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. 2017 Aug 24;7:9279. doi: 10.1038/s41598-017-09473-x

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The position of the FP-RK motif in CrRbcS TP is critical for its function in protein import into plant chloroplasts. (A and E) Sequences of AtRbcS TP, CrRbcS TP, and modified CrRbcS TPs. All constructs were fused to GFP. (B and F) Localization of reporter proteins. Protoplasts from Arabidopsis plants were transformed with the indicated constructs, and GFP patterns were observed 12 h after transformation. Green, red, and yellow signals represent GFP, autofluorescence of chlorophyll, and the overlap between green and red signals, respectively. Scale bar = 20 μm. (C and G) Western analysis of reporter proteins. Total protein extracts from transformed protoplasts were analyzed by western blotting with anti-GFP antibody. Pre, precursor form; P1, processed form 1; P2, processed form 2. Signal intensity of protein bands was measured using LAS3000 imager (FUJI FILM) software, and import efficiency was defined as the amount of P2 relative to the total amount of expressed protein. Three independent transformation experiments were performed, and the data represent means with standard deviation (SD). (D) Thermolysin sensitivity of reporter proteins. Protoplasts transformed with the indicated constructs were gently lysed and treated with thermolysin. Protein extracts were analyzed by western blotting using anti-GFP antibody. Pre, precursor form; P1, processed form 1; P2, processed form 2.