 24 h AT (Fig. 4C, a and b), indicating that motifs in T3 and T6 were important for the binding to chloroplasts. In agreement with data from image analysis, T3A/T6A:GFP primarily yielded the Pr with only a small portion of M at 12 h AT (Fig. 4C, c). The amount of Pr was greatly reduced at 24 h AT and barely detectable at 36 h AT. However surprisingly, the M increased only slightly with time, suggesting that Pr was degraded rapidly in the cytoplasm. 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. The amount of RFP increased gradually with time (Fig. 4C, d), confirming that the proteolysis was specific for T3A/T6A:GFP. The reason for the proteolysis was unclear. It is possible that Ala substitutions in T3 and T6 render the reporter protein susceptible to a protease. 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. In addition, aleurain and Toc75 were detected by anti-aleurain and anti-Toc75 antibodies, respectively. In agreement with the image analysis, most of Pr was detected in the supernatant (Fig. 4C, e). 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. However, we cannot rule out the possibility that these regions are involved in binding to the lipid membranes of chloroplasts (Pilon et al., 1995
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). Proteins are unfolded during translocation across the chloroplast envelope membrane (Guera et al., 1993
With T3A/T8A:GFP at 12 h AT, GFP signals accumulated at discrete sites on or along the surface of the chloroplasts (Fig. 4C, j). At 24 h AT, the GFP signals were present in the chloroplasts (Fig. 4C, k). 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. The import of T3A/T8A:GFP was analyzed by western blotting using anti-GFP antibody. At 12 h AT, approximately 60% of the total T3A/T8A:GFP protein was detected as M, with the remainder of the protein in the 37-kD form (Fig. 4C, l). The intermediate of T3A/T8A:GFP was reduced gradually over time as M increased, indicating that import occurred slowly. Samples taken at 12 h AT were fractionated by ultracentrifugation and the supernatant and pellet were analyzed by western blotting using anti-GFP antibody. Both the 37-kD form and the M were detected in the pellet but not in the supernatant (Fig. 4C, m), indicating that both forms remained associated with envelope membranes. 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
Next, double-T mutants T4A/T6A, T4A/T7A, and T4A/T8A were generated and fused to GFP (Fig. 5A). These constructs were transiently expressed in protoplasts to examine their targeting to chloroplasts. The targeting efficiency of another group of mutants, T4A/T6A:GFP, T4A/T7A:GFP, and T4A/T8A:GFP, was examined. 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. 5B, a and b). The GFP pattern of T4A/T6A:GFP was quite similar to that of T3A/T6:GFP (Fig. 5B, a and b). Consistent with the image analysis, western-blot analysis using anti-GFP antibody showed that T4A/T6A:GFP was present in the 39-kD Pr form with barely detectable amounts of M, up to 36 h AT (Fig. 5B, c). These results indicated that Ala substitution in T4 and T6 resulted in complete inhibition of protein import at very early stages. 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. When protein extracts were fractionated, Pr was equally distributed in the supernatant and pellet fractions (Fig. 5B, d), indicating that chloroplast association was defective, but not to the extent observed with T3A/T6A:GFP. 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.
Similar to T3A/T7A:GFP, T4A/T7A:GFP produced no GFP signal (data not shown). When detected by immunohistochemistry using anti-GFP antibody, T4A/T7A:GFP displayed a ring pattern around the red autofluorescent chlorophyll signal (Fig. 5B, e and f) that was clearly different from the diffuse pattern of cytoplasmic GFP (Fig. 5B, n), indicating that the reporter protein accumulated at the envelope membrane, but was not imported into the chloroplasts. Western-blot analysis confirmed this observation: T4A/T7A:GFP was present as the 37-kD intermediate at 12 h AT (Fig. 5B, g). 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. 4C, c). When RFP was cotransformed with T4A/T7A:GFP, the RFP level rose gradually (Fig. 5B, h), suggesting that the proteolysis was specific for T4A/T7A:GFP. When protein extracts were fractionated, the 37-kD form of T4A/T7A:GFP was detected in the pellet (Fig. 5B, i). 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. The 37-kD form of T4A/T7A:GFP was resistant to solubilization by Triton X-100 (Fig. 5C, a). Under the same conditions, the integral membrane AtVSR1 protein was completely solubilized (Fig. 5C, c). 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. 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. 5D, a). 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. 5D, b). Under the same conditions, RbcL was also resistant to thermolysin (Fig. 5D, c). GFP signals from T4A/T8A:GFP overlapped with the red autofluorescent signal of chlorophyll at 12 and 24 h AT (Fig. 5B, j and k), indicating that it was imported efficiently into chloroplasts. 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. 5B, l). 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. When protein extracts obtained at 12 h AT were fractionated, the 37-kD form of T4A/T8A:GFP was detected primarily in the supernatant. This was slightly different from the fractionation pattern of T4A:GFP that yielded 50% of the 37-kD form in the pellet.
