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An endogenous green fluorescent protein-photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer.

Fourrage C, Swann K, Gonzalez Garcia JR, Campbell AK, Houliston E - Open Biol (2014)

Bottom Line: Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria.Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms).Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universités, UPMC Univ Paris 06, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), Observatoire Océanologique, 06230 Villefranche-sur-mer, France.

ABSTRACT
Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria. It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

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Related in: MedlinePlus

Diversification of GFP and clytin genes. (a) Amino acid alignments of the N terminus of deduced Clytia GFP and clytin protein sequences. GFP2a, GFP2c and clytin2 sequences show an N terminal presequence, predicted with high probability to mediate targeting to mitochondria (Mitoprot software). Predicted cleaved sequences are shown in brackets. Black shading: identical amino acids; grey shading, similar amino acids. (b) Phylogenetic relationships of representative cnidarian GFP and clytin genes, deduced by ML from the alignments provided in the electronic supplementary material, figure S1. Sequences of arthropod and cephalochordate GFPs were used as an out-group for the GFPs tree (a), and sequences of calmodulin protein were used as an out-group for the clytin tree (b). Superscript M indicates the presence of a predicted mitochondrial targeting sequence. Bootstrap percentages (500 replicates) over 50% are shown. The scale bar indicates the number of amino acid substitutions per site. Acoe, Aequorea coerulescens; Amac, Aequorea macrodactyla; Avic, Aequorea Victoria; Amil, Acropora millepora; Ant, Anthomedusae species; Bf, Branchiostoma floridae; Cgra, Clytia gracilis; Cgre, Clytia gregarium; Che, Clytia hemisphaerica; Dis, Discosoma species; Laes, Labidocera aestiva; Mcav, Montastraea cavernosa; Mcel, Mitrocoma cellularia; Nv, Nematostella vectensis; Olon, Obelia longissima; Phy, Phialidium species; Pplu, Pontellina plumata; Rren, Renilla reniformis. Sequence accession numbers are given in the electronic supplementary material, table S2.
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RSOB130206F7: Diversification of GFP and clytin genes. (a) Amino acid alignments of the N terminus of deduced Clytia GFP and clytin protein sequences. GFP2a, GFP2c and clytin2 sequences show an N terminal presequence, predicted with high probability to mediate targeting to mitochondria (Mitoprot software). Predicted cleaved sequences are shown in brackets. Black shading: identical amino acids; grey shading, similar amino acids. (b) Phylogenetic relationships of representative cnidarian GFP and clytin genes, deduced by ML from the alignments provided in the electronic supplementary material, figure S1. Sequences of arthropod and cephalochordate GFPs were used as an out-group for the GFPs tree (a), and sequences of calmodulin protein were used as an out-group for the clytin tree (b). Superscript M indicates the presence of a predicted mitochondrial targeting sequence. Bootstrap percentages (500 replicates) over 50% are shown. The scale bar indicates the number of amino acid substitutions per site. Acoe, Aequorea coerulescens; Amac, Aequorea macrodactyla; Avic, Aequorea Victoria; Amil, Acropora millepora; Ant, Anthomedusae species; Bf, Branchiostoma floridae; Cgra, Clytia gracilis; Cgre, Clytia gregarium; Che, Clytia hemisphaerica; Dis, Discosoma species; Laes, Labidocera aestiva; Mcav, Montastraea cavernosa; Mcel, Mitrocoma cellularia; Nv, Nematostella vectensis; Olon, Obelia longissima; Phy, Phialidium species; Pplu, Pontellina plumata; Rren, Renilla reniformis. Sequence accession numbers are given in the electronic supplementary material, table S2.

Mentions: A further strong indication that GFP2 and clytin2 are targeted to egg mitochondria came from sequence analysis (figure 7). The predicted amino acid sequence of clytin2 and of some CheGFP2 variants displayed N-terminal leaders of 30–50 amino acids, which were shown using Mitoprot (http://ihg.gsf.de/ihg/mitoprot.html) [31] or iPsort (http://ipsort.hgc.jp/) [32] protein localization prediction software to have high probability (more than 0.8) to direct protein targeting and internalization within the mitochondrial lumen. For GFP2, pre-sequences with putative cleavage sites were present in sequences of two of the three variants from our EST collection (figure 7a). The three variants were otherwise quasi-identical at the nucleotide level, suggesting that the putative targeting sequence may derive from an alternative spliced N terminal exon. This was confirmed by the analysis of preliminary raw genomic sequence data from the ongoing C. hemisphaerica genome sequence project. Concerning clytin2 protein, sequence analysis predicted targeting to mitochondria with 0.9 probability. In contrast to Clytia GFP2, no leaderless cDNA variants were identified in our transcriptomic data, while genome sequence analysis confirmed that the 5′ leader sequence to this independent gene is not coded by a separate exon.Figure 7.


