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The Drosophila mitochondrial translation elongation factor G1 contains a nuclear localization signal and inhibits growth and DPP signaling.

Trivigno C, Haerry TE - PLoS ONE (2011)

Bottom Line: Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality.We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity.Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, Florida, United States of America.

ABSTRACT
Mutations in the human mitochondrial elongation factor G1 (EF-G1) are recessive lethal and cause death shortly after birth. We have isolated mutations in iconoclast (ico), which encodes the highly conserved Drosophila orthologue of EF-G1. We find that EF-G1 is essential during fly development, but its function is not required in every tissue. In contrast to mutations, missense mutations exhibit stronger, possibly neomorphic phenotypes that lead to premature death during embryogenesis. Our experiments show that EF-G1 contains a secondary C-terminal nuclear localization signal. Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality. We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity. Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low.

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Related in: MedlinePlus

The C-terminal tail of EF-G1 functions as a nuclear localization signal.(A–I) Subcellular localization of GFP tagged EF-G1 (CG4567) and EF-G2 (CG31159) proteins in living S2 cells. (A) GFP is found in the cytoplasm and the nucleus. (B) GFP-tagged EF-G1 that lacks the N-terminal mitochondrial target sequence predominantly localizes to the nucleus. (C) The mutant CG4567-G538E also mainly localizes to the nucleus. (D) In contrast, when the C-terminal tail of EF-G1 is removed, the GFP-tagged protein is found again in the cytoplasm. (E) Similarly, Drosophila EF-G2 is cytosolic and nuclear. (F) Addition of the CG4567 C-tail to EF-G2 enhances its nuclear localization. (G) Addition of the N-terminal mitochondrial target protein sequence (TP) of EF-G1 results in punctate GFP staining. (H) A similar punctate pattern is seen with a GFP-tagged EF-G1 protein that contains the TP. (I) While punctate staining is also seen with the mutant CG4567-G538E, one can detect a faint signal of the protein in the nucleus (arrow, compare to H). (J–O) Co-localization of GST-tagged EF-G1 and EF-G2 proteins (green) with DAPI (blue, nucleus) and MitoTracker (red, mitochondria) in fixed S2 cells. (J) A GST-tagged EF-G1 protein without the TP exclusively localizes to the nucleus. (K) Removal of the C-tail of EF-G1 changes the subcellular localization from the nucleus to the cytoplasm. (L) Unlike EF-G1, EF-G2 without the TP is found in the cytoplasm. (M) Addition of the EF-G1 C-tail to EF-G2 alters the subcellular localization from the cytoplasm to the nucleus. (N) A GST-tagged EF-G1 protein with the N-terminal TP co-localizes with MitoTracker in mitochondria that aggregate due to fixation (green+red = yellow). (O) The mutant TP-CG4567-G538E also localizes to mitochondrial aggregates (yellow). However, in contrast to the wild type protein, significant amounts of the mutant protein are also detected outside of mitochondria (green).
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pone-0016799-g006: The C-terminal tail of EF-G1 functions as a nuclear localization signal.(A–I) Subcellular localization of GFP tagged EF-G1 (CG4567) and EF-G2 (CG31159) proteins in living S2 cells. (A) GFP is found in the cytoplasm and the nucleus. (B) GFP-tagged EF-G1 that lacks the N-terminal mitochondrial target sequence predominantly localizes to the nucleus. (C) The mutant CG4567-G538E also mainly localizes to the nucleus. (D) In contrast, when the C-terminal tail of EF-G1 is removed, the GFP-tagged protein is found again in the cytoplasm. (E) Similarly, Drosophila EF-G2 is cytosolic and nuclear. (F) Addition of the CG4567 C-tail to EF-G2 enhances its nuclear localization. (G) Addition of the N-terminal mitochondrial target protein sequence (TP) of EF-G1 results in punctate GFP staining. (H) A similar punctate pattern is seen with a GFP-tagged EF-G1 protein that contains the TP. (I) While punctate staining is also seen with the mutant CG4567-G538E, one can detect a faint signal of the protein in the nucleus (arrow, compare to H). (J–O) Co-localization of GST-tagged EF-G1 and EF-G2 proteins (green) with DAPI (blue, nucleus) and MitoTracker (red, mitochondria) in fixed S2 cells. (J) A GST-tagged EF-G1 protein without the TP exclusively localizes to the nucleus. (K) Removal of the C-tail of EF-G1 changes the subcellular localization from the nucleus to the cytoplasm. (L) Unlike EF-G1, EF-G2 without the TP is found in the cytoplasm. (M) Addition of the EF-G1 C-tail to EF-G2 alters the subcellular localization from the cytoplasm to the nucleus. (N) A GST-tagged EF-G1 protein with the N-terminal TP co-localizes with MitoTracker in mitochondria that aggregate due to fixation (green+red = yellow). (O) The mutant TP-CG4567-G538E also localizes to mitochondrial aggregates (yellow). However, in contrast to the wild type protein, significant amounts of the mutant protein are also detected outside of mitochondria (green).

