The Drosophila Mitochondrial Translation Elongation Factor G1 Contains a Nuclear Localization Signal and Inhibits Growth and DPP Signaling

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 null 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

Intron of the mouse Hoxa-7 gene contains conserved homeodomain binding sites that can functionas an enhancer element in Drosophila

The 5′ flanking sequences and the intron of the mouse Hoxa-7 gene were searched for regulatory elements that can function in Drosophila. Only the intron is able to activate a lacZ fusion gene in various tissues of Drosophila embryos. This enhancer function requires a cluster of three homeodomain binding sites (HB1-element) that are also found in the introns of other Hox genes as well as in a putative autoregulatory element of Ultrabithorax (Ubx), the Drosophila homolog of Hoxa-7. If a single binding site in the autoregulatory element of fushi tarazu (ftz) is replaced by the HB1-element of Hoxa-7, the expression pattern is altered and newly controlled by the homeotic gene caudal (cad). These data suggest that HB1 is a potential target for different homeodomain proteins of both vertebrates and invertebrates

Expression of mutant EF-G1 forms reduces growth and affects patterning in wings.

<p>(A) Heterozygous <i>A9</i>-GAL4/+ female wing with wild type patterning. Longitudinal veins L1-L5 and the anterior and posterior crossveins are indicated (AC, PC). (B) Wings that express four <i>CG4567-G538E</i> transgenes encoding the EF-G1 protein of <i>II032</i> mutants with <i>ptc</i>-GAL4 show normal proportions but exhibit reduced growth between L3 and L4 where the mutant protein is expressed. They also lack the anterior crossvein (arrow). (C) Overexpression of two wild type <i>ico</i> transgenes in wings using <i>A9</i>-GAL4 does not affect patterning but results in smaller wings. (D) In comparison, ubiquitous expression of two <i>CG4567-G538E</i> transgenes reduces growth and interferes with proper vein formation. (E) Expression of three EF-G1 transgenes that lack the C-terminal tail has no apparent effects on growth or patterning. (F) Compared to two full-length mutant transgenes (D), expression of three tailless mutant CG4567-G538E transgenes results in no major pattern defects but reduces the size of wings.</p

Clonal analysis of <i>ico</i> mutants in the eye.

<p>Clones of homozygous <i>ico</i> alleles were generated with <i>eyeless</i>-Flipase in the eyes of females. Homozygous <i>ico</i> clones do not contain eye pigment and are white. Pigmented cells are heterozygous for <i>ico</i>. (A) Clones of the mutation <i>II032</i> do not survive to adulthood. (B–D) Clones of the other three missense mutations exhibit a small numbers of white cells. In contrast, homozygous clones of the two null mutations develop normally (E and F), indicating that EF-G1 function is not required in the eye and that clones expressing C-terminally truncated proteins do not affect growth and patterning. The cause for the black patches of cells that can be clearly seen in <i>G^A1</i>, <i>B^A18</i>, <i>and G^A25</i> but are also present in the other alleles is not entirely clear. However, their presence does not affect the interpretation of the results. See text for details.</p

Rescue of <i>ico</i> mutants by fly and human EF-G1 proteins.

<p>Females with <i>ico</i> mutations and fly or human transgenes on the X or third chromosome <i>(UAS-EF-G1/UAS-EF-G1</i>; <i>ico/CyO</i>; <i>+/+</i> or <i>w/w</i>; <i>ico</i>; <i>UAS-EF-G1/CyO-TM6B)</i> were crossed to males with <i>ico</i> mutations and the ubiquitous GAL4 drivers <i>armadillo</i>-GAL4 and <i>daughterless</i>-GAL4 balanced over chromosomes with dominant curly wing and tubby body markers <i>(w/Y</i>; <i>ico</i><b><i>^{Del EY2}</i></b>; <i>arm+da-GAL4/CyO-TM6B)</i>. The numbers of the rescued homozygous <i>ico</i> mutant females, which have straight wings and are not tubby, were compared to their heterozygous <i>ico</i> sisters, which have curly wings. (Row 3) <i>TP-GFP-CG4567</i> transgenes that express a GFP-tagged <i>Drosophila</i> EF-G1 protein inserted after the N-terminal target sequence (TP) encode a functional protein that can rescue <i>ico</i> mutants. (Row 4, 9, and 10) Transgenes that express the <i>II032</i> mutant EF-G1 or <i>Drosophila</i> EF-G2 (CG31159) proteins do not rescue <i>ico</i> mutants. (Row 5) <i>CG4567-ΔCstop</i> is a transgene with a premature stop codon that lacks the C-terminal tail. (Row 7 and 8) The adults rescued with <i>hEF-G1-N174N</i> and <i>hEF-G1-S321P</i> hatched at least 3-5 days later than their siblings did. (Row 8 and10) The number of the animals rescued with the <i>hEF-G1-S321P</i> or <i>CG31159</i> transgenes was compared to curly and tubby animals with the darker eye color of the <i>arm+da-GAL4</i>.</p

