Bidirectional Tet Expression Plasmids

Clontech's fluorescent protein-Mito constructs contain the N-terminal 29 amino acids of COX 8 (including the start codon; see Persic et al., 1997, Gene 187:1-8) which targets the fluorescent protein (FP) to mitochondria. The mitochondrial signal peptide of COX 8 should take the FP to the MATRIX of mitochondria since COX 8 is a protein located to the inner membrane of mitochondria, but our construct does not contain the membrane region of COX 8. We assume (like the authors who used the same or similar constructs: (Rizzuto et al. 1995, Current Biology 5:635); (Rizzuto et al., 1992, Nature 358:325); (Persic et al., 1997, Gene 187:1-8) that the FP in our FP-Mito vectors is located to the matrix. Theoretically, the signal peptide should be cleaved off after the Lys (amino acid 25) (Rizzuto et al., 1989, J. Biol. Chem. 264:10595). If cleavage occurs, there should be 4 amino acids of the mature N-terminus of COX 8 left fused to the FP. It hasn't been proven that this occurs with the FP-Mito constructs, as it would be expected from theory.