Nucleus-Encoded Light-Harvesting Chlorophyll a/b Proteins are Imported Normally into Chlorophyll b-Free Chloroplasts of Arabidopsis  Sabine Nick, Jörg Meurer, Jürgen Soll, Elisabeth Ankele  Molecular Plant  Volume 6, Issue 3, Pages 860-871 (May 2013) DOI: 10.1093/mp/sss113 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 LHC Protein Expression in Wild-Type (Col0) and cao-1 Mutant Plants.Chloroplasts equivalent to 20 µg of protein were fractionated by SDS–PAGE, blotted onto membranes which were used for immunoassays of LHC proteins using specific antibodies to analyze steady-state levels of (A) LHCA1–4 and (B) LHCB1–6. To determine protein abundance of LHCB1, LHCB4 and LHCB5 in cao-1, increasing amounts of protein concentration were loaded for comparison (C). The 100% loading control corresponds to 10 µg of chloroplast proteins. (D) Coomassie stained gel as loading control for (C). M, Molecular weight marker in kDa. Molecular Plant 2013 6, 860-871DOI: (10.1093/mp/sss113) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Import of LHC Proteins into Chloroplasts of Wild-Type (Col0) and cao-1.35S-labeled precursor proteins of LHCB1, LHCB4 and LHCB5 were imported for 20 min into isolated chloroplasts of (A) wild-type and (B) cao-1. Chloroplast samples were analyzed before (Im) or after (Th) thermolysin treatment to remove surface-attached proteins. As a control, 10% of the translation product was loaded (TL). Precursor (p) and mature proteins (m) are indicated. The asterisk (*) indicates a second translation product resulting from a second methionine in the amino acid sequence of Lhcb1.3 and Lhcb4.1. (C) Coomassie gels used for autoradiography are given as loading control. Molecular weight of protein markers are given in kDa as indicated on the left. (D) The amount of mature protein was calculated using ImageQuant (Version 5.2, GE Healthcare). Mature wild-type LHCB was set to 100%. The values given represent the mean of four independent experiments. Molecular Plant 2013 6, 860-871DOI: (10.1093/mp/sss113) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Import Efficiencies of Plastocyanin (PC) and LHCB4 in cao-1 Plants.35S-labeled precursor proteins of plastocyanin (A, B) or LHCB4 (C, D) were imported into chloroplasts isolated from (A, C) wild-type (Col0) or (B, D) cao-1. During a time course of 12.5 min, samples were taken every 2.5 min, separated by SDS–PAGE and processed proteins monitored by radiography. As a control, 10% of the translation product was loaded (TL). Precursor (p) and mature proteins (m) are indicated. The asterisk (*) indicates a second translation product resulting from a second methionine in the amino acid sequence Lhcb4.1. Molecular weight of protein markers are given in kDa as indicated on the left. Molecular Plant 2013 6, 860-871DOI: (10.1093/mp/sss113) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Stability of Imported and Processed LHC Proteins.35S-labeled precursor proteins of (A) LHCB1, (B) LHCB4 and (C) LHCB5 were imported for 10 min into isolated chloroplasts of wild-type and cao-1. The stability of the mature proteins was monitored over the course of 60 min. During this period, chloroplasts were kept at 25°C, and samples were harvested immediately after import (0 min.) and after further incubation for 5, 15, 30 and 60 min. Precursor (p), mature proteins (m) and translation products (TL) are indicated. Membranes were exposed to the same X-ray film. To confirm the stability of mature (D) LHCB1, (E) LHCB4 and (F) LHCB5, radioactively labeled bands were quantified by liquid scintillation counting. The amount of measured radioactivity at time point 0 was set to 100%. The values given for each time point represent the mean of three independent experiments. Molecular weight of protein markers are given in kDa as indicated on the left. Molecular Plant 2013 6, 860-871DOI: (10.1093/mp/sss113) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Assembly of In Vivo Synthesized LHC Proteins in cao-1 Mutants. Assembly of complexes synthesized in vivo was analyzed by BN/SDS-PAGE. Thylakoids were isolated from 35S-labeled leaves of wild-type and cao-1 and separated by BN-PAGE in the first dimension and SDS-PAGE in the second dimension. (A) The autoradiographs show the subunit composition of thylakoid protein complexes in the second dimension. mLHCB, D1 proteins are indicated. Proteins marked with asterisks (*) are CP43 and CP47. (B) Thylakoids separated on SDS-PAGES for second dimensions were blotted and decorated with antibodies recognizing all LHCA and LHCB proteins. Signals obtained from high molecular weight complexes are indicated by arrows. HMW: high-molecular-weight complexes; LMW: low-molecular-weight complexes. Molecular weight of protein markers are given in kDa as indicated on the left. Molecular Plant 2013 6, 860-871DOI: (10.1093/mp/sss113) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Turnover Rates of LHC Proteins Synthesized In Vivo in the cao-1 Mutant. In vivo pulse-chase experiments were separated by SDS–PAGE and monitored by radiography. (A) Leaves of wild-type and cao-1 were pre-incubated with chloramphenicol for 30 min followed by pulse-labeling with 35S-methionine for 30 min in the light (lanes 1–2). Thereafter, the samples were incubation with 10 mM unlabeled methionine (chase) for three different time points: chase 1 for 3 h in the light (lanes 3–4), chase 2 for 4 h in the light (lanes 5–6), and chase 3 for 3 h in the light followed by an additional hour in the dark (lanes 7–8). (B) The same samples as in (A) but blotted onto a PVDF-membrane and decorated with specific LHCB4 antibody. (C) To determine protein abundance of LHCB4 in cao-1, increasing amounts of protein concentration were loaded for comparison. The 100% loading control corresponds to 20 µg of chloroplast proteins. Coomassie stained gel as loading control for the immunoblot in (c). Molecular weight of protein markers are given in kDa as indicated on the left. Molecular Plant 2013 6, 860-871DOI: (10.1093/mp/sss113) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions