The framework of YAO.Channel consists of TOC and TIC complex, which originates from chloroplast translocase complex on outer and inner membrane respectively. The YAO.Channel then enables the selective transit for enzymes or transcriptional factors equipped with YAO.Channel - targeting signal peptides, into YAO. The existing mitochondrial protein transport machine is TOM and TIM complex, and they are employed to assemble the TOC and TIC complex - the new transport machinery, called YAO.Channel. Signal peptides play a key role in this process, determining whether a protein can be targeted to mitochondria, and where a protein will be located in organelle.

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    Figure 1. Three stages towards a new transport machinery.

Signal Peptide BioBricks

        To achieve the goal that various proteins can be equipped with the proper signal peptides so as to be assembled onto membranes of mitochondria or be targeted into the matrix, first we standardized the BioBricks for signal peptides. By referring to dozens of documents, we picked out 3 kinds of signal peptides for mitochondrion-targeting proteins:

        1. Directing proteins onto outer membrane, which can be used to help assemble the TOC proteins onto mitochondria outer membrane;

        2. Directing proteins onto inner membrane, which can be used to help assemble the TIC (Translocase of the Inner membrane of Chloroplast)proteins onto mitochondria inner membrane;

        3. Directing proteins into matrix, which can be used to guide the enzymes or transcriptional factors into matrix of YAO.

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    Figure 2. Signal peptides directing proteins to mitochondria. 1) Three kinds of signal peptides. 2) Directing proteins toward TOM and TIM complex. 3) Protein transported to different destinations.

        Accordingly, 3 kinds of signal peptide BioBricks were constructed, each consists of sequences of signal peptide and the adapter.

        These BioBricks can be confirmed to be functional When they are linked with YFP. Precise location of YFP can be determined by in suite hybridization and electron microscopy.

Assembly of TOC and TIC Complex

        TOC and TIC proteins are the components of TOC (Translocase of the Outer membrane of Chloroplast)and TIC (Translocase of the Inner membrane of Chloroplast) complex. It’s been demonstrated that Cyanidioschizon merolae, a red alga, possesses the most primary transport machinery, with only two Toc proteins (Toc75 and Toc34) in TOC complex and 4 TIC proteins in TIC complex. As the signal peptides targeting to mitochondria and chloroplast are distinct thus ensuring the specificity of YAO.Channel, we decided to use TIC and TOC proteins as the new transport machinery for YAO.

        1. Toc75 is the outer membrane translocation channel in chloroplast, like the Tom40 in mitochondrion, and they both belong to the transmembrane β-barrel protein family, sharing the similar structure. When Toc75 is equipped by Tom40 targeting peptides, it will be recognized by mitochondrion at TOM complex, and then after the passage of TOM complex, it will theoretically be assembled onto the mitochondrial outer membrane with the assistance of SAM complex.

        2. Toc34 is a GTPase protein that functions as signal peptides receptor, N-terminally anchored at chloroplast outer membrane, just as Tom20 and Tom70 do in mitochondria. When equipped with Tom20 or Tom70 targeting peptide, it will theoretically be assembled onto mitochondrial outer membrane in the same way as Toc75, but with the assistance of Mim1 instead of SAM complex.

        3. Presently we only focus on the approaches for assembly of TOC proteins as it’s much easier than the assembly of TIC proteins. Hopefully we would come up with a feasible way in future time to assemble TIC complex.

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    Figure 3. Assembling Toc75 & Tic110 onto mitochondria membranes. 1) Adding mitochondrial signal peptides to TOC & TIC proteins, directing of TOM & TIC proteins toward mitochondria. 2) TOC & TIC installed onto the membranes of mitochondria.

A New Transport Machinery

         When the TOC and TIC proteins were successfully assembled onto the membranes of mitochondria, basically speaking the goal for building up a new transport machinery is achieved. The TOC and TIC proteins will act in tandem to transport proteins across the two membranes of YAO into the matrix, as long as the proteins to be transported are equipped with the signal peptides that can be recognized by Toc34 (Toc34-targeting signal peptides BioBricks are not available presently).

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    Figure 4. Test of the new transport machinery. 1) Adding signal peptides recognized by TOC & TIC to reporters, and directing towards TOC & TIC complex. 2) Reporters transported into mitochondrial matrix. 3) Removal of the original TOM & TIM complex.

        Moreover, to endue YAO with independency in protein transit, i.e. all the proteins for YAO should be transported via only YAO.Channel rather than the original one - TOM complex, we will try to knock down all Tom proteins, and finally select the yeast whose transport machinery is YAO.Channel.