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Cross-Species Interaction between Rapidly Evolving Telomere-Specific Drosophila Proteins.

Vedelek B, Blastyák A, Boros IM - PLoS ONE (2015)

Bottom Line: The accelerated evolution of terminin components may indicate the involvement of these proteins in the process by which new species arise, as the resulting divergence of terminin proteins might prevent hybrid formation, thus driving speciation.Our results suggest formation of stable subcomplexes of terminin, but not of the whole complex in vitro.We found that the accelerated evolution is restricted to definable regions of terminin components, and that the divergence of D. melanogaster Drosophila Telomere Loss and D. yakuba Verrocchio proteins does not preclude their stable interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary.

ABSTRACT
Telomere integrity in Drosophila melanogaster is maintained by a putative multisubunit complex called terminin that is believed to act in analogy to the mammalian shelterin complex in protecting chromosome ends from being recognized as sites of DNA damage. The five proteins supposed to form the terminin complex are HP1-ORC associated protein, HP1-HOAP interacting protein, Verrocchio, Drosophila Telomere Loss/Modigliani and Heterochromatic Protein 1. Four of these proteins evolve rapidly within the Drosophila genus. The accelerated evolution of terminin components may indicate the involvement of these proteins in the process by which new species arise, as the resulting divergence of terminin proteins might prevent hybrid formation, thus driving speciation. However, terminin is not an experimentally proven entity, and no biochemical studies have been performed to investigate its assembly and action in detail. Motivated by these facts in order to initiate biochemical studies on terminin function, we attempted to reconstitute terminin by co-expressing its subunits in bacteria and investigated the possible role of the fast-evolving parts of terminin components in complex assembly. Our results suggest formation of stable subcomplexes of terminin, but not of the whole complex in vitro. We found that the accelerated evolution is restricted to definable regions of terminin components, and that the divergence of D. melanogaster Drosophila Telomere Loss and D. yakuba Verrocchio proteins does not preclude their stable interaction.

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Co-expression of interacting partners increases the solubility of terminin proteins.(A) Presumed interactions among terminin proteins based on GST pull-down assays [17–19]. (B) Solubility of terminin components expressed individually in Arctic Express cells. Arrowheads point to bands corresponding to specific terminin proteins. HipHop expression cannot be observed on Coomassie-stained gel. On panel B images of different parts of the same gel are shown. (C) Co-expression of terminin components from polycistronic constructs. On panel C different parts of the same gel are shown except for the part with DTL/Moi-Ver data. L: molecular weight marker ladder, W: whole cell extract, S: supernatant, P: pellet, NC: control supernatant without any heterologous protein.
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pone.0142771.g003: Co-expression of interacting partners increases the solubility of terminin proteins.(A) Presumed interactions among terminin proteins based on GST pull-down assays [17–19]. (B) Solubility of terminin components expressed individually in Arctic Express cells. Arrowheads point to bands corresponding to specific terminin proteins. HipHop expression cannot be observed on Coomassie-stained gel. On panel B images of different parts of the same gel are shown. (C) Co-expression of terminin components from polycistronic constructs. On panel C different parts of the same gel are shown except for the part with DTL/Moi-Ver data. L: molecular weight marker ladder, W: whole cell extract, S: supernatant, P: pellet, NC: control supernatant without any heterologous protein.

Mentions: Previous studies have indicated interactions among terminin components as summarized in Fig 3A. In brief: Ver interacts with DTL/Moi and HOAP [18], and DTL/Moi interacts with Ver, HOAP and HP1 [19]. However, HipHop does not interact directly with Ver or DTL/Moi [17, 18, 19]. To verify these interactions and to explore more connections between specific terminin proteins, furthermore to uncover signs of co-evolution which might play a role in speciation, we studied heterologously expressed terminin proteins. Our strategy to obtain recombinant terminin components for complex assembly studies involved cloning cDNA sequences into expression vector(s) and producing the proteins in bacteria. For each of the five proteins we attempted to express, we used cDNA fragments encoding the complete coding regions, nonetheless in some cases we observed expression of shorter products resulting from degradation (see later in more detail). The use of monocistronic constructs revealed that HP1, HOAP, Ver and DTL/Moi were expressed at high level upon induction in BL21DE3 Codon Plus RIL cells. HP1 appeared in denaturing gels as two bands, the lower being an N-terminal truncation, which was present even if the cells were lysed directly in SDS sample buffer after harvesting. HipHop expression was consistently low, and despite various attempts which included alterations in construct designs, conditions of induction and choices of host cells and as well trials of co-expression with other terminin proteins, we could not achieve notable expression. The expression of HipHop at low level was, however, verified by immunodetection of the heterologously expressed HA tag-labelled protein [data not shown].


