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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.


Energy requirement for OEP80tr import in vitro. Translation products including the radiolabeled proteins indicated at left (tl) were incubated without (–) or with (+) apyrase at room temperature for 15 min in the light. The resultant samples were incubated with isolated chloroplasts without (–) or with (+) 3 mM MgATP for 30 min at room temperature in the dark. Chloroplasts were re-isolated, fractionated and examined as described in Figure 4A.
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Figure 5: Energy requirement for OEP80tr import in vitro. Translation products including the radiolabeled proteins indicated at left (tl) were incubated without (–) or with (+) apyrase at room temperature for 15 min in the light. The resultant samples were incubated with isolated chloroplasts without (–) or with (+) 3 mM MgATP for 30 min at room temperature in the dark. Chloroplasts were re-isolated, fractionated and examined as described in Figure 4A.

Mentions: We also tested the ATP requirement for the import of chloroplast Omp85 homologs. Excluding ATP from the reaction disrupted import of Toc75 as evidenced by the increased level of the precursor (pr) and the decreased level of the intermediate (i) and mature form (m) (Figure 5, panel Toc75, compare lanes 2 and 6), but did not affect membrane integration of OEP80tr and Toc75-IV (Figure 5, panels OEP80tr and Toc75-IV, compare lanes 5 and 9). Interestingly, apyrase treatment disrupted import of Toc75 and Toc75-IV but did not affect that of OEP80tr (Figure 5, compare lanes 6 and 10; Figure S2). Again, our result with Toc75-IV appeared to contradict the conclusion of the previous study that chloroplast-association of Toc75-IV did not require ATP (Baldwin et al., 2005). This discrepancy may be due to the use of different methods to deplete nucleoside triphosphates (NTPs) in the reaction. The previous work used gel filtration to remove NTPs from the translation products (Baldwin et al., 2005). By contrast, our assay used apyrase which should remove NTPs not only from the translation products but also from the chloroplasts used for the assay. Thus, our data suggest that a slight amount of ATP is required for Toc75-IV targeting while it is not the case for the targeting of OEP80tr.


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

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

Energy requirement for OEP80tr import in vitro. Translation products including the radiolabeled proteins indicated at left (tl) were incubated without (–) or with (+) apyrase at room temperature for 15 min in the light. The resultant samples were incubated with isolated chloroplasts without (–) or with (+) 3 mM MgATP for 30 min at room temperature in the dark. Chloroplasts were re-isolated, fractionated and examined as described in Figure 4A.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Energy requirement for OEP80tr import in vitro. Translation products including the radiolabeled proteins indicated at left (tl) were incubated without (–) or with (+) apyrase at room temperature for 15 min in the light. The resultant samples were incubated with isolated chloroplasts without (–) or with (+) 3 mM MgATP for 30 min at room temperature in the dark. Chloroplasts were re-isolated, fractionated and examined as described in Figure 4A.
Mentions: We also tested the ATP requirement for the import of chloroplast Omp85 homologs. Excluding ATP from the reaction disrupted import of Toc75 as evidenced by the increased level of the precursor (pr) and the decreased level of the intermediate (i) and mature form (m) (Figure 5, panel Toc75, compare lanes 2 and 6), but did not affect membrane integration of OEP80tr and Toc75-IV (Figure 5, panels OEP80tr and Toc75-IV, compare lanes 5 and 9). Interestingly, apyrase treatment disrupted import of Toc75 and Toc75-IV but did not affect that of OEP80tr (Figure 5, compare lanes 6 and 10; Figure S2). Again, our result with Toc75-IV appeared to contradict the conclusion of the previous study that chloroplast-association of Toc75-IV did not require ATP (Baldwin et al., 2005). This discrepancy may be due to the use of different methods to deplete nucleoside triphosphates (NTPs) in the reaction. The previous work used gel filtration to remove NTPs from the translation products (Baldwin et al., 2005). By contrast, our assay used apyrase which should remove NTPs not only from the translation products but also from the chloroplasts used for the assay. Thus, our data suggest that a slight amount of ATP is required for Toc75-IV targeting while it is not the case for the targeting of OEP80tr.

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.