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Evolution and targeting of Omp85 homologs in the chloroplast outer envelope membrane.

Day PM, Potter D, Inoue K - Front Plant Sci (2014)

Bottom Line: Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85), Toc75, and OEP80 are inconsistent.The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80.Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, University of California at Davis Davis, CA, USA.

ABSTRACT
Translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75) is the core component of the chloroplast protein import machinery. It belongs to the Omp85 family whose members exist in various Gram-negative bacteria, mitochondria, and chloroplasts of eukaryotes. Chloroplasts of Viridiplantae contain another Omp85 homolog called outer envelope protein 80 (OEP80), whose exact function is unknown. In addition, the Arabidopsis thaliana genome encodes truncated forms of Toc75 and OEP80. Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85), Toc75, and OEP80 are inconsistent. The bipartite targeting sequence-dependent sorting of Toc75 has been demonstrated but the targeting mechanisms of other chloroplast Omp85 homologs remain largely unexplored. This study was aimed to address these unresolved issues in order to further our understanding of chloroplast evolution. Sequence alignments and recently determined structures of bacterial Omp85 homologs were used to predict structures of chloroplast Omp85 homologs. The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80. Phylogenetic analyses using Omp85 homologs from various cyanobacteria and chloroplasts provided strong support for the grouping of Toc75 and OEP80 sister to cyanoOmp85. However, this support was diminished when the analysis included Omp85 homologs from other bacteria and mitochondria. Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.

No MeSH data available.


Related in: MedlinePlus

Import of chloroplast Omp85 homologs in vitro. (A) Chloroplasts isolated from pea seedlings were incubated with radiolabeled proteins indicated at left in the import condition for 10 min in the light. After the reaction, chloroplasts were re-isolated and divided into two samples. The first sample was loaded directly on SDS-PAGE (C). The second sample was lysed hypotonically and separated into supernatant (S1) and pellet fractions by centrifugation. The pellet fraction was further resuspended into 0.1M Na2CO3 and separated into the supernatant (S2) and pellet (P) fractions by another centrifugation. The obtained S1, S2, and P fractions contained soluble, peripheral membrane, and integral membrane proteins, respectively. Radiolabeled proteins recovered in each fraction were separated by SDS-PAGE and visualized by phosphorimager analysis. Each lane was loaded with the sample equal to chloroplasts containing 3 μg chlorophylls used for the import assay, and tl was loaded with 10% of the translation products equivalent to those used for the import assay containing 3 μg chlorophyll-equivalent chloroplasts. For OEP80, the 74-kD OEP80 precursor, and the 66–71 kD import products are indicated with x and y, respectively. For others, pr, i, and m indicate precursor, import intermediate, and mature forms, respectively. The Coomassie Brilliant Blue (CBB)-stained gel for OEP80tr is shown in the bottom. The major soluble protein of 50 kD (LSU = ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) and the major membrane protein of 25 kD (LHCP = light-harvesting chlorophyll a/b binding protein) are indicated with arrowheads. (B) Chloroplast isolated from pea seedlings were incubated with radiolabeled proteins indicated at left in the import condition for 30 min in the light. After the reaction, chloroplasts were re-isolated and treated for 30 min without (–) or with (+) thermolysin on ice or trypsin at room temperature. Digestion control was done by including detergent (Triton X-100 = TX100) in the reaction. After the reactions were quenched with EDTA (for thermolysin) or trypsin inhibitor (for trypsin), chloroplasts were re-isolated by a 40% Percoll cushion and the radiolabeled proteins in the resultant samples were examined as described in the legend to panel (A). CBB-stained gels for OEP80tr are shown. See legend to panel (A) for descriptions of the labels.
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Figure 4: Import of chloroplast Omp85 homologs in vitro. (A) Chloroplasts isolated from pea seedlings were incubated with radiolabeled proteins indicated at left in the import condition for 10 min in the light. After the reaction, chloroplasts were re-isolated and divided into two samples. The first sample was loaded directly on SDS-PAGE (C). The second sample was lysed hypotonically and separated into supernatant (S1) and pellet fractions by centrifugation. The pellet fraction was further resuspended into 0.1M Na2CO3 and separated into the supernatant (S2) and pellet (P) fractions by another centrifugation. The obtained S1, S2, and P fractions contained soluble, peripheral membrane, and integral membrane proteins, respectively. Radiolabeled proteins recovered in each fraction were separated by SDS-PAGE and visualized by phosphorimager analysis. Each lane was loaded with the sample equal to chloroplasts containing 3 μg chlorophylls used for the import assay, and tl was loaded with 10% of the translation products equivalent to those used for the import assay containing 3 μg chlorophyll-equivalent chloroplasts. For OEP80, the 74-kD OEP80 precursor, and the 66–71 kD import products are indicated with x and y, respectively. For others, pr, i, and m indicate precursor, import intermediate, and mature forms, respectively. The Coomassie Brilliant Blue (CBB)-stained gel for OEP80tr is shown in the bottom. The major soluble protein of 50 kD (LSU = ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) and the major membrane protein of 25 kD (LHCP = light-harvesting chlorophyll a/b binding protein) are indicated with arrowheads. (B) Chloroplast isolated from pea seedlings were incubated with radiolabeled proteins indicated at left in the import condition for 30 min in the light. After the reaction, chloroplasts were re-isolated and treated for 30 min without (–) or with (+) thermolysin on ice or trypsin at room temperature. Digestion control was done by including detergent (Triton X-100 = TX100) in the reaction. After the reactions were quenched with EDTA (for thermolysin) or trypsin inhibitor (for trypsin), chloroplasts were re-isolated by a 40% Percoll cushion and the radiolabeled proteins in the resultant samples were examined as described in the legend to panel (A). CBB-stained gels for OEP80tr are shown. See legend to panel (A) for descriptions of the labels.

