Detection of Dual Targeting and Dual Function of Mitochondrial Proteins in Yeast.

ABSTRACT

Eukaryotic cells are defined by the existence of subcellular compartments and organelles. The localization of a protein to a specific subcellular compartment is one of the most fundamental processes of a living cell. It is well documented that in eukaryotic cells molecules of a single protein can be located in more than one subcellular compartment, a phenomenon termed dual targeting, bimodal targeting, or dual localization. Recently, growing evidence started to accumulate for abundant dual targeting of mitochondrial proteins, which are localized to a second location in the cell, besides this specific organelle. We have termed these dual localized proteins echoforms or echoproteins (echo in Greek denotes repetition). As the research on dual targeting of proteins is developing and evidence is accumulating for high abundance of the phenomenon, there is a growing need for new methods that would allow the identification of dual localized proteins and analysis of their functions in each subcellular compartment. This is particularly critical for single translation products that are encoded by the same gene and are actually derived from the same protein but nevertheless distribute between different subcellular compartments. The above considerations have led us to develop several approaches for studying dual localized proteins and their dual function. These include an α-complementation-based assay, specific depletion, and selection of the individual echoproteins.