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Results: 5

1.
Fig. 5.

Fig. 5. From: An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts.

Radicicol inhibits the import of a diverse set of nucleus-encoded chloroplast preproteins. (AD) In vitro translated [35S] preatE1α (A), [35S] preatAPG1 (B), [35S] pre-LHCP (C), or [35S] preatTic20 (D) were incubated with pea chloroplasts and 1 mM ATP or 5 mM ATP in the presence or absence of radicicol at 26 °C for 20 min as described in the legend of Fig. 4A. Lane 1 contains 10% of the [35S] preprotein added to each reaction (IVT). Each data bar represents the mean ± SEM (n = 3).

Hitoshi Inoue, et al. Proc Natl Acad Sci U S A. 2013 February 19;110(8):3173-3178.
2.
Fig. 3.

Fig. 3. From: An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts.

atHsp90C is essential for viability. (A) Schematic representation of the AtHSP90C gene (AT2g04030). The position of the T-DNA insertion in athsp90c-1 (SALK_120525) is indicated by an open triangle. Protein-coding exons are depicted by black boxes, and untranslated regions are depicted by white boxes. LB, the T-DNA left border. (B) Micrographs of dissected siliques from AtHSP90C/athsp90c-1 heterozygous plants. Aborted embryos/seeds are indicated by arrowheads. (C) Complementation of homozygous of hsp90c-1 with the atHsp90C cDNA under control of a 1185-bp genomic fragment from upstream of the AT2g04030 coding region (atHsp90C). Visual phenotype and genotypes of wild type and hsp90c-1 plants transformed with the atHsp90C cDNA were confirmed by PCR analysis of genomic DNA from plants with primer sets specific for the cDNA, T-DNA insertion, and endogenous gene (SI Materials and Methods and Table S1).

Hitoshi Inoue, et al. Proc Natl Acad Sci U S A. 2013 February 19;110(8):3173-3178.
3.
Fig. 1.

Fig. 1. From: An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts.

Hsp90C is associated with preprotein import intermediates. (A) Diagrams of the preproteins used in this study. (B) Urea-denatured preatSSU–3xFLAG and preatTic40–3xFLAG were incubated with isolated pea chloroplasts under import conditions. After the reaction, the chloroplasts were isolated and treated with 1 mM DSP in the dark for 15 min. Chloroplasts (1 mg of chlorophyll) were dissolved in buffer containing 1% Triton X-100 and immunoprecipitated with M2 Flag antibody. Equivalent fractions of total chloroplast detergent extract (L), unbound material (UB), and immunoaffinity eluates (E), were resolved by SDS/PAGE and the resolved polypeptides were visualized by immunoblotting with antisera to chloroplast proteins as indicated to the Left of each panel. Lanes 4 and 8 of the Lower panels contain 10% of the preprotein added to each import reaction (Pr). Gels in the Lower panels were loaded with 10% of the material in the Upper panels and were probed with M2 FLAG antibody to detect preprotein intermediates.

Hitoshi Inoue, et al. Proc Natl Acad Sci U S A. 2013 February 19;110(8):3173-3178.
4.
Fig. 4.

Fig. 4. From: An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts.

Radicicol inhibits preprotein import into chloroplasts. (A and B) Isolated pea chloroplasts were incubated with in vitro translated [35S] preatSSU (A) or [35S] preatTic40 (B) and 1 or 5 mM ATP at 26 °C for 20 min (lanes 2–6) in the presence of different concentrations of radicicol as indicated. Lane 1 contains 10% of the [35S] preprotein added to each reaction (IVT). (C) Chloroplasts were incubated with urea-denatured preatSSU–3xFLAG in the presence or absence of radicicol and the indicated concentrations of nucleoside triphosphates (NTP). The chloroplasts were isolated, resolved by SDS/PAGE, and the precursor (pre) and mature (mat) forms of atSSU–3xFLAG were detected by immunoblotting with anti-FLAG. (D) Chloroplasts were incubated with urea-denature preatSS–3xFLAG in the presence of 0.1 mM ATP and GTP to promote formation of the early import intermediate (lane 2). In lanes 3–6, equivalent samples of chloroplasts to lane 2 were incubated with the indicated concentration of ATP and 500 μM radicicol to promote protein import. (C and D) Lane 1 contains 10% of the preprotein (pr) added to each reaction. (AD) Each data bar represents the mean ± SEM (n = 3).

Hitoshi Inoue, et al. Proc Natl Acad Sci U S A. 2013 February 19;110(8):3173-3178.
5.
Fig. 2.

Fig. 2. From: An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts.

Hsp90C is associated with TOC–TIC supercomplexes. (A) Isolated pea chloroplasts (200 μg of chlorophyll) from untreated chloroplasts (−) or DSP-treated chloroplasts (+DSP) were dissolved in buffer containing 1% Triton X-100 and sequentially applied to preimmune IgG sepharose and anti-Hsp90C sepharose. The unbound fraction (UB) and eluates from the preimmune IgG (Pre) or anti-Hsp90C (E) sepharose were resolved by SDS/PAGE and immunoblotted with antibodies to the proteins indicated at the Left of the panels. (B) Total membrane fraction from DSP-treated chloroplasts was subjected to immunoaffinity chromatography and immunoblot analysis as in A. (C) DSP-treated chloroplasts were subjected to immunoaffinity chromatography using preimmune IgG sepharose, anti-Tic110 sepharose, or anti-Tic40 sepharose and immunoblotted as in A. (D) Isolated chloroplasts (200 μg of chlorophyll) were incubated with urea-denatured preatSSU–3xFLAG (lanes 6–9). Chloroplasts were treated with 1 mM DSP and subjected to sequential immunoaffinity chromatography as described in A. Lane 5 contains 10% of the preprotein added to each import reaction (Pr). (E) Quantification of proteins coeluting with Hsp90C in lanes 3 and 8 of D. Mean of the amounts measured in the absence of preatSSU–3xFLAG (D, lane 3) was set at 1 for each sample (white bars). Chemilumenescence signals from the immunoblots in the presence of preatSSU–3xFLAG (D, lane 8) were quantified and plotted as a ratio to the amount coeluting in the absence of preatSSU–3xFLAG (black bars). Error bars represent the SE (n = 3).

Hitoshi Inoue, et al. Proc Natl Acad Sci U S A. 2013 February 19;110(8):3173-3178.

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