Team:Hong Kong HKUST/Project/module3

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Revision as of 20:29, 27 September 2013

Modules

Glyoxylate Shunt

Protein Trafficking
FA Sensing Mechanism

Protein Trafficking

Overview

In our project, we introduced bacterial glyoxylate enzymes into mammalian cells to create alternative metabolic pathway. However, unlike their native environment in bacteria, the two enzymes needed to find their way through the high compartmentalized system in order to reach the citric acid cycle where they could act on.
To guide the glyoxylate enzymes into the mitochondria where mammalian citric acid cycle resides, we produced recombinant glyoxylate enzymes by attaching the Mitochondrial Leader Sequence (MLS) to their N-termini.
We have also constructed the MLS in its own into standard BioBricks (BBa_K1119000 & BBa_K1119001), and we quantitatively characterized their behavior using GFP reporter.

Biology of Mitochondrial Leader Sequence (MLS)

In eukaryotes, the signal sequence guides the translocation of the newly synthesized peptide.

The story starts with the MLS attached to the N-terminus of the protein of interest. Once the protein of interest is synthesized, this preprotein would remain unfolded by associating with chaperons. The preprotein will stay in the cytosol until the MLS gets recognized by receptor of the TOM complex on the outer mitochondrial membrane. Binding of the MLS to the receptor will trigger the feeding of the peptide through the translocation channel. Afterwards, the MLS will then be handed over to a TIM complex which sits on the inner membrane, which will then open up the channels on the inner membrane and allow the peptide to pass through. Once the peptide is through the double membranes, mitochondrial chaperone will be involved in pulling the peptide into the mitochondria and refold the protein. Lastly, the MLS will be cleaved by signal peptidase and dissociate from the transported peptide. For the MLS we used four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein after the cleavage.(Alberts, 2002)

Reference

Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2010). Essential cell biology. (3rd ed., p. 505). UK: Garland Science.

Bohnert, M., & Pfanner, N. (2007). A dynamic machinery for import of mitochondrial precursor proteins. FEBS Letters, 581, 2802-2810. Retrieved from http://ac.els-cdn.com/S0014579307002530/1-s2.0-S0014579307002530-main.pdf?_tid=95ba5a1a-27a7-11e3-b268-00000aab0f01&acdnat=1380308827_541781deb3d3577ca5de6f02f5152202

Alberts, B. (2002). Molecular biology of the cell. (4th ed., pp. 1050-1061). New York:Garland Science.

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 					<h3>Biology of Mitochondrial Leader Sequence (MLS)</h3>
 <p>In eukaryotes, the signal sequence guides the translocation of the newly synthesized peptide.</p>
-<p>The story starts with the MLS attached to the N-terminus of the protein of interest. Once the protein of interest is synthesized, this preprotein would remain unfolded by associating with chaperons. The preprotein will stay in the cytosol until the MLS gets recognized by receptor of the TOM complex on the outer mitochondrial membrane. Binding of the MLS to the receptor will trigger the feeding of the peptide through the translocation channel. Afterwards, the MLS will then be handed over to a TIM complex which sits on the inner membrane, which will then open up the channels on the inner membrane and allow the peptide to pass through. Once the peptide is through the double membranes, mitochondrial chaperone will be involved in pulling the peptide into the mitochondria and refold the protein. Lastly, the MLS will be cleaved by signal peptidase and dissociate from the transported peptide. For the MLS we used four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein after the cleavage.</p>
+<p>The story starts with the MLS attached to the N-terminus of the protein of interest. Once the protein of interest is synthesized, this preprotein would remain unfolded by associating with chaperons. The preprotein will stay in the cytosol until the MLS gets recognized by receptor of the TOM complex on the outer mitochondrial membrane. Binding of the MLS to the receptor will trigger the feeding of the peptide through the translocation channel. Afterwards, the MLS will then be handed over to a TIM complex which sits on the inner membrane, which will then open up the channels on the inner membrane and allow the peptide to pass through. Once the peptide is through the double membranes, mitochondrial chaperone will be involved in pulling the peptide into the mitochondria and refold the protein. Lastly, the MLS will be cleaved by signal peptidase and dissociate from the transported peptide. For the MLS we used four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein after the cleavage.(Alberts, 2002)</p>
 <img src="https://static.igem.org/mediawiki/2013/8/87/MLS_mechanism.png" >
 				</div>