Alpha-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 beta-galactosidase alpha-complementation to study dual distribution of proteins in yeast cells. Using a simple colony color phenotype, we show that alpha-complementation depends on co-compartmentalization of alpha and omega 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 alpha 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 alpha-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.
Karniely
Sharon
S
Department of Molecular Biology, Hebrew University Medical School, Jerusalem 91120, Israel.
Rayzner
Adi
A
Sass
Ehud
E
Pines
Ophry
O
D001483:Base Sequence; D002451:Cell Compartmentation; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005649:Fumarate Hydratase / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D001616:beta-Galactosidase; D005649:Fumarate Hydratase