Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (<i>Nicotiana tabacum L.</i>), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, indicating the possibility of two tandem targeting signals. In this work the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) to generate a series of constructs and introduced into tobacco leaves by <i>Agrobacterium</i>-mediated transient transfection. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined under the confocal microscope. Constructs predicted to be targeted to the chlroplast all localized to the chloroplast. However, this was not the case for constructs that were predicted to be mitochondrial targeted. While some constructs localized to mitochondria, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein which was present in those constructs. In addition, during the process of generating these constructs our sequence analysis indicated a stop codon present at amino acid position 161 of the mature NADP+-ICDH protein from both Xanthi and Petit Havana cultivars of tobacco. This was confirmed by multiple sequencing reactions and created discrepancies with the reported sequence present in the database. The results of this study raise interesting questions with regard to the targeting and processing of NADP+-ICDH.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-03272009-131441 |
| Date | 01 April 2009 |
| Creators | McKinnon, John David |
| Contributors | Smith, Mathew, Gray, Gordon, Bett, Kirstin, Harkness, Troy, Coulman, Bruce |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-03272009-131441/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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