Mentions: We hypothesized that comparing targeting mechanisms of chloroplast Omp85 homologs may provide us with hints about their evolution. The immediate questions we had were as follows: Is OEP80tr targeted to the chloroplast outer membrane? Does the targeting of OEP80 and OEP80tr include processing and require ATP as in the case of Toc75? To test these, we conducted import assays using radiolabeled proteins synthesized by in vitro transcription and translation and chloroplasts isolated from pea seedlings. For OEP80, we used the DNA construct lacking the sequence encoding the first 52 residues for in vitro transcription because this portion is dispensable for proper expression and functioning of endogenous OEP80 (Patel et al., 2008; Hsu et al., 2012). After the import reaction, intact chloroplasts were re-isolated and analyzed either directly (C) or after separation by lysis and centrifugation into soluble (S1), peripheral-membrane (S2), and integral-membrane (P) fractions. The fractionation was monitored by distribution of the abundant endogenous proteins located in the stroma [the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (LSU)] and thylakoids [light harvesting chlorophyll a/b binding protein (LHCP)] by Coomassie Brilliant Blue staining (Figure 4A, panel CBB), as well as the distribution of the newly-imported peripheral membrane protein Tic22 (Kouranov et al., 1998) (Figure 4A, panel Tic22). In order to test the location of the imported proteins, re-isolated chloroplasts were also treated with thermolysin, which has access only to the surface of the outer membrane (Cline et al., 1981), or trypsin, which can penetrate the outer but not the inner membrane of the chloroplast envelope (Jackson et al., 1998).


Evolution and targeting of Omp85 homologs in the chloroplast outer envelope membrane.

Day PM, Potter D, Inoue K - Front Plant Sci (2014)

Import of chloroplast Omp85 homologs in vitro. (A) Chloroplasts isolated from pea seedlings were incubated with radiolabeled proteins indicated at left in the import condition for 10 min in the light. After the reaction, chloroplasts were re-isolated and divided into two samples. The first sample was loaded directly on SDS-PAGE (C). The second sample was lysed hypotonically and separated into supernatant (S1) and pellet fractions by centrifugation. The pellet fraction was further resuspended into 0.1M Na2CO3 and separated into the supernatant (S2) and pellet (P) fractions by another centrifugation. The obtained S1, S2, and P fractions contained soluble, peripheral membrane, and integral membrane proteins, respectively. Radiolabeled proteins recovered in each fraction were separated by SDS-PAGE and visualized by phosphorimager analysis. Each lane was loaded with the sample equal to chloroplasts containing 3 μg chlorophylls used for the import assay, and tl was loaded with 10% of the translation products equivalent to those used for the import assay containing 3 μg chlorophyll-equivalent chloroplasts. For OEP80, the 74-kD OEP80 precursor, and the 66–71 kD import products are indicated with x and y, respectively. For others, pr, i, and m indicate precursor, import intermediate, and mature forms, respectively. The Coomassie Brilliant Blue (CBB)-stained gel for OEP80tr is shown in the bottom. The major soluble protein of 50 kD (LSU = ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) and the major membrane protein of 25 kD (LHCP = light-harvesting chlorophyll a/b binding protein) are indicated with arrowheads. (B) Chloroplast isolated from pea seedlings were incubated with radiolabeled proteins indicated at left in the import condition for 30 min in the light. After the reaction, chloroplasts were re-isolated and treated for 30 min without (–) or with (+) thermolysin on ice or trypsin at room temperature. Digestion control was done by including detergent (Triton X-100 = TX100) in the reaction. After the reactions were quenched with EDTA (for thermolysin) or trypsin inhibitor (for trypsin), chloroplasts were re-isolated by a 40% Percoll cushion and the radiolabeled proteins in the resultant samples were examined as described in the legend to panel (A). CBB-stained gels for OEP80tr are shown. See legend to panel (A) for descriptions of the labels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4195282&req=5

