α-Complementation as a probe for dual localization of mitochondrial proteins

There are a growing number of proteins which are reported to reside in multiple compartments within the eukaryotic cell. However, lack of appropriate methods limits our knowledge on the true extent of this phenomenon. In this study, we demonstrate a novel application of β-galactosidase α-complementation to study dual distribution of proteins in yeast cells. Using a simple colony color phenotype, we show that α-complementation depends on co-compartmentalization of α and ω fragments and exploit this to probe dual localization of proteins between the cytosol and mitochondria in yeast. The quality of our assay was assessed by analysis of the known dual targeted enzyme fumarase and several mutant derivatives, which are exclusively localized to one or the other of these subcellular compartments. Addition of the α fragment did not abolish the enzymatic activity of the tagged proteins nor did it affect their localization. By examining 10 yeast gene products for distribution between the cytosol and the mitochondria, we demonstrate the potential of α-complementation to screen the mitochondrial proteome for dual distribution. Our data indicate the distribution of two uncharacterized proteins - Bna3 and Nif3 - between the cytosol and the mitochondria.