To determine the critical amino acid residues in T3, T6, and T7, the mutants shown in Figure 6A were generated. The original sequences of subregions of T3, T6, or T7 were restored in each of the double-T mutants. The targeting efficiency of T3A + APFNG/T6A:GFP, T3A + LKSSA/T6A:GFP, T3A/T6A + GGRVN:GFP, and T3A/T6A + CMQVW:GFP was examined in protoplasts. Protein extracts were analyzed by western blotting using an anti-GFP antibody. The targeting efficiency of T3A + LKSSA/T6A improved greatly and was comparable to that of T3A or T6A (Fig. 6B, b), whereas the targeting efficiency of T3A + APFNG/T6A improved moderately, to 40% of that of T3A (Fig. 6B, c). These data indicated that LKSSA was the critical motif in T3. In addition, the targeting efficiency of T3A/T6A + CMQVW was equivalent to that of T3A or T6A (Fig. 6B, e), whereas the targeting efficiency of T3A/T6A + GGRVN improved marginally, to 30% to 40% of that of T3A (Fig. 6B, d), indicating that CMQVW was the critical motif in T6. 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 targeting efficiency of T3A + APFNG/T7A, T3A + LKSSA/T7A, T3A/T7A + PPIGK, and T3A/T7A + KKFET was examined by western-blot analysis (Fig. 6C). The targeting efficiencies of T3A + LKSSA/T7A and T3A/T7A + KKFET were similar to those of T3A and T7A (Fig. 6C, b and e), indicating that LKSSA in T3 or KKFET in T7 completely rescued the targeting deficiency of T3A/T7A. 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. 6C, c and d). These results suggested that the KKFET motif in T7 was critical for targeting a protein to chloroplasts. Furthermore, these results suggested that the KKFET motif in T7 was functionally redundant or compensatory to the LKSSA motif in T3 for protein targeting to chloroplasts.
Ala substitutions inevitably introduced a new secondary structure into the transit peptide that could affect targeting efficiency. Therefore, as an alternative approach, serial deletion mutants,
The double-T deletion mutants were examined for their ability to target a protein to the chloroplast. With these double-T deletion mutants, the processed M was almost undetectable throughout the time course, indicating that targeting was almost completely abolished (Fig. 7C). In addition, the intermediate form of  T4/T7:GFP was also sensitive to a protease (Fig. 7C, d) as observed with T4A/T7A:GFP (Fig. 5B, g). However, close inspection of the western-blot patterns showed that T2/T3:GFP, T3/T4:GFP, T3/T6:GFP, and T6/T7:GFP produced Pr, whereas T4/T7:GFP produced the 34-kD intermediate form (Fig. 7D). Thus, although the absence of T3, T6, or T7 alone was tolerated, the absence of the double-T blocks, T3 and T4, T3 and T6, T4 and T7, or T6 and T7 completely abolished targeting of the transit peptide (Fig. 7C), as observed with double-T mutants with Ala substitutions in the corresponding regions.
To further characterize the behavior of these reporter proteins, the subcellular distribution of the double-T deletion mutants was determined by ultracentrifugation.
Next, we examined whether the sequence motifs identified in the transit peptide of RbcS are also present in the transit peptides of other proteins. Sequence alignments revealed that identical or similar sequence motifs are present in the transit peptides of ferredoxin-2 and Asp carbamoyltransferase (ATCase; Fig. 8; Williamson and Slocum, 1994
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 relationships among the eight domains of the transit peptide are very complex. Under the conditions used in these experiments, only Ala substitution of T4 significantly affects the targeting efficiency. However, among the various double-T Ala substitution mutants, many exhibit 50% to 100% loss of targeting efficiency. The data herein clearly demonstrate that the transit peptides have multiple sequence elements with complex relationships. In addition, the data obtained with Ala substitution mutants were confirmed with individual- or double-T deletion mutants.