Figure 4: Import of chloroplast Omp85 homologs in vitro. (A) Chloroplasts isolated from pea seedlings were incubated with radiolabeled proteins indicated at left in the import condition for 10 min in the light. After the reaction, chloroplasts were re-isolated and divided into two samples. The first sample was loaded directly on SDS-PAGE (C). The second sample was lysed hypotonically and separated into supernatant (S1) and pellet fractions by centrifugation. The pellet fraction was further resuspended into 0.1M Na2CO3 and separated into the supernatant (S2) and pellet (P) fractions by another centrifugation. The obtained S1, S2, and P fractions contained soluble, peripheral membrane, and integral membrane proteins, respectively. Radiolabeled proteins recovered in each fraction were separated by SDS-PAGE and visualized by phosphorimager analysis. Each lane was loaded with the sample equal to chloroplasts containing 3 μg chlorophylls used for the import assay, and tl was loaded with 10% of the translation products equivalent to those used for the import assay containing 3 μg chlorophyll-equivalent chloroplasts. For OEP80, the 74-kD OEP80 precursor, and the 66–71 kD import products are indicated with x and y, respectively. For others, pr, i, and m indicate precursor, import intermediate, and mature forms, respectively. The Coomassie Brilliant Blue (CBB)-stained gel for OEP80tr is shown in the bottom. The major soluble protein of 50 kD (LSU = ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) and the major membrane protein of 25 kD (LHCP = light-harvesting chlorophyll a/b binding protein) are indicated with arrowheads. (B) Chloroplast isolated from pea seedlings were incubated with radiolabeled proteins indicated at left in the import condition for 30 min in the light. After the reaction, chloroplasts were re-isolated and treated for 30 min without (–) or with (+) thermolysin on ice or trypsin at room temperature. Digestion control was done by including detergent (Triton X-100 = TX100) in the reaction. After the reactions were quenched with EDTA (for thermolysin) or trypsin inhibitor (for trypsin), chloroplasts were re-isolated by a 40% Percoll cushion and the radiolabeled proteins in the resultant samples were examined as described in the legend to panel (A). CBB-stained gels for OEP80tr are shown. See legend to panel (A) for descriptions of the labels.
Mentions: We hypothesized that comparing targeting mechanisms of chloroplast Omp85 homologs may provide us with hints about their evolution. The immediate questions we had were as follows: Is OEP80tr targeted to the chloroplast outer membrane? Does the targeting of OEP80 and OEP80tr include processing and require ATP as in the case of Toc75? To test these, we conducted import assays using radiolabeled proteins synthesized by in vitro transcription and translation and chloroplasts isolated from pea seedlings. For OEP80, we used the DNA construct lacking the sequence encoding the first 52 residues for in vitro transcription because this portion is dispensable for proper expression and functioning of endogenous OEP80 (Patel et al., 2008; Hsu et al., 2012). After the import reaction, intact chloroplasts were re-isolated and analyzed either directly (C) or after separation by lysis and centrifugation into soluble (S1), peripheral-membrane (S2), and integral-membrane (P) fractions. The fractionation was monitored by distribution of the abundant endogenous proteins located in the stroma [the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (LSU)] and thylakoids [light harvesting chlorophyll a/b binding protein (LHCP)] by Coomassie Brilliant Blue staining (Figure 4A, panel CBB), as well as the distribution of the newly-imported peripheral membrane protein Tic22 (Kouranov et al., 1998) (Figure 4A, panel Tic22). In order to test the location of the imported proteins, re-isolated chloroplasts were also treated with thermolysin, which has access only to the surface of the outer membrane (Cline et al., 1981), or trypsin, which can penetrate the outer but not the inner membrane of the chloroplast envelope (Jackson et al., 1998).

Bottom Line: Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85), Toc75, and OEP80 are inconsistent.The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80.Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, University of California at Davis Davis, CA, USA.

ABSTRACT
Translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75) is the core component of the chloroplast protein import machinery. It belongs to the Omp85 family whose members exist in various Gram-negative bacteria, mitochondria, and chloroplasts of eukaryotes. Chloroplasts of Viridiplantae contain another Omp85 homolog called outer envelope protein 80 (OEP80), whose exact function is unknown. In addition, the Arabidopsis thaliana genome encodes truncated forms of Toc75 and OEP80. Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85), Toc75, and OEP80 are inconsistent. The bipartite targeting sequence-dependent sorting of Toc75 has been demonstrated but the targeting mechanisms of other chloroplast Omp85 homologs remain largely unexplored. This study was aimed to address these unresolved issues in order to further our understanding of chloroplast evolution. Sequence alignments and recently determined structures of bacterial Omp85 homologs were used to predict structures of chloroplast Omp85 homologs. The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80. Phylogenetic analyses using Omp85 homologs from various cyanobacteria and chloroplasts provided strong support for the grouping of Toc75 and OEP80 sister to cyanoOmp85. However, this support was diminished when the analysis included Omp85 homologs from other bacteria and mitochondria. Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.

No MeSH data available.


Related in: MedlinePlus