One idea is, use the mt-specific signal. A protein I have cloned, cardiolipin synthase, is localized EXCLUSIVELY in the inner mt membrane. At4g04870 has 38AA signal peptide, which you can use for yeast expression (look up my FEBS paper and PhD thesis). After this sequence, you could clone-in regular FLAG sequence to ensure you can remove the targeting sequence.

GL and let me know how it worked!

I heard it from my colleague and found the following article that may be useful to you.

Gerard G.M. D'Souza, Sarathi V. Boddapati, Volkmar Weissig

Mitochondrial leader sequence-plasmid DNA conjugates delivered into mammalian cells by DQAsomes co-localize with mitochondria.

Mitochondrion Volume 5, Issue 5, October 2005, Pages 352–358

I think if you can replace the leader sequence of the targeting protein with a leader sequence of a known mitochondrial matrix protein while cloning, then it can lead protein directly to mitochondrial matrix and you may get its expression in that particular compartment.

Good luck with your experiments !

Agreed- if you add a signal sequence you can get it to go directly to the mito. For a matrix-targeted protein see my paper attached

Del Gaizo V, Payne RM. Mol Ther. 2003 Jun;7(6):720-30.PMID: 12788645.

I agree with the fact you can use the presequence of a mitochondrial sequence. However, be sure the protein you take the leader sequence from is really located in the matrix and not a membrane protein like cardiolipin synthase as previously suggested. As far as i know innermembrane proteins only pass the outermembrane and are then incorporated from the intermembrane space into the innermembrane so they actually do not enter the matrix. So better safe then sorry and use a presequence of a real mitochondrial matrix protein.

Good luck!