The C-terminal tail of EF-G1 functions as a nuclear localization signal.

<p>(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, <i>Drosophila</i> 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).</p

Sequence alignment of <i>Drosophila</i> and human EF-G1 and EF-G2 proteins.

<p>Sequence alignment of the <i>Drosophila</i> and human EF-G1 and EF-G2 proteins (dEF-G1, hEF-G1, dEF-G2, hEF-G2). The positions of the identified fly and human <i>EF-G1</i> mutations shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016799#pone-0016799-g003" target="_blank">Figure 3</a> are indicated by numbers (1–11) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016799#pone.0016799-Coenen1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016799#pone.0016799-Antonicka1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016799#pone.0016799-Valente1" target="_blank">[6]</a>.</p

The identified mutations in EF-G1 are located in various domains.

<p>Similarities between EF-G1 and bacterial G-factors indicate that the proteins likely share a similar three-dimensional structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016799#pone.0016799-Czworkowski1" target="_blank">[20]</a>. The first and largest domain of EF-G1 is its GTP-GDP binding domain. Six of the identified mutations are located in this domain. The second domain shares homology to the elongation factor EF-Tu. Domains IV and V show similarities with other RNA binding proteins, and structural analysis suggests that Domains III, IV, and V form an interface mimicking the shape of a tRNA that is used to interact with ribosomes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016799#pone.0016799-Nissen1" target="_blank">[13]</a>. In addition to bacterial G-factors and mitochondrial EF-G2 proteins, EF-G1 contains a positively charged C-terminal tail. The deletion EY1 removes 1888 nucleotides, which creates a stop codon six amino acids after P314 and encodes a truncated protein that only consists of the first domain. Similarly, deletion EY2 removes 3663 nucleotides, which creates a stop codon 19 residues past C367 and encodes a protein truncated within domain II.</p

EMS-mutant <i>ico</i> alleles are embryonic lethal and interact with DPP signaling.

<p>(A–D) Cuticle preparations of dead embryos. (A and B) Homozygous or transheterozygous combinations of the <i>ico</i> alleles <i>II032</i>, <i>G^A1</i>, <i>G^A25</i>, <i>and B^A18</i> die as embryos with head involution defects. (C and D) Animals heterozygous for <i>ico</i> EMS-mutant alleles are embryonic lethal in combination with <i>dpp^hr4</i>, indicating that they exhibit reduced DPP signaling.</p

lolal Is an Evolutionarily New Epigenetic Regulator of dpp Transcription during Dorsal-Ventral Axis Formation

Secreted ligands in the Dpp/BMP family drive dorsal-ventral (D/V) axis formation in all Bilaterian species. However, maternal factors regulating Dpp/BMP transcription in this process are largely unknown. We identified the BTB domain protein longitudinals lacking-like (lolal) as a modifier of decapentaplegic (dpp) mutations. We show that Lolal is evolutionarily related to the Trithorax group of chromatin regulators and that lolal interacts genetically with the epigenetic factor Trithorax-like during Dpp D/V signaling. Maternally driven Lolal(HA) is found in oocytes and translocates to zygotic nuclei prior to the point at which dpp transcription begins. lolal maternal and zygotic mutant embryos display significant reductions in dpp, pMad, and zerknullt expression, but they are never absent. The data suggest that lolal is required to maintain dpp transcription during D/V patterning. Phylogenetic data revealed that lolal is an evolutionarily new gene present only in insects and crustaceans. We conclude that Lolal is the first maternal protein identified with a role in dpp D/V transcriptional maintenance, that Lolal and the epigenetic protein Trithorax-like are essential for Dpp D/V signaling and that the architecture of the Dpp D/V pathway evolved in the arthropod lineage after the separation from vertebrates via the incorporation of new genes such as lolal.Peer reviewe