Mentions: To express the fusion proteins in S2 cells, the pUAST2 constructs were co-transfected with a plasmid that expresses GAL4 under the control of the actin5C promoter and enhancer. Four days after transfection, we were able to detect robust expression of GFP without a TP in the cytosol as well as in the nucleus of S2 cells using a confocal microscope (Figure 6A). Compared to GFP alone, expression of GFP-CG4567 or the mutant GFP-CG4567-G538E without the TP is predominantly nuclear (Figure 6B and C). In contrast, removal of the C-tail reverts to a mixed cytosolic and nuclear expression pattern (Figure 6D). An EF-G2 fusion protein (GFP-CG31159) that lacks the TP shows a similarly mixed expression pattern (Figure 6E). However, when the tail of CG4567 is added to the C-terminus of CG31159, we again observe a predominantly nuclear localization of the protein (Figure 6F). When the TP is added to GFP or GFP-CG4567, the expression pattern dramatically changes to a punctate pattern that likely corresponds to mitochondria (Figure 6G and H), since TP-GFP-CG4567 encoding transgenes are able to rescue ico mutant flies (Table 1). A similar pattern is seen with the mutant TP-GFP-CG4567-G538E (Figure 6I). However, in contrast to the wild type TP-GFP-CG4567 protein, a faint signal of the mutant protein can be detected in the nucleus (Figure 6H and I, arrows). The finding that only the mutant protein can be detected in the nucleus suggests that the presence of the mutant proteins affects the efficiency of EF-G1 import into mitochondria in wild type cells.


The Drosophila mitochondrial translation elongation factor G1 contains a nuclear localization signal and inhibits growth and DPP signaling.

Trivigno C, Haerry TE - PLoS ONE (2011)

The C-terminal tail of EF-G1 functions as a nuclear localization signal.(A–I) Subcellular localization of GFP tagged EF-G1 (CG4567) and EF-G2 (CG31159) proteins in living S2 cells. (A) GFP is found in the cytoplasm and the nucleus. (B) GFP-tagged EF-G1 that lacks the N-terminal mitochondrial target sequence predominantly localizes to the nucleus. (C) The mutant CG4567-G538E also mainly localizes to the nucleus. (D) In contrast, when the C-terminal tail of EF-G1 is removed, the GFP-tagged protein is found again in the cytoplasm. (E) Similarly, Drosophila EF-G2 is cytosolic and nuclear. (F) Addition of the CG4567 C-tail to EF-G2 enhances its nuclear localization. (G) Addition of the N-terminal mitochondrial target protein sequence (TP) of EF-G1 results in punctate GFP staining. (H) A similar punctate pattern is seen with a GFP-tagged EF-G1 protein that contains the TP. (I) While punctate staining is also seen with the mutant CG4567-G538E, one can detect a faint signal of the protein in the nucleus (arrow, compare to H). (J–O) Co-localization of GST-tagged EF-G1 and EF-G2 proteins (green) with DAPI (blue, nucleus) and MitoTracker (red, mitochondria) in fixed S2 cells. (J) A GST-tagged EF-G1 protein without the TP exclusively localizes to the nucleus. (K) Removal of the C-tail of EF-G1 changes the subcellular localization from the nucleus to the cytoplasm. (L) Unlike EF-G1, EF-G2 without the TP is found in the cytoplasm. (M) Addition of the EF-G1 C-tail to EF-G2 alters the subcellular localization from the cytoplasm to the nucleus. (N) A GST-tagged EF-G1 protein with the N-terminal TP co-localizes with MitoTracker in mitochondria that aggregate due to fixation (green+red = yellow). (O) The mutant TP-CG4567-G538E also localizes to mitochondrial aggregates (yellow). However, in contrast to the wild type protein, significant amounts of the mutant protein are also detected outside of mitochondria (green).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045377&req=5

