Targeting of E. coli beta-galactosidase to the nucleus in yeast.
In order to identify determinants governing nuclear protein localization, we constructed a set of hybrid genes by fusing the S. cerevisiae gene, MAT alpha 2, coding for a presumptive nuclear protein, and the E. coli gene, lacZ, coding for beta-galactosidase. The resultant hybrid proteins contain 3, 13, 25, 67, or all 210 amino acids of wild-type alpha 2 protein at the amino terminus and a constant, enzymatically active portion of beta-galactosidase at the carboxy terminus. Indirect immunofluorescence and subcellular fractionation studies with yeast cells containing the alpha 2-LacZ hybrid proteins indicate that the alpha 2 segment can direct localization of beta-galactosidase to the nucleus. A segment as small as 13 amino acids from alpha 2 is sufficient for this localization. Comparison of amino acid sequences of other nuclear proteins with this region of alpha 2 reveals a sequence that may be necessary for nuclear targeting. Production of some alpha 2-LacZ hybrid proteins causes cell death, perhaps as a result of improper or incomplete localization. These studies also indicate that the alpha 2 protein, argued on genetic grounds to be a negative regulator, acts in the yeast nucleus.
D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology*; D003433:Crosses, Genetic; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005804:Genes, Lethal; D005814:Genes, Viral; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics*
D004262:DNA Restriction Enzymes; D005696:Galactosidases; D001616:beta-Galactosidase