In plant cells, there are two organelles which were once freely living prokaryotes. Due to two independent endosymbiotic events, they became semi-autonomous cell organelles. Mitochondria are thought to be derived from α-proteobacteria and chloroplasts evolved from cyanobacteria. Indeed those organelles have kept their prokaryotic biochemistry but plastidal and mitochondrial genomes have been reduced to plasmid size (Timmis et al. 2004). This is an effect of intracellular gene transfer in which genes from the organelle are transferred to the nucleus. The driving force of this transfer is not proofed yet but there are several theories. Mutational pressure on the organelle genome, such as DNA damage and lack of recombination, might lead to an accumulation of deleterious mutations and therefore to a relocation of essential genes to the nucleus (McFadden 1999). Once transferred to the nucleus, the gene must be expressed and translocated back to the respective organelle. Therefore, the protein has to be translocated over several membranes and through different suborganellar compartments.
Generally, mitochondria contain an inner and an outer membrane, the intermembrane space and the matrix. Chloroplasts are more complex and contain outer and inner envelopes, the inter membrane space, the stroma, the thylakoid membranes and the thylakoid lumen. Complex import mechanisms are known for both organelles. The chloroplast import machinery has an independent origin similar but different to the mitochondrial import machinery (McFadden 1999), but in general chloroplasts and mitochondria share a common principle of protein translocation over their membranes, ToC/TiC and ToM/TiM complexes. The ToC/TiC complexes are the translocators over the outer and inner envelope of the chloroplast and the ToM/TiM complexes translocate proteins over the outer and inner membrane of mitochondria (Teixeira and Glaser 2013). Protein import into the correct organelle and the correct suborganellar location is essential for its proper function. A critical step for the relocation of the protein is to acquire a so called target sequence, which allows a correct sorting to the target organelle. Therefore, proteins contain an N-terminal extension, the so called transit peptide, which is responsible for sorting into either the chloroplast or the mitochondria (McFadden 1999, Emanuelsson et al. 2000). For further sorting within the organelle an additional target sequence is necessary. This can be added to adjacent to the original trageting sequence, e.g. for thylakoid targeted proteins, or in other regions of the protein (Emanuelsson et al. 2000).
The aim of this study was to check for an import of the radioactive marked protein monodehydroascorbate reductase (MDHAR), translated in vitro from an Arabidopsis thaliana gene, into chloroplasts and mitochondria isolated from pea. MDHAR is an oxidoreductase, which participates in different metabolic pathways, especially in the ascorbate and alderate metabolism. The protein consists of 441 amino acids, which correlates to a molecular weight of 48.4 kDa. The biological role of MDHAR is related to salt stress, water deprivation and symbiosis with fungi (TAIR).
Material and Methods
Experiments were performed as described in the script, except the following aspects:
- Mitochondria concentration was determined by protein concentration measurement via Bradford assay
- After import mitochondria were resuspend in 15µL 2x sample buffer for SDS-PAGE
- For mitoplast preparation import reaction was centrifuged by 16 100 x g at 4°C
- Stromal proteins were precipitated by adding 500 µL 50% TCA
- Stromal proteins were centrifuged for 5 minutes at 16 000 x g after washing with 0.5% TCA
- Western Blot blocking buffer contained 5% milk powder
An in vitro translation was performed on provided MDHAR mRNA. To check whether the translation was successful, a 12.5% SDS-PAGE was performed and the gel was stained with Coomassie. Due to the staining some proteins of the in vitro translation were visible (fig.1A). For detection of the radioactive labelled proteins, the gel was exposed to a radioactivity screen (fig.1B).
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Figure 1: In vitro translation of MDHAR. A) Coomassie stained 12.5 % SDS-PAGE gel B) Radioactivity screen with [35S] labelled methionine.
For mitochondrial isolation, a continuous gradient form 4.4% (w/v) PVP (at the bottom) raising to 0% (w/v) PVP (at the top) was prepared in a centrifuge tube. The grinded plant material could be loaded at the gradient for high speed centrifugation, so that the organelles were able to arrange themselves in the density layer. Mitochondria were visible in the bottom (4% (w/v) PVP) by forming a green-yellow layer. Afterwards, the mitochondrial concentration was measured via Bradford assay at 595nm and calculated. Mitochondria referring to a protein amount of 100 µg was used for import assay.
Import assays were performed with two samples, one with Valinomycin (V+) and one sample without Valinomycin (V-). Valinomycin blocks the potassium channels in the mitochondrial membrane, which inhibits the mitochondrial membrane potential. Therefore Valinomycin samples serve as a negative control. Both samples were divided after import and to one proteinase K was added to digest proteins sticking to the outer membrane of the mitochondria (fig.2, left part).
Chloroplast import assay was performed in a similar way but without blocking the membrane potential. For a digestion of membrane sticking proteins, one sample was treated with thermolysin (fig.2, right part).
The radioactivity screen shows that in all mitochondrial samples a band is visible, even if valinomycin or proteinase K were added (fig. 2 left part), whereas in chloroplast samples no bands are visible (fig. 2, right part).