pone-0016799-g006: The C-terminal tail of EF-G1 functions as a nuclear localization signal.(A–I) Subcellular localization of GFP tagged EF-G1 (CG4567) and EF-G2 (CG31159) proteins in living S2 cells. (A) GFP is found in the cytoplasm and the nucleus. (B) GFP-tagged EF-G1 that lacks the N-terminal mitochondrial target sequence predominantly localizes to the nucleus. (C) The mutant CG4567-G538E also mainly localizes to the nucleus. (D) In contrast, when the C-terminal tail of EF-G1 is removed, the GFP-tagged protein is found again in the cytoplasm. (E) Similarly, Drosophila EF-G2 is cytosolic and nuclear. (F) Addition of the CG4567 C-tail to EF-G2 enhances its nuclear localization. (G) Addition of the N-terminal mitochondrial target protein sequence (TP) of EF-G1 results in punctate GFP staining. (H) A similar punctate pattern is seen with a GFP-tagged EF-G1 protein that contains the TP. (I) While punctate staining is also seen with the mutant CG4567-G538E, one can detect a faint signal of the protein in the nucleus (arrow, compare to H). (J–O) Co-localization of GST-tagged EF-G1 and EF-G2 proteins (green) with DAPI (blue, nucleus) and MitoTracker (red, mitochondria) in fixed S2 cells. (J) A GST-tagged EF-G1 protein without the TP exclusively localizes to the nucleus. (K) Removal of the C-tail of EF-G1 changes the subcellular localization from the nucleus to the cytoplasm. (L) Unlike EF-G1, EF-G2 without the TP is found in the cytoplasm. (M) Addition of the EF-G1 C-tail to EF-G2 alters the subcellular localization from the cytoplasm to the nucleus. (N) A GST-tagged EF-G1 protein with the N-terminal TP co-localizes with MitoTracker in mitochondria that aggregate due to fixation (green+red = yellow). (O) The mutant TP-CG4567-G538E also localizes to mitochondrial aggregates (yellow). However, in contrast to the wild type protein, significant amounts of the mutant protein are also detected outside of mitochondria (green).
Mentions: To express the fusion proteins in S2 cells, the pUAST2 constructs were co-transfected with a plasmid that expresses GAL4 under the control of the actin5C promoter and enhancer. Four days after transfection, we were able to detect robust expression of GFP without a TP in the cytosol as well as in the nucleus of S2 cells using a confocal microscope (Figure 6A). Compared to GFP alone, expression of GFP-CG4567 or the mutant GFP-CG4567-G538E without the TP is predominantly nuclear (Figure 6B and C). In contrast, removal of the C-tail reverts to a mixed cytosolic and nuclear expression pattern (Figure 6D). An EF-G2 fusion protein (GFP-CG31159) that lacks the TP shows a similarly mixed expression pattern (Figure 6E). However, when the tail of CG4567 is added to the C-terminus of CG31159, we again observe a predominantly nuclear localization of the protein (Figure 6F). When the TP is added to GFP or GFP-CG4567, the expression pattern dramatically changes to a punctate pattern that likely corresponds to mitochondria (Figure 6G and H), since TP-GFP-CG4567 encoding transgenes are able to rescue ico mutant flies (Table 1). A similar pattern is seen with the mutant TP-GFP-CG4567-G538E (Figure 6I). However, in contrast to the wild type TP-GFP-CG4567 protein, a faint signal of the mutant protein can be detected in the nucleus (Figure 6H and I, arrows). The finding that only the mutant protein can be detected in the nucleus suggests that the presence of the mutant proteins affects the efficiency of EF-G1 import into mitochondria in wild type cells.

Bottom Line: Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality.We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity.Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, Florida, United States of America.

ABSTRACT
Mutations in the human mitochondrial elongation factor G1 (EF-G1) are recessive lethal and cause death shortly after birth. We have isolated mutations in iconoclast (ico), which encodes the highly conserved Drosophila orthologue of EF-G1. We find that EF-G1 is essential during fly development, but its function is not required in every tissue. In contrast to mutations, missense mutations exhibit stronger, possibly neomorphic phenotypes that lead to premature death during embryogenesis. Our experiments show that EF-G1 contains a secondary C-terminal nuclear localization signal. Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality. We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity. Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low.

Show MeSH
Related in: MedlinePlus