A rapid screen for putative mislocalization mutations by using a GAL4-hybrid protein.
A GAL4 one-hybrid system was used to develop an indirect assay for intracellular localization. The Kar1 protein, a component of the yeast spindle pole body (SPB), was shown to be targeted to the SPB by electron microscopic analysis, or indirect immunofluorescence methods. To assay localization of the Kar1p by measuring the reporter gene expression on solid media, we constructed Kar1-Gal4 hybrid proteins with or without the SPB localization domain. The long fusion Kar1(299)-Gal4 with the localization domain led to non-growth on SC-His + AT media, and quite a low level of the lacZ reporter gene expression. The short fusion Kar1(107)-Gal4 without the localization domain caused a full activation of the reporter genes, HIS3 and lacZ, indicating that the protein remains in the nucleoplasm. These results suggest that the Kar1(299)-Gal4p fusion protein, localized to the SPB, can be clearly differentiated with the mislocalized protein by assaying the reporter gene expression. By utilizing the Kar1(299)-Gal4 construct, we isolated ten spontaneous mutations that were defective in the Kar1-Gal4p localization. Four of these mutations were in the same complementation group. We propose, therefore, that the Gal4-hybrid localization assay can be utilized in cases where the target organelle or the structure is too small for microscopic analysis, or in the initial screening for mutations defective in localization.
Lee
M Y
MY
Department of Microbiology, College of Natural Sciences, Chungnam National University, Taejeon, Korea.
Kim
J
J
D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics*; D017930:Genes, Reporter; D005820:Genetic Testing / Q000379:methods*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C074559:KAR1 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase