Limits...
Dual Organellar Targeting of Aminoacyl-tRNA Synthetases in Diatoms and Cryptophytes.

Gile GH, Moog D, Slamovits CH, Maier UG, Archibald JM - Genome Biol Evol (2015)

Bottom Line: In cryptophytes, translation also takes place in the periplastid compartment (PPC), which is the reduced cytoplasm of the plastid's red algal ancestor and which retains a reduced red algal nucleus.We searched the organelle and nuclear genomes of the cryptophyte Guillardia theta and the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana for aaRS genes and found an insufficient number of genes to provide each compartment with a complete set of aaRSs.We tested four of the predicted dual targeted aaRSs with green fluorescent protein fusion localizations in P. tricornutum and found evidence for dual targeting to the mitochondrion and plastid in P. tricornutum and G. theta, and indications for dual targeting to the PPC and cytosol in G. theta.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada g.gile@dal.ca.

Show MeSH

Related in: MedlinePlus

Fluorescence localization of aaRSs. Constructs of N-terminal targeting sequences from aaRSs fused to eGFP were expressed in the diatom P. tricornutum. (A) Localization of aaRS proteins from P. tricornutum, arginyl-tRNA synthetase 2 (Pt_argRS2) and asparaginyl-tRNA synthetase 2 (Pt_asnRS2). GFP fluorescence can be seen in a region adjacent to the plastid as well as colocalizing with PAF, indicative of dual targeting to the mitochondrion and the plastid. (B) Localization of tyrosyl-tRNA synthetase proteins from the cryptophyte G. theta (Gt_tyrRS1 and Gt_tyrRS2). The Gt_tyrRS1 construct shows diffuse fluorescence throughout the cytosolic area as well as in a small concentrated region characteristic of PPC localization (the “blob-like structure”), seen here as a small, hourglass-shaped, bright green spot. The Gt_tyrRS2 construct shows GFP fluorescence adjacent to and colocalizing with PAF, indicative of dual targeting to the mitochondrion and plastid. TL, transmitted light; Scale bars: 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494062&req=5

evv095-F3: Fluorescence localization of aaRSs. Constructs of N-terminal targeting sequences from aaRSs fused to eGFP were expressed in the diatom P. tricornutum. (A) Localization of aaRS proteins from P. tricornutum, arginyl-tRNA synthetase 2 (Pt_argRS2) and asparaginyl-tRNA synthetase 2 (Pt_asnRS2). GFP fluorescence can be seen in a region adjacent to the plastid as well as colocalizing with PAF, indicative of dual targeting to the mitochondrion and the plastid. (B) Localization of tyrosyl-tRNA synthetase proteins from the cryptophyte G. theta (Gt_tyrRS1 and Gt_tyrRS2). The Gt_tyrRS1 construct shows diffuse fluorescence throughout the cytosolic area as well as in a small concentrated region characteristic of PPC localization (the “blob-like structure”), seen here as a small, hourglass-shaped, bright green spot. The Gt_tyrRS2 construct shows GFP fluorescence adjacent to and colocalizing with PAF, indicative of dual targeting to the mitochondrion and plastid. TL, transmitted light; Scale bars: 10 μm.

Mentions: To test this prediction, we chose two P. tricornutum putatively dual-targeted aaRSs for localization experiments: asnRS2 (Phatr2 protein ID 42274, GenBank accession number KR017890) because it had the most strongly predicted signal peptide from the first M and the most strongly predicted mitochondrial presequence from the next M, and argRS2 (Phatr2 protein ID 36013, GenBank accession number KR017888) because it was the only signal-bearing aaRS without a downstream M residue before the conserved aaRS domain (fig. 2 and supplementary table S1, Supplementary Material online). We transformed P. tricornutum cells with vectors encoding the asnRS2 and argRS2 N-terminal extensions fused to eGFP. After expression of the fusion constructs, in both cases we were able to detect fluorescence not only overlying the autofluorescence of the plastid but also clearly extended alongside the plastid, a pattern that is strongly indicative of dual targeting to the plastid and mitochondrion (fig. 3A). Because the intensity of GFP fluorescence in the plastid was rather faint in comparison to the mitochondrion-localized GFP signal, and because it is known that mitochondria are usually located in close proximity to the plastid in P. tricornutum (Prihoda et al. 2012), we additionally stained the positive clones with mitotracker. We observed a clear colocalization of mitotracker with GFP fluorescence, as well as colocalization of GFP with PAF where mitotracker was absent (supplementary fig. S1, Supplementary Material online). However, it should be noted that the plastid-localized GFP fluorescence was not seen in all clones or in all cells of a clone, in which case the localization appeared to be mitochondrial only.Fig. 2.—


Dual Organellar Targeting of Aminoacyl-tRNA Synthetases in Diatoms and Cryptophytes.

Gile GH, Moog D, Slamovits CH, Maier UG, Archibald JM - Genome Biol Evol (2015)

Fluorescence localization of aaRSs. Constructs of N-terminal targeting sequences from aaRSs fused to eGFP were expressed in the diatom P. tricornutum. (A) Localization of aaRS proteins from P. tricornutum, arginyl-tRNA synthetase 2 (Pt_argRS2) and asparaginyl-tRNA synthetase 2 (Pt_asnRS2). GFP fluorescence can be seen in a region adjacent to the plastid as well as colocalizing with PAF, indicative of dual targeting to the mitochondrion and the plastid. (B) Localization of tyrosyl-tRNA synthetase proteins from the cryptophyte G. theta (Gt_tyrRS1 and Gt_tyrRS2). The Gt_tyrRS1 construct shows diffuse fluorescence throughout the cytosolic area as well as in a small concentrated region characteristic of PPC localization (the “blob-like structure”), seen here as a small, hourglass-shaped, bright green spot. The Gt_tyrRS2 construct shows GFP fluorescence adjacent to and colocalizing with PAF, indicative of dual targeting to the mitochondrion and plastid. TL, transmitted light; Scale bars: 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494062&req=5

evv095-F3: Fluorescence localization of aaRSs. Constructs of N-terminal targeting sequences from aaRSs fused to eGFP were expressed in the diatom P. tricornutum. (A) Localization of aaRS proteins from P. tricornutum, arginyl-tRNA synthetase 2 (Pt_argRS2) and asparaginyl-tRNA synthetase 2 (Pt_asnRS2). GFP fluorescence can be seen in a region adjacent to the plastid as well as colocalizing with PAF, indicative of dual targeting to the mitochondrion and the plastid. (B) Localization of tyrosyl-tRNA synthetase proteins from the cryptophyte G. theta (Gt_tyrRS1 and Gt_tyrRS2). The Gt_tyrRS1 construct shows diffuse fluorescence throughout the cytosolic area as well as in a small concentrated region characteristic of PPC localization (the “blob-like structure”), seen here as a small, hourglass-shaped, bright green spot. The Gt_tyrRS2 construct shows GFP fluorescence adjacent to and colocalizing with PAF, indicative of dual targeting to the mitochondrion and plastid. TL, transmitted light; Scale bars: 10 μm.
Mentions: To test this prediction, we chose two P. tricornutum putatively dual-targeted aaRSs for localization experiments: asnRS2 (Phatr2 protein ID 42274, GenBank accession number KR017890) because it had the most strongly predicted signal peptide from the first M and the most strongly predicted mitochondrial presequence from the next M, and argRS2 (Phatr2 protein ID 36013, GenBank accession number KR017888) because it was the only signal-bearing aaRS without a downstream M residue before the conserved aaRS domain (fig. 2 and supplementary table S1, Supplementary Material online). We transformed P. tricornutum cells with vectors encoding the asnRS2 and argRS2 N-terminal extensions fused to eGFP. After expression of the fusion constructs, in both cases we were able to detect fluorescence not only overlying the autofluorescence of the plastid but also clearly extended alongside the plastid, a pattern that is strongly indicative of dual targeting to the plastid and mitochondrion (fig. 3A). Because the intensity of GFP fluorescence in the plastid was rather faint in comparison to the mitochondrion-localized GFP signal, and because it is known that mitochondria are usually located in close proximity to the plastid in P. tricornutum (Prihoda et al. 2012), we additionally stained the positive clones with mitotracker. We observed a clear colocalization of mitotracker with GFP fluorescence, as well as colocalization of GFP with PAF where mitotracker was absent (supplementary fig. S1, Supplementary Material online). However, it should be noted that the plastid-localized GFP fluorescence was not seen in all clones or in all cells of a clone, in which case the localization appeared to be mitochondrial only.Fig. 2.—

Bottom Line: In cryptophytes, translation also takes place in the periplastid compartment (PPC), which is the reduced cytoplasm of the plastid's red algal ancestor and which retains a reduced red algal nucleus.We searched the organelle and nuclear genomes of the cryptophyte Guillardia theta and the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana for aaRS genes and found an insufficient number of genes to provide each compartment with a complete set of aaRSs.We tested four of the predicted dual targeted aaRSs with green fluorescent protein fusion localizations in P. tricornutum and found evidence for dual targeting to the mitochondrion and plastid in P. tricornutum and G. theta, and indications for dual targeting to the PPC and cytosol in G. theta.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada g.gile@dal.ca.

Show MeSH
Related in: MedlinePlus