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Import-associated translational inhibition: novel in vivo evidence for cotranslational protein import into dictyostelium discoideum mitochondria

Ahmed, Afsar U., Beech, Peter, Lay, Sui T., Gilson, Paul R. and Fisher, Paul R. 2006, Import-associated translational inhibition: novel in vivo evidence for cotranslational protein import into dictyostelium discoideum mitochondria, Eukaryotic cell, vol. 5, no. 8, pp. 1314-1327, doi: 10.1128/EC.00386-05.

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Title Import-associated translational inhibition: novel in vivo evidence for cotranslational protein import into dictyostelium discoideum mitochondria
Author(s) Ahmed, Afsar U.
Beech, Peter
Lay, Sui T.
Gilson, Paul R.
Fisher, Paul R.
Journal name Eukaryotic cell
Volume number 5
Issue number 8
Start page 1314
End page 1327
Publisher American Society for Microbiology
Place of publication Washington, D.C.
Publication date 2006-08
ISSN 1535-9778
1535-9786
Summary 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.

Language eng
DOI 10.1128/EC.00386-05
Field of Research 060199 Biochemistry and Cell Biology not elsewhere classified
Socio Economic Objective 970106 Expanding Knowledge in the Biological Sciences
HERDC Research category C1 Refereed article in a scholarly journal
Copyright notice ©2006, American Society for Microbiology
Persistent URL http://hdl.handle.net/10536/DRO/DU:30009056

Document type: Journal Article
Collections: School of Life and Environmental Sciences
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Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.