Cross-Species Interaction between Rapidly Evolving Telomere-Specific Drosophila Proteins.

Vedelek B, Blastyák A, Boros IM - PLoS ONE (2015)

Co-expression of interacting partners increases the solubility of terminin proteins.(A) Presumed interactions among terminin proteins based on GST pull-down assays [17–19]. (B) Solubility of terminin components expressed individually in Arctic Express cells. Arrowheads point to bands corresponding to specific terminin proteins. HipHop expression cannot be observed on Coomassie-stained gel. On panel B images of different parts of the same gel are shown. (C) Co-expression of terminin components from polycistronic constructs. On panel C different parts of the same gel are shown except for the part with DTL/Moi-Ver data. L: molecular weight marker ladder, W: whole cell extract, S: supernatant, P: pellet, NC: control supernatant without any heterologous protein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643883&req=5

pone.0142771.g003: Co-expression of interacting partners increases the solubility of terminin proteins.(A) Presumed interactions among terminin proteins based on GST pull-down assays [17–19]. (B) Solubility of terminin components expressed individually in Arctic Express cells. Arrowheads point to bands corresponding to specific terminin proteins. HipHop expression cannot be observed on Coomassie-stained gel. On panel B images of different parts of the same gel are shown. (C) Co-expression of terminin components from polycistronic constructs. On panel C different parts of the same gel are shown except for the part with DTL/Moi-Ver data. L: molecular weight marker ladder, W: whole cell extract, S: supernatant, P: pellet, NC: control supernatant without any heterologous protein.
Mentions: Previous studies have indicated interactions among terminin components as summarized in Fig 3A. In brief: Ver interacts with DTL/Moi and HOAP [18], and DTL/Moi interacts with Ver, HOAP and HP1 [19]. However, HipHop does not interact directly with Ver or DTL/Moi [17, 18, 19]. To verify these interactions and to explore more connections between specific terminin proteins, furthermore to uncover signs of co-evolution which might play a role in speciation, we studied heterologously expressed terminin proteins. Our strategy to obtain recombinant terminin components for complex assembly studies involved cloning cDNA sequences into expression vector(s) and producing the proteins in bacteria. For each of the five proteins we attempted to express, we used cDNA fragments encoding the complete coding regions, nonetheless in some cases we observed expression of shorter products resulting from degradation (see later in more detail). The use of monocistronic constructs revealed that HP1, HOAP, Ver and DTL/Moi were expressed at high level upon induction in BL21DE3 Codon Plus RIL cells. HP1 appeared in denaturing gels as two bands, the lower being an N-terminal truncation, which was present even if the cells were lysed directly in SDS sample buffer after harvesting. HipHop expression was consistently low, and despite various attempts which included alterations in construct designs, conditions of induction and choices of host cells and as well trials of co-expression with other terminin proteins, we could not achieve notable expression. The expression of HipHop at low level was, however, verified by immunodetection of the heterologously expressed HA tag-labelled protein [data not shown].

Bottom Line: The accelerated evolution of terminin components may indicate the involvement of these proteins in the process by which new species arise, as the resulting divergence of terminin proteins might prevent hybrid formation, thus driving speciation.Our results suggest formation of stable subcomplexes of terminin, but not of the whole complex in vitro.We found that the accelerated evolution is restricted to definable regions of terminin components, and that the divergence of D. melanogaster Drosophila Telomere Loss and D. yakuba Verrocchio proteins does not preclude their stable interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Szeged, Szeged, Hungary.

ABSTRACT
Telomere integrity in Drosophila melanogaster is maintained by a putative multisubunit complex called terminin that is believed to act in analogy to the mammalian shelterin complex in protecting chromosome ends from being recognized as sites of DNA damage. The five proteins supposed to form the terminin complex are HP1-ORC associated protein, HP1-HOAP interacting protein, Verrocchio, Drosophila Telomere Loss/Modigliani and Heterochromatic Protein 1. Four of these proteins evolve rapidly within the Drosophila genus. The accelerated evolution of terminin components may indicate the involvement of these proteins in the process by which new species arise, as the resulting divergence of terminin proteins might prevent hybrid formation, thus driving speciation. However, terminin is not an experimentally proven entity, and no biochemical studies have been performed to investigate its assembly and action in detail. Motivated by these facts in order to initiate biochemical studies on terminin function, we attempted to reconstitute terminin by co-expressing its subunits in bacteria and investigated the possible role of the fast-evolving parts of terminin components in complex assembly. Our results suggest formation of stable subcomplexes of terminin, but not of the whole complex in vitro. We found that the accelerated evolution is restricted to definable regions of terminin components, and that the divergence of D. melanogaster Drosophila Telomere Loss and D. yakuba Verrocchio proteins does not preclude their stable interaction.

Show MeSH
Related in: MedlinePlus