Localization of specific peptides, proteins, and macromolecular complexes to the mitochondria in mammalian cells is generally accomplished through the use of peptide signals that mediate transport to the organelle. Recombinant plasmids have been constructed that contain a fusion protein consisting of the yellow-green variant (referred to as enhanced yellow fluorescent protein; EYFP) of the Aequorea victoria green fluorescent protein (GFP) coupled to the mitochondrial targeting nucleotide sequence from subunit VIII of human cytochrome C oxidase. Upon transcription and translation of the plasmid in transfected mammalian hosts, the mitochondrial localization signal is responsible for transport and distribution of the fluorescent protein chimera throughout the cellular mitochondrial network. Tubular mitochondria can be subsequently visualized using fluorescence microscopy, as illustrated for a variety of established adherent cell lines in Figure 1. The single bandpass emission filter featured by the Nikon YFP HYQ optical block, which was employed to capture these images, produces sharp contrast with little interference from autofluorescence or other fluorescent species.

Plasmid pEYFP-Mitochondria vector gene product expression in various cell types (from both transiently and stably transfected clones; see Figure 1) occurs due to the efficient intracellular translation of a fusion nucleotide sequence combining the enhanced yellow fluorescent protein domain with the mitochondria targeting sequence from subunit VIII of human cytochrome C oxidase, as discussed above. The addition of simian virus 40 (SV40) polyadenylation signals, which are inserted downstream from the chimeric EYFP-Mitochondria fusion sequence, assures proper processing of the transcribed messenger RNA 3' terminus. The fluorescence excitation maximum of EYFP is 513 nanometers and the corresponding emission maximum occurs at 527 nanometers, with a relatively high (approximately 0.60) fluorescence quantum yield. In addition to the four chromophore mutations that shift the fluorescence emission maximum, the nucleotide coding sequence of the EYFP gene contains over 190 silent base alterations, which correspond to human codon-usage preferences that increase translational efficiency.

The collection of specimens illustrated in Figure 1 demonstrates the effectiveness of the Nikon YFP HYQ filter combination for imaging a series of cell lines containing the chimeric EYFP plasmid vector that expresses a fluorescent fusion protein targeted at the intracellular mitochondrial network. The fusion protein is transported into the mitochondria to enable visualization of the subcellular structure in living and fixed cells. Susceptible adherent cell cultures were transfected with the appropriate vector using proprietary lipophilic reagents, and were then cultured for a period of at least 24 hours in nutrient medium supplemented with fetal bovine serum to allow high expression levels of the fluorescent fusion protein.

The enhanced yellow fluorescent protein gene used in these studies contains four amino acid substitutions that shift the emission maximum of green fluorescent protein (GFP) by 18 nanometers, from approximately 509 to 527 nanometers. The gene is optimized with human codons (as described above) and features a consensus Kozak translation initiation signal to achieve higher expression levels in mammalian cell cultures. In general, vectors targeted at specific subcellular organelles contain a fusion gene segment, which couples the EYFP gene to a peptide sequence or complete protein that is localized to a region of interest in living cells.