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. 2011 Feb 25;6(2):e16799. doi: 10.1371/journal.pone.0016799

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This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

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(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).