Andrew C Edmondson and Dr. Brent L Nielsen, Microbiology and Molecular Biology
DNA recombination is the exchange of segments between homologous pieces of DNA, resulting in variations in genetic diversity. Recombination provides essential genetic variation, but can also cause harmful rearrangements, resulting in genetic disorders in mammals, plants and yeast. DNA recombination in plant mitochondria is supported by a variety of evidence, but the mechanisms are poorly understood. Due to evidence of an evolutionary relationship between bacteria and mitochondria, a RecA mechanism has been proposed for mitochondrial recombination. This is supported by a similar mechanism in chloroplasts.
Mitochondria are vital components of both mammal and plant eukaryotic cells. They generate essential energy to carry out metabolic processes in cells. Mitochondria have their own genomes, but many of their vital proteins are nuclear encoded. These genes are translated in the cytoplasm and the proteins must then be transported to the mitochondria. These proteins carry an additional amino acid sequence which serves as a sorting tag (transit peptide) to informthe cell of its proper location. In theory, the cell’s recognition of the transit peptide should not be hindered by any attached protein. Thus, even a foreign protein attached to the transit peptide would be sorted as if it were the protein originally attached to the sorting tag.
Our laboratory has isolated and identified a RecA protein homolog in Arabidposis which is specifically targeted to mitochondria and is related to a RecA protein homolog in chloroplasts (1). Another vital protein for the proposed recombination mechanism is a Single Stranded DNA Binding protein (SSB). SSBs coat single stranded DNA and are vital for DNA replication and recombination. SSBs directly interact with the RecA protein and with single stranded DNA.
SSB bacterial protein sequences were obtained from NCBI (www.ncbi.nlm.nih.gov) and were compared with proteins in the Arabidopsis thaliana database at TAIR (www.arabidopsis.org). These were then analyzed with computer programs to predict the sub cellular localization of each protein. The selected SSB homolog in Arabidposis (At4g11060) was predicted by some programs to be targeted to chloroplasts and by others to mitochondria. An EST of the gene was obtained from Kazusa DNA Research Institute, Chiba, Japan (SQ130f07F). DNA sequencing confirmed that the EST contained the complete coding region for the protein.
A DNA construct containing the coding region for the sorting tag (transit peptide) from the SSB protein fused with the coding region for green fluorescent protein (GFP) was made. The GFP would be easy to recognize because it would shine a bright green color under UV light. The GFP would be targeted to the same organelles that the SSB would have been, allowing us to identify the localization of the SSB protein using a microscope. The localization prediction programs indicated that the tag should be, at most, 28 amino acids in length. We designed two sets of primers to amplify the 84 bases that encoded the predicted 28 amino acid residue transit peptide and engineer restriction sites on either end of the gene segment. The gene fragment amplified by the first set of primers was directionally ligated into the pGFPuv (Clontech) GFP expression vector for in vitro import assays. The gene fragment amplified by the second set of primers was directionally cloned into the pCAMBIA 1302 GFP expression vector for in vivo import assays. Both constructs were confirmed by restriction analysis, PCR, and sequencing.
Top 10 competent cells (Invitrogen) were used to express the recombinant GFP protein attached to the SSB’s transit peptide (TP-GFP). Cells were disrupted and the GFP was isolated as described (2). Chloroplasts and mitochondria were isolated from 50 g soybean leaves on percoll gradients according to (3) and (4), respectively. Organelles were incubated with the isolated TPGFP for 30 minutes at 25°C in import buffer (0.33 M Sorbitol, 50mM Tris-HCl pH 8.0, 2mM MgCl2, 2mM ATP, 2mM NADH, 1mM EDTA, 1mM DTT). Samples were then treated with 100ìg/ml proteinase K for 20 minutes at 25°C. Organelles were reisolated on percoll gradients and washed. They were then visualized with fluorescence and DIC microscopy at 1000X.
Chloroplasts glowed red under UV light, a natural occurrence termed autofluorescence, but did not show any green fluorescence from imported GFP (Figure 1). Mitochondria, however, have no autofluorescence, but our samples glowed green from imported GFP (Figure 2). This suggests that the TP-GFP was only imported into mitochondria, which in turn suggests that the transit peptide from our SSB protein targets specifically to mitochondria and not to chloroplasts.
The creation of our GFP constructs took longer than anticipated and we have not yet performed the in vivo import assays. We are currently awaiting the arrival of BY-2 tobacco cells for transformation with our recombinant pCAMBIA 1302 vector. We hope to complete the in vivo import assays within the next several weeks. Based on our results from the in vitro import assays, we anticipate that we will again see the TP-GFP imported specifically to mitochondria and not to other sub cellular compartments.
Our results provide evidence to support the currently proposed mechanism of mitochondrial recombination, indicating that SSB is imported into mitochondria where it would be available to work in concert with the mitochondria targeted RecA protein. They also provide us with some additional information about mitochondria targeted transit peptides. As indicated by the contradictory results from the sub cellular localization computer programs, functional genomics is still a beginning science which requires supporting evidence from the lab. With the human genome completely sequenced, functions for each protein must be determined. This is a daunting task, but will be accelerated as our understanding of functional genomics increases.
References
- Khazi, F., Edmondson, A., Nielsen, B., manuscript submitted for publication.
- Yakhnin, A. V., et al. (1998) Green Fluorescent Protein Purification by Organic Extraction. Protein Expression and Purification 14, 382-386.
- Cline, K. Isolation of Pea Chloroplasts for Protein Import Studies. www.hos.ufl.edu/clineweb/PeaIsol.html
- Leaver, C. J., et al. (1983) Protein Synthesis by Isolated Plant Mitochondria. Methods in Enzymology 97, 476-484.