An endogenous green fluorescent protein-photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer.

Fourrage C, Swann K, Gonzalez Garcia JR, Campbell AK, Houliston E - Open Biol (2014)

Diversification of GFP and clytin genes. (a) Amino acid alignments of the N terminus of deduced Clytia GFP and clytin protein sequences. GFP2a, GFP2c and clytin2 sequences show an N terminal presequence, predicted with high probability to mediate targeting to mitochondria (Mitoprot software). Predicted cleaved sequences are shown in brackets. Black shading: identical amino acids; grey shading, similar amino acids. (b) Phylogenetic relationships of representative cnidarian GFP and clytin genes, deduced by ML from the alignments provided in the electronic supplementary material, figure S1. Sequences of arthropod and cephalochordate GFPs were used as an out-group for the GFPs tree (a), and sequences of calmodulin protein were used as an out-group for the clytin tree (b). Superscript M indicates the presence of a predicted mitochondrial targeting sequence. Bootstrap percentages (500 replicates) over 50% are shown. The scale bar indicates the number of amino acid substitutions per site. Acoe, Aequorea coerulescens; Amac, Aequorea macrodactyla; Avic, Aequorea Victoria; Amil, Acropora millepora; Ant, Anthomedusae species; Bf, Branchiostoma floridae; Cgra, Clytia gracilis; Cgre, Clytia gregarium; Che, Clytia hemisphaerica; Dis, Discosoma species; Laes, Labidocera aestiva; Mcav, Montastraea cavernosa; Mcel, Mitrocoma cellularia; Nv, Nematostella vectensis; Olon, Obelia longissima; Phy, Phialidium species; Pplu, Pontellina plumata; Rren, Renilla reniformis. Sequence accession numbers are given in the electronic supplementary material, table S2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043110&req=5

RSOB130206F7: Diversification of GFP and clytin genes. (a) Amino acid alignments of the N terminus of deduced Clytia GFP and clytin protein sequences. GFP2a, GFP2c and clytin2 sequences show an N terminal presequence, predicted with high probability to mediate targeting to mitochondria (Mitoprot software). Predicted cleaved sequences are shown in brackets. Black shading: identical amino acids; grey shading, similar amino acids. (b) Phylogenetic relationships of representative cnidarian GFP and clytin genes, deduced by ML from the alignments provided in the electronic supplementary material, figure S1. Sequences of arthropod and cephalochordate GFPs were used as an out-group for the GFPs tree (a), and sequences of calmodulin protein were used as an out-group for the clytin tree (b). Superscript M indicates the presence of a predicted mitochondrial targeting sequence. Bootstrap percentages (500 replicates) over 50% are shown. The scale bar indicates the number of amino acid substitutions per site. Acoe, Aequorea coerulescens; Amac, Aequorea macrodactyla; Avic, Aequorea Victoria; Amil, Acropora millepora; Ant, Anthomedusae species; Bf, Branchiostoma floridae; Cgra, Clytia gracilis; Cgre, Clytia gregarium; Che, Clytia hemisphaerica; Dis, Discosoma species; Laes, Labidocera aestiva; Mcav, Montastraea cavernosa; Mcel, Mitrocoma cellularia; Nv, Nematostella vectensis; Olon, Obelia longissima; Phy, Phialidium species; Pplu, Pontellina plumata; Rren, Renilla reniformis. Sequence accession numbers are given in the electronic supplementary material, table S2.
Mentions: A further strong indication that GFP2 and clytin2 are targeted to egg mitochondria came from sequence analysis (figure 7). The predicted amino acid sequence of clytin2 and of some CheGFP2 variants displayed N-terminal leaders of 30–50 amino acids, which were shown using Mitoprot (http://ihg.gsf.de/ihg/mitoprot.html) [31] or iPsort (http://ipsort.hgc.jp/) [32] protein localization prediction software to have high probability (more than 0.8) to direct protein targeting and internalization within the mitochondrial lumen. For GFP2, pre-sequences with putative cleavage sites were present in sequences of two of the three variants from our EST collection (figure 7a). The three variants were otherwise quasi-identical at the nucleotide level, suggesting that the putative targeting sequence may derive from an alternative spliced N terminal exon. This was confirmed by the analysis of preliminary raw genomic sequence data from the ongoing C. hemisphaerica genome sequence project. Concerning clytin2 protein, sequence analysis predicted targeting to mitochondria with 0.9 probability. In contrast to Clytia GFP2, no leaderless cDNA variants were identified in our transcriptomic data, while genome sequence analysis confirmed that the 5′ leader sequence to this independent gene is not coded by a separate exon.Figure 7.

Bottom Line: Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria.Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms).Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universités, UPMC Univ Paris 06, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), Observatoire Océanologique, 06230 Villefranche-sur-mer, France.

ABSTRACT
Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria. It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

Show MeSH
Related in: MedlinePlus