In T1 of the transit peptide, the hydrophobic nature of the ML motif is important for the initial rate of targeting. 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
Ala substitution of T2 alone does not affect targeting. However, when T2A is combined with T4A, the reporter proteins accumulate at the envelope membrane as intermediate forms, indicating that T2 and T4 act together to translocate a protein into chloroplasts. In addition, Ala substitution of both T2 and T7 causes 40% to 50% of the total reporter protein to remain in the intermediate form, almost identical to the result with T4A alone. 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. The N-terminal region of pSSU (precursor of Rubisco small subunit) interacts with Toc159 with high affinity (Becker et al., 2004
As in the case of T2, Ala substitution of T3 or T6 alone does not affect targeting. However, when T3A is combined with T6A the mutant cannot target proteins into the chloroplast and precursors accumulate in the cytoplasm. A simple explanation for these results is that T3 and T6 work together to serve as initial binding sites, during an early stage of protein import into chloroplasts. However, T3 and T6 share no obvious sequence similarity; the sequences LKSSA in T3 and CMQVW in T6 are critical for targeting. 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 T3 also displays a functional relationship to T7. The targeting efficiency of the mutant transit peptide T3A/T7A is reduced by more than 80%. However, in contrast to T3A/T6A:GFP, T3A/T7A:GFP yields a 37-kD intermediate that is detected in the pellet. As mentioned above, T7 is thought to play a role in crossing the envelope membrane. 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, 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. Detailed analysis shows that the KKFET sequence in T7 is critical for this process.
The T4 region is the most critical for targeting to chloroplasts. Ala substitution or deletion of T4 causes targeting efficiency to drop to 40% to 50% of the wild-type level. T4 has two essential sequences, FP and RK. 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 T4 also has a functional relationship to T6. 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. 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.
As described above, among the individual-T Ala substitutions and deletions, only the T4 substitution or deletion noticeably reduces the targeting efficiency. One possible explanation for this is redundancy in the transit peptide motifs. Consistent with this idea, the T2 motif is functionally equivalent to or compensates for the T7 motif. In addition, the LKSSA sequence in T3 is functionally equivalent to or compensates for the CMQVW sequence in T6 and the KKFET sequence in T7. However, the motifs that are functionally equivalent or compensatory do not necessarily have any sequence similarity. These motifs likely serve as binding sites for proteins or membranes at various steps during translocation (Richter and Lamppa, 1999
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. The next step will be to identify factors that bind to these motifs. Components of the Toc and Tic complexes have been identified and characterized at the molecular level (Gutensohn et al., 2000
Growth of Plants 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. Leaf tissues were harvested from 2-week-old plants and used immediately for protoplast isolation.
Construction of RbcS-nt:GFP was described previously (Lee et al., 2002
All plasmids were purified with Qiagen (Basel) columns, according to the manufacturer's protocol. The plasmids were introduced into Arabidopsis protoplasts prepared from leaf tissues by polyethylene glycol-mediated transformation (Jin et al., 2001
Protein extracts were prepared from the transformed protoplasts as described previously (Jin et al., 2001 For subcellular fractionation, cell extracts were separated into soluble and membrane fractions by ultracentrifugation at 100,000g for 30 min. 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. These fractions were analyzed by western blotting. The protein blots were developed with an ECL kit (Amersham Pharmacia Biotech) and images were obtained using a LAS3000 image capture system (FUJIFILM). The amount of protein on the immunoblots was determined by measuring the intensity of the bands with LAS3000 software.
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
Transformed protoplasts were placed on poly-L-Lys-coated glass slides and fixed as described previously (Lee et al., 2003
The authors thank Dr. Hsou-min Li (Academi of Sinica, Nankang, Taiwan) for anti-Toc75 antibody. Received November 22, 2005; returned for revision November 27, 2005; accepted December 1, 2005.
1 This work was supported by a grant from the Creative Research Initiative program of the Ministry of Science and Technology (Korea).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Inhwan Hwang (ihhwang{at}postech.ac.kr).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.074575. * Corresponding author; e-mail ihhwang{at}postech.ac.kr; fax 82–54–279–8159.
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-COP, and anti-VSR (BP80) antibodies. T, Total; P, pellet fraction; S, supernatant fraction; Pr, precursor form; and M, mature form.
