This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ahmed, A. U. Articles by Fisher, P. R. Search for Related Content PubMed PubMed Citation Articles by Ahmed, A. U. Articles by Fisher, P. R.

 Previous Article  |  Next Article 

Eukaryotic Cell, August 2006, p. 1314-1327, Vol. 5, No. 8
1535-9778/06/$08.00+0     doi:10.1128/EC.00386-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Import-Associated Translational Inhibition: Novel In Vivo Evidence for Cotranslational Protein Import into Dictyostelium discoideum Mitochondria

Afsar U. Ahmed,¹ Peter L. Beech,² Sui T. Lay,¹ Paul R. Gilson,² and Paul R. Fisher^1*

Department of Microbiology, La Trobe University, Victoria 3086,¹ Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Victoria 3125, Australia²

Received 27 December 2005/ Accepted 29 May 2006

To investigate protein import into the mitochondria of Dictyostelium discoideum, green fluorescent protein (GFP) was fused as a reporter protein either to variable lengths of the N-terminal region of chaperonin 60 (the first 23, 40, 80, 97, and 150 amino acids) or to the mitochondrial targeting sequence of DNA topoisomerase II. The fusion proteins were expressed in AX2 cells under the actin-15 promoter. Fluorescence images of GFP transformants confirmed that Dictyostelium chaperonin 60 is a mitochondrial protein. The level of the mitochondrially targeted GFP fusion proteins was unexpectedly much lower than the nontargeted (cytoplasmic) forms. The distinction between targeted and nontargeted protein activities was investigated at both the transcriptional and translational levels in vivo. We found that targeting GFP to the mitochondria results in reduced levels of the fusion protein even though transcription of the fusion gene and the stability of the protein are unaffected. [³⁵S]methionine labeling and GFP immunoprecipitation confirmed that mitochondrially targeted GFP is translated at much slower rates than nontargeted GFP. The results indicate a novel phenomenon, import-associated translational inhibition, whereby protein import into the mitochondria limits the rate of translation. The simplest explanation for this is that import of the GFP fusion proteins occurs cotranslationally, i.e., protein synthesis and import into mitochondria are coupled events. Consistent with cotranslational import, Northern analysis showed that the GFP mRNA is associated with isolated mitochondria. This association occurred regardless of whether the GFP was fused to a mitochondrial leader peptide. However, the presence of an import-competent leader peptide stabilized the mRNA-mitochondria association, rendering it more resistant to extensive EDTA washing. In contrast with GFP, the mRNA of another test protein, aequorin, did not associate with the mitochondria, and its translation was unaffected by import of the encoded polypeptide into the mitochondria.

---

* Corresponding author. Mailing address: Department of Microbiology, Thomas Cherry Building, La Trobe University, Bundoora, Victoria 3086, Australia. Phone: 61 3 9479 2229. Fax: 61 3 9479 1222. E-mail: fisher{at}lumi.latrobe.edu.au.

---

Eukaryotic Cell, August 2006, p. 1314-1327, Vol. 5, No. 8
1535-9778/06/$08.00+0     doi:10.1128/EC.00386-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Francione, L., Smith, P. K., Accari, S. L., Taylor, P. E., Bokko, P. B., Bozzaro, S., Beech, P. L., Fisher, P. R. (2009). Legionella pneumophila multiplication is enhanced by chronic AMPK signalling in mitochondrially diseased Dictyostelium cells. DMM 2: 479-489 [Abstract] [Full Text]